studies on the antimicrobial and immunomodulating

Studies on the Antimicrobial and

Immunomodulating Properties of Plant Extracts on

Bacterial Pathogens

THESIS SUBMITTED

THE FULLFILLMENT OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

AMBER FAROOQUI

IMMUNOLOGY AND INFECTIOUS DISEASES RESEARCH

LABORATORY

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF KARACHI

APPROVAL SHEET

TITLE OF THESIS

Studies on the Antimicrobial and Immunomodulating Properties

of Plant Extracts on Bacterial Pathogens

NAME OF CANDIDATE Amber Farooqui

DEGREE OFFERED Doctor of Philosophy

SUPERVISOR EXTERNAL EXAMINER

Prof. Dr. Shahana Urooj Kazmi Dean Faculty of Science & Professor of Microbiology University of Karachi

In the Loving Memory of My Mother

“Naheed Shakoor Usmani”

Her strong perosnlity with soft heart is key inspiration and her prayers are priceless assets of my life

Acknowledgment

I am grateful to Allah (Subhan wa Tala) who gave me strength and passions to put continuous efforts making this dissertation in presentable form. I pray Him to give me more strength and wisdom and enables me to spread this light to others.

This is a great opportunity for me to thank my research supervisor, Dr. Shahana Urooj Kazmi, Dean-Faculty of Science and Professor of Microbiology, University of Karachi for allowing me freedom to purse my scientific thoughts throughout this study. Her immense guidance, continuous support and personal interest have made this thesis a success. I feel no hesitation to say that Prof. Kazmi’s broad vision, professional approach and friendly behavior are helpful in personality building process of her students including me.

I would like to pay my sincere gratitude to Prof. Dr. Salvatore Rubino Microbiology and Virology Division, Department of Biomedical Sciences, University of Sassari, Italy for giving me opportunity to be the part of his research group and providing a friendly working atmosphere in his lab- a home far from home.

My journey towards the accomplishment of this dissertation was never possible without my friend, Mr. Adnan Khan who was always there to help me. Especially his extreme support and care during ups and downs of my life enabled me to remain consistent towards my studies. I would like to express my sincere gratitude to my friend, Mr. Saeed Khan for his timely and valuable suggestions and help to make my way easier. I would like to avail this opportunity to thank my friend, Ms. Sania Siddiq for her continuous support from the day one when I stepped in IIDRL.

I am truly thankful to Dr. Nafisa Hassan Ali from whom I learn how to initiate lab work and to Ms. Nazia Bibi, my long running colleague and friend for great time we had together in IIDRL. I am also indebted to all members of IIDRL especially Dr. Nazir Ahmed, Dr. Naveed Faraz, Mrs. Mehmooda Kazmi and others for their support.

My special thank goes to Dr. Bianca Paglietti for her guidance and help. The other faculty members of Sassari University Prof. Sergio Uzzau, Prof. Stefania Zanetti, Prof. Paola Rappelli, Dr. Ana Maria and all staff members of Micro-Sassari University for their technical expertise and help. Also, many thank goes to Dr. Viviana Santercole and Dr. Salvatore Corbu of Porto Conte Research-Italy for their cooperation in getting MALDI-TOF work done. I would like to thank Dr. Rehana Afzal and Ms. Fahima Rashid of Chemistry Department-KU

for helping me to carry out chemistry portion. I would like to extend my thanks Dr. Qurban Ali for providing space in NVL-Islamabad to work on animal pathogens. Mr. Aftab Ahmed Khan and Dr. Aamir Ahmed of HEJ also deserve my sincere gratitude.

Above all, I would never be able to make it without prayers and support of my father, Mr. M. Qamar udduja Farooqi from whom I learn how to be patient and have strong believe in Allah. My brother, Mr. Saad Shahid Farooqi deserves my especial thanks for his support, love and sufferings he had through during my study period.

Finally, I am thankful to Higher Education Commission of Pakistan for giving financial support to carry out this work.

CONTENTS

Title Summary Urdu Translation of Summary CHAPTER ONE: INTRODUCTION 1 Antimicrobial Resistance 1.1 Development of Antibiotics‐History

1.2 Antibiotic Resistance‐ Current Global Status A: Staphylococcus aureus B: Escherichia Coli C: Salmonella D: Pasteurella multocida E: Mycobacterium tuberculosis 1.3 How Bacteria Become Resistant 1.3.1 Intrinsic Resistance 1.3.2 Acquired Resistance A: Mutation and Selection B: Exchange of Genes 1.3.3 Physiological Mechanisms 1.4 Other Factors contributing towards Drug Resistance 2 Herbal medicines 2.1 Problems and Challenges 2.1.1 Slow Methodologies 2.1.2 Limited Availability of Plant Material 2.1.3 Low Investment 2.1.4 Decreasing Plant Resources 2.2 Methods/ Approaches in Herbal Medicine

A Selection of Plant B Extraction C Screening of Crude Extracts D Bio‐assay guided Fractionation E Purification and Chemical Characterization of Bioactive

Components

2.3 Types of Biological Activities 2.3.1 2.3.1 Anti‐cancer Activity 2.3.2 2.3.2 Nervous System Suppressing / Activating or Analgesic

Activity

2.3.3 2.3.3 Cardiovascular/ Metabolic 3 Antimicrobial Activity 3.1 Antibacterial Activity 3.2 Strategies for Eradication of Bacterial Infection 3.3 Synergistic Antibacterial Combinations 3.4 Antimycobacterial Activity 3.5 Anti‐parasitic Activity 4 Immunomodulation 4.1 Immunomodulation and Phagocytosis

4.2 Immunomodulation and Humoral Immune Response 4.3 Immunomodulation and Oxidative Challenge

LITERATURE REVIEW OF THE PLANTS USED IN THIS STUDY

A Camellia sinensis B Juglans regia C Hippophae rhamnoides CHAPTER TWO: MATERIAL AND METHODS

2.1 Collection, Isolation and Characterization of Bacterial Pathogens

2.1.1 Characterization of Bacterial Pathogens by Conventional Methods

A Identification B Antibiotic Susceptibility Pattern

2.1.2 Characterization by Molecular Methods

A Plasmid Analysis i) Bacterial DNA Extraction ii) Incompatibility grouping of plasmids by PCR B Determination of Class 1 Integron C Analysis of Conserved Region of Class 1 Integron D PCR for dfrA7

2.1.3 Pulse Field Gel Electrophoresis

Collection, Preparation and Characterization of Plants

2.2.1 Plants Collection 2.2.2 Preparation of Aqueous Extracts 2.2.3 Preparation of Organic Extracts 2.2.4 Bioassay‐guided Chemical Analysis of Extracts A Thin Layer Chromatography B Bioautography C MALDI‐TOF‐MS 2.2.5 Isolation of newly purified compound from

Camellia sinensis

2.3 Antimicrobial Activity of Plants and Plant derived Substances

2.3.1 Agar Well Diffusion Method

2.3.2 Determination of MIC of Plants and Plant derived Substances by Agar Dilution Method

2.3.3 Determination of MIC of Plants and Plant derived Substances by Microbroth Dilution Method

2.3.4 Determination of MIC of Plants and Plant derived Substances by Tube Dilution Method

2.3.5 Determination of Minimum Bactericidal Concentration (MBC) of Plants and Plant derived Substances

2.3.6 Effect of Plants and Plant derived Substances on Time Kill Kinetics of Bacterial Pathogens

2.3.7 Antimicrobial activity of Plant Extracts in combination with

Antibiotics A Checkerboard Titration Method for Synergistic Studies B Disc Diffusion/ Agar incorporation Method for Synergistic

Studies

C Etest strip/ Agar incorporation Method for Synergistic Studies

D Effect of Synergistic Antimicrobial Combinations on Time Kill Kinetics of Bacterial Pathogens

2.3.8 Effect of Plant Extracts on Bacterial cell Morphology

2.3.9 Effect of Plant Extracts on Protein Profiles of Bacterial Pathogens

2.3.10 Antimycobacterial Activity of Plant Extracts 2.3.11 Anti‐Trichomonas Activity of Plant Extracts 2.4

In‐Vitro Toxicity Studies of Plants

2.4.1 Hemolytic Activity of Plants and Plant derived Substances 2.4.2 Cytotoxicity Plant Extracts against Human Vascular

Endothelial cells

2.4.3 Free Radical Scavenging Activity of plant Extracts 2.5

Immunopharmacological Studies of Plants

2.5.1 2.5.1 Animal Toxicity Studies of Plant Extracts 2.5.2 2.5.2 In Vivo Antimicrobial Activity 2.5.3 Intracellular Killing in Phagocytic Cells in the Presence of

Plants

2.5.4 Effect of Plants on Humoral Immune Response

3 CHAPTER THREE: RESULTS

3.1 Collection, Isolation and Characterization of Bacterial isolates 3.1.1 Characterization of bacterial strains by conventional

method

3.1.2 Characterization of Salmonella enterica 3.1.3 DNA fingerprinting by pulse Gel Electrophoresis 3.2 Bioassay‐guided chemical characterization of Plants 3.2.1 Camellia sinensis (Green Tea) 3.2.2 Juglans regia (Dandasa) 3.2.3 Hippophae rhamnoides (Sea buckthorn) 3.3 Antimicrobial Activity of Plants and Plant derived Substances 3.3.1 Camellia sinensis (Green Tea) 3.3.2 FA‐CS II, a newly purified compound from Green Tea 3.3.3 Juglans regia (Dandasa) 3.3.4 Hippophae rhamnoides (Sea buckthorn) 3.3.5 Synergistic Antimicrobial Combinations 3.3.6 Effect of Plant Extracts on Bacterial Cell Morphology 3.3.7 Effect of Plant Extracts on Protein Profiles of Bacteria 3.3.8 Antimycobacterial Activity of Plant Extracts 3.3.9 Anti‐Trichomonas Activity of Plant Extracts

3.4 In‐Vitro Toxicity Studies of Plants 3.4.1 Hemolytic activity of Plants and Plant derived Substances 3.4.2 Cytotoxicity of Plant Extracts against Human Vascular

Endothelial cells

3.4.3 Free Radical Scavenging Activity of Plant Extracts 3.4.4 Effect of Plant Extracts on Cell Proliferation by 3H Thymidine

Incorporation

3.5 Immunopharmacological Studies 3.5.1 Animal Toxicity Studies of Plant Extracts 3.5.2 In‐vivo Antimicrobial Activity 3.5.3 Intracellular killing in Phagocytic Cells in the Presence of

Plants

3.5.4 Effect of Plants on Humoral Immune Response

CHAPTER FOUR: DISCUSSION

References Appendix List of Abbreviation

List of Tables

S # Title

1 List of Anti Cancer Drugs in Clinical Trials

2 a List of Clinical Bacterial Isolates

b List of Reference Bacterial Strains

3 a Identification Scheme for Gram Positive Cocci

b Identification Scheme for Gram Positive Rods

c Identification Scheme for Gram Negative Rods

4 Genotypic Characterization of Escherichia Coli Isolates

5 Oligonucleotides used for Identification of Resistant Genes in Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A

6 Solvent Mixtures Used in Thin Layer Chromatography

7 List of Matrixes used for MALDI-TOF-MS

8 Parameters for MALDI-TOF Acquisitions

9 List of Isolates of Mycobacterium species

10 Preparation of Challege Dose of Pasteurella multocida for LD50Determination

11 Reference Interpretive Standards and MIC Breakpoints of Antibiotics against Staphylococcus aureus

12 Reference Interpretive Standards and MIC Breakpoints of Antibiotics against Enterobacteriacae

13 a Molecular Charaterization of Salmonella enterica serovar Typhi

b Molecular Characterization of Salmonella enterica serovar Paratyphi A

14 List of Plants

15 Summary of Bioassay-guided Chemical Analysis of Bioactive Compound(s) of Camellia sinensis

16 SummaryofBioassay-guided Chemical Analysis of Bioactive Compound(s) of Juglans regia

17 Summary of Bioassay-guided hemicalAnalysis of Bioactive Compound(s) of Hippophae rhamnoides

18 Antimicrobial Activity of camellia sinensis against a wide range of intracellular and extracellular bacterial pathogens

19 Antimicrobial activity of juglans regia against intracellular and extracellular pathogens

20 Antimicrobial Activity of Hippophae rhamnoides

21 Synergistic Antimicrobial Activity of Juglans regia with Oxacillin against MRSA

22 Synergistic Antimicrobial Activity of Camellia sinensis with Nalidixic acid against Salmonella enterica serovar Typhi by Disc Diffusion/ Agar Incorporation Method

23 Synergistic Antimicrobial Activity of Camellia sinensis with Nalidixic acid against Salmonella enterica serovar Typhi by Checkerboard Titration Method

24 Antimycobacterial Activity of Plant Extracts

25 Hematological and Biochemical Parameters during Acute Animal Toxicity Studies of Camellia sinensis

26 Hematological and Biochemical Parameters during Sub-acute Animal Toxicity Studies of Camellia sinensis

27 Hematological parameters during acut eanimal

toxicity studies of juglans regia

28 Biochemical Parameters during Acute Animal Toxicity Studies of Juglans regia

29 Hematological Parameters during Sub-acute Animal Toxicity Studies of Juglans regia

30 Biochemical parameters during sub acute animal toxicity studies of juglans regia

List of Figures

SNo Title 1 Schematic Diagram of class 1 integron 2 Widely Used Chemical Fractionation Scheme for Plants 3 Function of Phagocytic Cells 4 Effect of Antibiotics on Phagocytosis 5 Caspase Activation pathway 6 Major Green Tea Catechins 7 Schematic Diagram of the Principle of MALDI-TOF-

8 Antimicrobial Susceptibility Pattern of Staphylococcus aureus

9 Antimicrobial Susceptibility Pattern of Diarrheal Isolates of Escherichia coli

10 Antibiotic Susceptibility Pattern of Uropathogenic Escherchia coli

11 Antibiotics Susceptibility Pattern of Salmonella enterica serovar Paratyphi A

12 Antibiotics Susceptibility Pattern of Salmonella enterica serovar Typhi

13 Plasmid Analysis of Salmonella enterica Isolates 14 Plasmid Incompatibility Grouping of Salmonella

enterica

15 100bp DNA ladder 16 Analysis of Class I Integron in Salmonella enterica 17 Analysis of 3´ conserved variable segment (CS 5´3´) of

intI1 gene

18 Analysis of the Presence of Trimethoprim Resistance Cassette (dfrA7)

19 DNA fingerprinting of Salmonella enterica by Pulse Field Gel Electrophoresis

20 Pattern of Different Pulsotypes of Salmonella Typhi 21 Chemical Characterization of Plant Extracts by Thin

Layer Chromatography

22 Bioautography of Plant Extracts run in Solvent System F

23 MALDI-TOF-MS Analysis of Camellia sinensis 24 Structure of FA-CS II 25 Bioautography of Plant Extracts run in Solvent System

26 MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. E) of Juglans regia

27 MALDI-TOF-MS Analysis of Bioactive Spot # 5 (Sol. E) of Juglans regia

28 MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. F) of Juglans regia

29 MALDI-TOF-MS Analysis of Bioactive Spot # 5 (Sol. F) of Juglans regia

30 MALDI-TOF-MS Analysis of Bioactive Spot # 1 (Sol. E) of Hippophae rhamnoides

33 MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. F) of Hippophae rhamnoides

34 MALDI-TOF-MS Analysis of Bioactive Spot # 6 (Sol. F) of Hippophae rhamnoides

35 Susceptibility Profile of Camellia sinensis 36 Antimicrobial Activity of Camellia sinensis against

MDR Salmonella enterica serovar Typhi strains

Effect of Camellia sinensis on Time Kill Kinetics of MRSA

Effect of Camellia sinensis on Time Kill Kinetics of MSSA

39 Effect of Camellia sinensis on Time Kill Kinetics of Staphylococcus aureus ATCC 29213

40 Effect of Camellia sinensis on Time Kill Kinetics of ETEC

41 Effect of Camellia sinensis on Time Kill Kinetics of EPEC

42 Effect of Camellia sinensis on Time Kill Kinetics of EAggEC

43 Effect of Camellia sinensis on Time Kill Kinetics of Uropathogenic E. coli

44 Susceptibility Profile of Juglans regia on Intracellular and Extracellular Pathogens

45 Effect of Juglans regia on Time Kill Kinetics of MRSA

46 Effect of Juglans regia on Time Kill Kinetics of MSSA 47 Effect of Juglans regia on Time Kill Kinetics of

Staphylococcus aureus ATCC 29213

48 Effect of Juglans regia on Time Kill Kinetics of ETEC 49 Effect of Organic Fractions of Juglans regia on Time

Kill Kinetics of MRSA

50 Antimicrobial Activity of Hippophae rhamnoides against Pasteurella multocida

51 Synergistic Antimicrobial Activity of Juglans regia with Oxacillin against MRSA

52 Effect of Juglans regia on Cell Morphology of MRSA

53 Effect of Plant Extracts on Protein Profiles of MRSA 54 Effect of Plant Extracts on Protein Profiles of ETEC 55 Immunoblot Analysis 56 Anti-Trichomonas Activity of Plant Extracts 57 Hemolytic Activity of Plants and Plant derived

Substances

58 Cytotoxicity of Plant Extracts against Human Vascular Endothelial Cells

59 Free Radical Scavenging Activity of Hippophae rhamnoides

60 Effect of Plant Extracts on Cell Proliferation 61 In Vivo Antimicrobial Activity of Camellia sinensis

against MRSA in Experimental Murine Septicemia (a-b)

62 Effect of Hippophae rhamnoides on Organ Dislocation of Pasteurella multocida(A-F)

63 Intracellular Killing in Phagocytic Cells in the presence of Camellia sinensis and FA-CS II

64 Intracellular Killing in Phagocytic Cells in the presence of Juglans regia

65 Effect of Plants on Humoral Immune Response

Summary

A number of life threatening infections due to various intracellular and extracellular

bacterial pathogens are major cause of death in Pakistan. In the situation where it is

already difficult for the people, living with low socio-economic status and limited

health facilities to get better treatment options, emergence of antibiotics resistance

among these serious pathogens is a new challenge for the medical community.

Organisms with multi-drug resistance pattern like MRSA, MDR Salmonella, MDR

and XDR Mycobacterium tuberculosis are increasingly reported. Various

approaches from the use of vaccines to the discovery of novel drug targets have

been undertaken to combat this situation. It is therefore important to look for more

effective, safer and less toxic alternate options of treatment. Natural sources like

plants are rich in great variety of bioactive components and have long been used as

alternate regime in traditional medicine. WHO has also recommended member

countries to set up strategies for the safe use of traditional medicine. In view of

present scenario, we decided to explore antimicrobial properties of some famous

Pakistani plants of medicinal value.

Three different indigenous plants, included in this study due to their easy

availability and common use as food and cosmetic product were Camellia sinensis

dried leaves or Green Tea, a commonly used beverage, Juglans regia or dried bark

of Persian Walnut tree (locally known as Dandasa), a very famous teeth brightening

and lip decorating substance among the females of NWFP region of Pakistan and

Hippophae rhamnoides or Sea buckthorn berries, an abundantly found shrub in

Northern Areas of Pakistan and used as major ingredient of jam, jelly and juices. A

total of 377 clinical and 11 reference isolates of different intracellular and

extracellular bacterial pathogens were isolated from different clinical specimens,

identified, characterized and screened for antimicrobial susceptibility against

standard antibiotics , aqueous and organic extracts of these plants and plant derived

substances. After preliminary examinations, plants were subjected to bio-assay

guided chemical analysis and compound purification. Later on, studies on in-vivo

antimicrobial activity, mechanism of antimicrobial action and

immunopharmacological properties were determined carried out.

In order to locate plants bioactive component(s), a novel combinatorial approach of

bioautography and MALDI-TOF-MS was undertaken in addition with conventional

chromatography techniques. Bioactive spots located by bioautography of crude

extracts were directly subjected to MALDI-TOF-MS. This method, due to its

ability to analyze complex samples, gave clear spectra that can be directly linked

with antimicrobial activity. Several low molecular weight compounds with m/z

416, 444, 655, 860, 861, 862 in case of Juglans regia, 416, 440 in Hippophae

rhamnoides and 416, 438, 854 and 861 in Camellia sinensis were observed. During

the course of study, we were successful to isolate a new purine class alkaloid, FA

CS-II from Camellia sinensis of Pakistani origin. The compound was later found to

be antimicrobial against wide range of pathogens which suggested the possible

synergistic contribution of this component with major catechins in exhibition of

antimicrobial activity.

Among the extracellular organisms, Camellia sinensis showed antimicrobial

activity in bactericidal manner whereas static effect was observed against gram

negatives like ETEC. On the basis of higher MIC levels, complete inhibition of

ETEC exoproteins and persistence of 37kda protein, presumably ompA- a porin

which persists under stressed condition, due to the presence of Camellia sinensis,

we can give hypothesis that green tea may inhibit the virulence factors of gram

negative bacteria completely, leaving the organism alive. However, among gram

positives, it was interesting to note that Camellia sinensis exhibited better activity

against MRSA (MIC 0.19mg/ml). The bactericidal activity of FA-CS II with MIC

125µg/ml and MIC/MBC 0.5 against MRSA was comparable to epigallocatechin

gallate-the main tea catechin (MIC 100µg/ml as per previous reports). Our results

were further confirmed by observing drastic effect of Camellia sinensis on cell

morphology like thick intracellular material under Transmission electron

microscope. Moreover, status of MRSA virulence factors under stressed conditions

due to treatment with green tea was checked that clearly indicates the inhibition of

surface expressed proteins and supports electron microscopy observations. In-vivo

efficacy was proved by the significant reduction in organ bio-load in murine model

of disseminated septicemia that confirms the capability of Camellia sinensis to treat

systemic MRSA infections.

Juglans regia also found to be more effective against MRSA among all

extracellular pathogens. Significant antimicrobial activity was recorded in aqueous

(MIC 0.31mg/ml), n-hexane fraction (MIC 32µg/ml) and sub-fraction PP 1 (MIC

25µg/ml).Swollen, de-shaped and completely hollow bacterial cells seen in the

electron micrograph as well as inhibition of cell associated / structural proteins of

MRSA strains indicated the presence of anti-staphylococcal component in this

plant targeting bacterial cell wall. Juglans regia extract also showed strong synergy

with oxacillin (FICI 0.193) with all strains of Staphylococcus aureus, irrespective

of methicillin resistance. Though, to better understand mechanism of synergy, it is

important to identify nature of bioactive component(s) and their interaction with

other inhibitors of peptidoglycan synthesis.

Our third plant, Hippophae rhamnoides revealed most promising activity in

aqueous crude extract (MIC 50µg/ml and MBC 100µg/ml) against Pasteurella

maltocida serotype B2, isolated from water buffloes with Hemorrhagic septicemia

(HS) infection. In-vitro findings were further confirmed in mice model of HS

where Hippophae rhamnoides was found to protect mice infected with 104 x LD50

of virulent strain by exerting effect on multiple organ dislocation of pathogen. This

observation strongly suggested the possible use of Hippophae rhamnoides as

prophylactic supplement in animal feed to prevent HS among large ruminants, a

major economic loss of livestock and dairy industry.

The intracellular Mycobacterium species were also tested against aqueous and

methanolic extracts of these plants. Methanolic extracts showed better activity than

aqueous extracts. MICs of Camellia sinensis against Mycobacterium tuberculosis

H37Rv and clinical strains of XDR TB, MDR TB were 0.75mg/ml, 1.25 mg/ml and

2.5 mg/ml respectively whereas Hippophae rhamnoides and Juglans regia gave

MICs 0.75mg/ml against all strains. Salmonella enterica serovar Typhi (S. Typhi)

and Paratyphi A were another facultative intracellular pathogen included in our

study. Most of the isolates were MDR that was further confirmed by the presence

of INCH1 plasmid and class 1 integron containing various resistance cassettes.

Camellia sinensis was found to be most promising candidate against genetically

diverse variety of Salmonellae (confirmed by PFGE) alone at MIC 1.56mg/ml and

in combination with nalidixic acid (FICI 0.37) against MDR Salmonellae (R-type:

AmpCSxtTNA). Generalized behavior of green tea was again proved when we

observed lower MIC level against MDR strains as compare to antibiotic sensitive

strains. On the basis of strong antimicrobial activity of Camellia sinensis and

Juglans regia against MDR pathogens generally, we may suggest to check the

presence of efflux pump inhibiting compounds in these plants.

It is an undeniable fact that host immune response plays a pivotal role in the

eradication of infection. Keeping this in mind, a portion of this study was focused

on the evaluation of these plants to modulate various aspects of host immune

response. First was phagocytosis in which green tea and FA-CS II were found to be

inhibitory for intracellular growth of MRSA inside polymorphonuclear leucocytes.

Especially FA-CS II also found to be successful to stop the growth of intracellular

MRSA at concentrations below MIC. Second aspect was antibody production and

antibody producing B-lymphocytes. By using Sheep RBCs as antigen,

hemagglutination and hemolytic plaque methods were undertaken to evaluate the

effect of plant extracts on above mentioned important aspects of humoral immune

response. Among all plants, Camellia sinensis was found to be more promising. A

two-fold increase in PFCs and eight-fold increase in antibody titer of the animals

primed with multiple doses of aqueous extract of Camellia sinensis was observed

that suggest its possible role in B cell differentiation, however, FA-CS II increased

antibody producing cells but was unable to exert significant effect on antibody

production. Since it is preliminary data, further studies are required to reach on

final conclusion. Anti-oxidant activity of plants was also analyzed. A

concentration dependent free radical scavenging activity of Hippophae rhamnoides

with no adverse effect on thymidine incorporation was observed.

Plants were found to be non-toxic by toxicity studies carried out at three different

levels; human RBCs, human vascular endothelial cell line (ECV304) and in

BALB/C mice. Absence of any significant hemolytic activity on human RBCs

excludes the presence of direct membrane toxicity. In addition, all plants were also

found to be non-toxic on other mammalian cells and in animals.

In conclusion, our study not only proves antimicrobial and immunomodulating

potential of our indigenous flora but also provides a scientific basis for their

traditional use. In order to move towards drug development process, it is important

to carry out purification of bioactive components for mechanistic and

pharmodynamic studies.

INTRODUCTION

(Literature Review of Antimicrobials and Host Defense Systems)

1) Antimicrobial Resistance It is an unavoidable fact that antibiotics have long been considered as “Wonder

Weapon” that play a pivotal role to revolutionize the treatment of common

bacterial infections and ultimately lead to reduction in mortality. Shiny start of

antibiotic era, on one hand, saved the lives of humans, animals and plants but

was also bundled with a lot of threats in terms of antibiotic resistance. As many

antibiotics are developed, as many ways bacteria find to combat them.

Progressive increase in antibiotic resistance has now become a global concern.

Common bacterial infections now have become cause of death due to no or very

less treatment choices left. Easy availability, unregulated use, animal feed-

heavily loaded with antibiotics and emergence of new infectious diseases are

listed among major contributors of present situation. In order to understand the

recent global threat and factors contributing towards development of antibiotic

resistance, it is important to first look at the history of antibiotic development.

1.1) Development of Antibiotics-History From where do the drugs come? The question comes in mind first. There are

only two known sources, one includes synthetic chemicals, such as

sulfonamides and quinolones, and the second, natural products i.e. fermentation

products of microorganisms, extracts/ compounds of higher plants of marine

and terrestrial origin. The trend in recent years has been more focused on

rational drug design thus favors the synthetic chemical approach but structural

diversity of natural products still has its charm.

Concept of antimicrobial therapy came from ancient times, when natural

products are used for the treatment of different ailments, without any scientific

basis. In 1619, people used cinchona bark (quinine) to treat malaria and the

roots of emetine to treat amoebic dysentery (1, 2). After first few reports, the

term ‘Antibiosis’ was introduced by Paul Vuillemin, in 1889 which, was later

considered as parental term for antibiotics. 19th centaury started with many

developments in this field. Probable use of dyes as antimicrobial agents was

first reported by Paul Ehrlich in early 1900s. In 1909, Salvarsan was discovered

(3). Alexander Fleming was honored Nobel prize for his great discovery

Penicillin (4). In 1932, Mietzsch and Klarer, synthesized a dye bound to

sulfonamide group, named Prontosil red which was further tested for

antibacterial activity (5). In 1948, Brotzu et al purified some penicillin like

compounds from a fungus. The compounds were then known as cephalosporins

and then a series of antibiotics were introduced over the period of 60 years. In

last two decades, more efforts were employed on synergistic combination of

drugs rather introducing novel class. The approach of bacterial genome

sequencing, designing of synthetic drugs according to their variation have not

been fruitful yet and emergence of multidrug resistance organisms even worsens

the scenario.

1.2) Antibiotic Resistance- Current Global Status Antibiotic resistance started with the time of its development. However,

resistance is on heights since last two decades and it seems that now we are in

post-antibiotics era, which was predicted almost a decade ago. The

consequences of resistance among bacterial pathogens can be measured by

increased magnitude of morbidity, higher rates of mortality and greater costs of

hospitalization for patients infected with resistant bacteria relative to those

infected with sensitive strains. In developing countries, infectious diseases

remain the main cause of death (6). Some specific examples of extracellular

and intracellular microbial species that have developed significant resistance

over the years are as follows,

A. Staphylococcus aureus Staphylococcus aureus has been reported as a major cause of community and

hospital acquired infections. Infections causes by S. aureus used to respond to

β-lactam antibiotics. However, development of methicillin resistance amongst S

.aureus isolates (MRSA) left very little choices for treatment. In 2007, resistant

profile of Ethopian strains of S. aureus strains revealed 80% resistance against

tetracycline and 53% corimoxazole and chloramphenicol (7). In a survey 51%

S. aureus were found to be MRSA (8) whereas from China 63.1% resistance

was reported recently (9). MRSA is not limited to hospital associated infection

but it also made its way in community. There are many reports confirmed their

presence in community acquired infections. (10-12). This is matter of great

because these isolates may migrate into the health-care settings, and thus create

a two-way flow of MRSA (13).

At times, vancomycin was thought to be last resort for the treatment of MRSA

infections despite their high toxicity. Aggressive vancomycin dosing and

prolonged administration is associated with greater risk for renal toxicity in

patients already infected with MRSA (14). High prevalence of MRSA has led

the physicians to enhance use of vancomycin especially in chronic and seriously

ill patients that has resulted in the emergence of MRSA with reduced or no

susceptibility to glycopeptides. According to the Clinical and Laboratory

Standards Institute (CLSI), vancomycin-intermediate S. aureus (VISA) are now

those isolates with minimum inhibitory concentrations (MICs) between 4-8

µg/ml, whilst heterogeneous VISA (hVISA) strains appear to be susceptible to

vancomycin but contain a subpopulation of cells with reduced susceptibility to

vancomycin (MICs > 4µg/ml). Vancomycin-resistant S. aureus (VRSA) are

defined as those having MICs > 16µg/ml (15). The emergence of VISA, hVISA

and VRSA leads to the occurrence of severe therapeutic problems worldwide.

The resistance to vancomycin was reported to be inducible and transferable

In Pakistan, rate of the isolation of MRSA is not much different from rest of the

world. However, it is difficult to find published data about it. In 2004, Anwar et

al reported the results of their 2 year study about the nasal carriage of MRSA

among general population lived in Lahore and areas nearby. He found that

19.51% isolates were MRSA amongst all S. aureus isolated from nasal swabs.

nasal carriage of MRSA was more commonly found in children up to 9 years

(17). Data from a hospital in Rawalpindi showed the presence of methicillin

resistance in 42.01% cases considered to be positive for S.aureus among

hospitalized patients (18). In another report 43% MRSA were isolated from

various clinical samples submitted in a tertiary care hospital during one year

period (19). A multicentre study, conducted in Lahore also reported presence of

27.9% MRSA cases in hospitalized individuals (20).

B. Escherichia coli Escherichia coli is the most common facultative bacterium of intestinal normal

flora of humans and many animals but are also frequently associated with

bacterial sepsis, neonatal meningitis, nephritis, cystitis, and gastroenteritis to

infants and travelers to countries with poor hygiene. Β-lactamase production-

the major defense of gram negative bacteria against beta lactam antibiotics is

increasingly common among this species. Predominance of Extended-spectrum

beta-lactamase (ESBL) producing E.coli is not hidden anymore. The emergence

of CTX-M types of ESBLs in E.coli has also been reported isolated from a

tertiary care urology setting in Pakistan (21).

Another interesting mechanism increase the survival of E.coli in the presence

of antibiotics is recently studied. According to the group of scientist at The

Pennsylvania State University, the regulation of capsular synthesis (Rcs)

phosphorelay which was activated by inhibition of PBPs results in

peptidoglycan damage, independently of capsule synthesis and contribution to

the intrinsic resistance of E.coli to beta-lactam antibiotics (22).

According to epidemiological studies carried out in 2002 in Vietnam,

Escherichia coli is most commonly isolated in clinical samples from patients

with diarrhea and shows a high prevalence of resistance to antibiotics (23, 24).

An attention-grabbing study was carried out lately in China that indicated the

presence of two strains with resistance against 10 different antibiotics (25). 70%

of E. coli strains isolated from diarrheal cases were carried antibiotic resistant

genes, a report from Iran said (26). In Bangladesh, where Enterotoxigenic E.

coli (ETEC) is a common cause of acute watery diarrhea in infants and young

children, prevalence of MDR strains were observed among ETEC isolated from

surface water samples (27). In 2004, Sabir et al noticed a steady increase in the

number of ofloxacin resistant E. coli isolated from urinary samples over the

period of seven years i.e. from 24% in 1995 to 55% in 2002 (28). Outbreaks due

to E.coli O157-H7 are also major health concern.

C. Salmonella Salmonella species are found in animals and human beings. Serotypes highly

associated with man include Salmonella enterica serovar Typhi and Paratyphi A

and B. Typhoid fever is an acute systemic disease caused by these species. In

2006, The World Health Organization (WHO) gave an estimate of 16 to 33

million typhoid fever cases each year, with 500,000 to 600,000 deaths (a case

fatality rate of between 1.5 and 3.8%) (29). The Indian sub-continent is the most

commonly reported region of acquisition of typhoid since 1996 up to 2005

where more than 80% cases occurred. Incidences were on height in India and

Pakistan in 2005 (30).

Echo of drug resistant typhoid outbreaks came in almost four decades ago when

world faced chloramphenicol resistant typhoid. Since 1980s, strains of S. Typhi

resistant to chloramphenicol, ampicillin, and trimethoprim, now declared as

multidrug-resistant (MDR), have been responsible for numerous outbreaks in

countries in the Indian subcontinent, Southeast Asia, and Africa (31). The

plasmids conferring resistance generally belongs to the H1 incompatability

group, however, in Pakistan different groups of plasmids have been identified

(32). According to the estimates in 1995, forty seven percent of Salmonella

Typhi isolates were MDR (33).

Increased emergence of MDR cases had to play a role in the widespread use of

fluoroquinolones as the treatment of choice. Unfortunately in last few years,

unregulated use of quinolones results in chromosomally acquired quinolone

resistant S.Tphi and S. Paratyphi A (7, 34-36). A single mutation in gyrA was

established to be associated with reduced susceptibility to ciprofloxacin. In

2006, Giand et al first time reported an additional mutation in parC gene and its

connection with an increase in ciprofloxacin MIC (37).

In Pakistan, prevalence of MDR typhoid fever was 67.2% among hospitalized

children to a high failure rate with conventional therapy (38). Few more reports

about increasing MDR and fluoroquinolone resistance came up from Pakistan

D. Pasteurella multocida Pasteurella multocida is well known as commensal as well as pathogen in many

animal species but less frequenlty encountered in human infections. Many

systemic and cutansous infections have been reported in humans because of

possibility to acquire Pasteurella either from saliva of animals or surrounding

environment.

Hemmorhagic septimecia and pneumonia due to this organism are the major

problems of animal health that further contributes to lowering down livestock

and diary industry in Pakistan. Information based on field observations of

Veterinary Officers in nine districts of Punjab, Pakistan showed 11% incidence,

9% mortality and 78% case fatality rates of haemorrhagic septicemia in buffalo

and 4%, 2.5% and 62% respectively in cattle (40). This is very unfortunate that

surveillance of resistance in this exclusive animal pathogen is not properly done

compared with surveillance of zoonotic pathogens. Due to the negligence in

animal care, limited budget allocated for their health, it is difficult to bear cost

of culture and sensitivity. Available data shows the resistance of isolates against

clindamycin, sulfonamides and streptomycin due to the presence of R plasmids

(41). A novel trimethoprim resistance gene, designated dfrA20 has also been

observed in Pasteurella multocida (42). Another plasmid borne gene floR gene

responsible for chloramphenicol and florfenicol resistance was observed by the

same group of researchers in an isolate caused bovine pneumonia (43). A

comprehensive study was done for the characterization of plasmids with

antimicrobial resistant genes in avian isolates of Pasteurella multocida which

described the presence of sulII, tetG, catB2, aadA1, and blaP1 genes encode for

sulfonamide, tetracycline, chloramphenicol, aminoglycosides (streptomycin and

spectinomycin) and β-lactam (ampicillin and carbenicillin) respectively. Genes

were organized into an integron structure which

might facilitate the spreading of antibiotic resistance genes between P.

multocida and other gram-negative bacteria (44).

E. Mycobacterium tuberculosis Among intracellular bacterial pathogens, Mycobacterium species hold a place of

most threatening. From leprosy to Tuberculosis (TB), the organism has been a

major problem. Latest estimates indicate that one third of the world’s population

is infected with TB. The disease is responsible for infecting nearly 9 million

people and causing 2 million deaths worldwide annually. The World Health

Organization (WHO) declared South-East Asia region, the most vulnerable for

new TB cases in 2005 with 34% of the global incidents happened. Resistance

against isoniazid and rifampicin (MDR-TB), two main counter stones in anti-

TB therapy, has been an old fact now. Risk of the pandemic of drug resistant

TB is most worrisome. The global extent of the problem of MDR-TB becomes

evident in 1996 when World Health Organization (WHO) and the International

Union against Tuberculosis and Lung Disease coordinated for a global survey.

Lately, WHO organized another surveillance program to find out recent

developments. Latest findings show the 7% prevalence of MDR-TB among

previously treated cases. MDR-TB was found in all regions of the world with

exceptionally high rates in central Asian countries, China and Israel, in contrast,

Central Europe and Africa have the lowest levels of drug resistance (45).

After MDR-TB, world is now in the dilemma of extremely drug resistant

Tuberculosis (XDR-TB). XDR-TB can be defined as the Tuberculosis caused

by Mycobacterium tuberclosis resistance to Isoniazid and Rifampicin (MDR-

TB), plus to any fluroquinolones, and any one of the second-line anti-TB

injectable drugs (Amikacin, Kanamycin or Capreomycin). The situation raises

concerns about the future TB epidemic with very limited treatment options that

jeopardizes the major gains made in TB control and progress. In developing

countries like Pakistan, TB burden is exceptionally high. In 2004, data from

Butt et al showed 15% of the pulmonary TB isolates with mono-drug resistance,

28% with multi-drug resistance. Overall resistance against individual drugs was

rifampicin 32%, isoniazid 37%, streptomycin 19% and ethambutol 17%.

Approximately 7% of the isolates resistant to all four drugs (46). How much a

previous antibiotic treatment can affect the susceptibility profile especially in

our settings? The question starts another debate. A report from Karachi proved

this association where most of MDR-TB cases were observed in patients with

previous anti-tuberculous treatment. In addition, overall MDR cases were also

on height i.e. 47% (47). One more study was designed last year to evaluate the

rate of pulmonary TB cases in peri-urban neighbourhoods of Karachi, Pakistan.

Study revealed a higher prevalence of pulmonary tuberculosis than current

national estimates and exposed the poor operational performance of country’s

current approach to tuberculosis control (48). Drug resistance rate is

considerably lower among miliary TB cases in Pakistan for example, isoniazid

resistance was observed only in 9% cases whereas 0% in case of other first line

drugs i.e. rifampicin, ethambutol, pyrazinamide and streptomycin (49).

1.3) How Bacteria Become Resistant

Drug resistance is a condition in which there is insensitivity or decreased

sensitivity to drugs that normally inhibit cell growth. Bacteria may become

sensitive by two ways.

1.3.1) Intrinsic Resistance Bacteria can be resistant to an antibiotic due to certain phenotypic characters

e.g. slow growing/ dead bacteria or lack of drug receptor. It might be possible

that bacteria possess drug receptor but do not respond because of inadequate

concentration of antibiotic at the target site. However, the key reason behind

intrinsic resistance is the absence of drug target. For example, the difference in

the permeability barrier of gram positive and gram negative organisms decides

the spectrum of penicillin (50).

Another way of having inherent resistance is to overcome mimic, in certain

conditions, produced by certain drugs. Sulfonamide prevents the synthesis of

various compounds e.g. purines, thymidine, methionine etc required for the

growth of bacteria. But if these compounds are present in the medium or in

surroundings, bacteria may be able to utilize them and escape the inhibitory

pathway of sulfonamide.

1.3.2) Acquired Resistance

Bacteria can undergo changes that results in the resistance or insensitivity of an

organism. Acquired resistance is occurred by two genetic processes (A)

mutation and selection (also known as vertical evolution) (B) exchange of genes

between strains and species (named as horizontal gene transfer).

A. Mutation and Selection

Spontaneous mutation in single bacterium leads to the resistance of whole

population. A population of organisms can acquire resistance to a drug during

the therapy of patient. In the presence of antibiotic, abundance of resistant

mutants preferably eliminates the sensitive cells in other words we can say,

selective environment of an antibiotic exerts pressure on organisms which is in

favor of resistant organisms. This process proves the Darwanian theory of

natural selection.

B. Exchange of Genes

Transfer of a DNA segment containing resistant genes from one organism to

other; pilot the sensitive organism to resistant. Bacteria may develop genetic

resistance through the process mutation and selection but then transmit the

responsible elements like plasmids to sensitive strains by genetic recombination.

Other elements involves in resistance mechanisms includes transposons and

integrons.

In 1986, another type of mobile elements-integrons was reported. These are the

DNA sequences, interestingly common in upstream and downstream of various

antibiotic resistance genes. These are the mobile elements with the ability to

capture genes, notably those encoding antibiotic resistance, by site-specific

recombination. Their presence on plasmid suggested their mobile nature like

transposons. Because of the presence of site-specific integrase gene of the same

family and lack of many gene products associated with transposition, they were

not grouped with transposons. Intergase gene (int) is usually present nearby

recombination site (attI), and a promoter responsible of transciption of genes.

Class 1 integrons also have a variable region bordered by 5' and 3' conserved

regions where cassettes of drug resistant genes flanked. The 3' region consists of

an ethidium bromide resistance locus (qacED1), a sulfonamide resistance gene

(sulI), and an open reading frame containing a gene of unknown function.

1.3.3) Physiological Mechanisms

Apart from the above two methods, there are a number of mechanisms inside

the bacterium, responsible for drug resistance. In recent years, efflux pump

proteins have been reported to be responsible for the resistance of a number of

structurally unrelated antibiotics e .g. Acr AB TolC efflux pump in gram

negative rods. Recently, there are reports stressed on the presence of functional

AcrB and TolC efflux pump in S. enterica serovar Typhimurium for the

selection of ciprofloxacin-resistant mutants (51).

1.4) Other Factors contributing towards Drug Resistance A number of factors are involved in high drug resistance rate in our country;

lack of education and poor socioeconomic conditions are also among them (52).

In rural areas where people don’t have enough food for their survival,

malnutrition and compromised immune status welcome a number of pathogens

to colonize and grow. Moreover, expensive medical treatment in hospitals and

clinics don’t allow them to get proper treatment, therefore, they rely on self

medication. Over the counter availability of antibiotics make this work easier.

We don’t have any nationwide survey report on the status of self-medication

and its consequences in Pakistan but some reports proved its contribution in

increased drug resistance (28, 53, and 54). Another factor was highlighted few

years back when a report stated the ineffectiveness of some trade brands of

quinolones that are commonly available (55). In developing countries like ours,

poor diagnostic facilities and recruitment of untrained and nonqualified staff

especially in Microbiology results in the false negative microbiological reports

that leave an infected patient untreated. As a result he has to undergo the blind

antibiotic trials from physicians and ultimately become a reservoir of MDR

infectious diseases for others (56).

Problem of increase drug resistance also affect our agriculture and livestock but

contributing factors are more due to the involvement of animals. Since last three

decades, antibiotics are used as growth promoting and disease preventive

supplement in the feed of food animals. High amount of antibiotics in feed

results in accumulation of antibiotic residues in the tissue and high frequency of

resistant bacteria in the gut flora of food animals. When humans ingest these

animal commensals, they may transfer their resistance elements to other strains

or species that are pathogenic to humans. In this case, bacteria from zoonotic

sources serve as vectors that transmit resistance genes to the human bacterial

flora. In this case, bacteria from zoonotic sources serve as vectors that transmit

resistance genes to the human bacterial flora. (57). In developing countries like

Pakistan where human and animals live together especially in rural areas, their

concentration in proximity enhances potential transmission of microorganisms

in surroundings. A number of reports confirmed this (58-60). In 2003, WHO

recommended the sell of human and veterinary antimicrobial agents only under

prescription. According to their advice, all countries should establish

monitoring programs for tracking use and resistance to antimicrobials. They

have also called for a rapid phase-out of the use of antimicrobial growth

promoting agents. However, it should be responsibility of concerned authorities

to ensure the practice of WHO guidelines in developing world.

2) Herbal medicines

From the random use of plants in ancient times, herbal medicines go through

many developmental stages and finally gave a new subject to present day

scientists. This area of study brings together a range of disciplines from social

sciences to biological and chemical sciences. Integrated efforts are required

from every discipline to not only test the hypothesis but also towards the

isolation, identification of bioactive components, their pharmacology,

toxicology and of course testing of their potential to help mankind. Isolation of

bioactive compounds from plants provides a scientific basis of their traditional

use and a hope towards discovery of novel drugs. The success story of herbal

medicines can be hindered by a number of questions about their safety, toxicity

and side effects. Therefore, researchers working in this area need to work on

calculation of appropriate dosage, their in-vivo effects and clinical trials.

Tale of herbal medicine starts from the primitive age of man. In the earlier days

people used to find out remedies from the herbs and shrubs in near by areas

where they lived. It can be estimated that there were thousands of plants used in

different therapies in prehistory. They first used plants as food and if results of

ingestion were in favor, they linked them with some sedative and curative

properties (61), for example, there are leftovers of hollyhock plant, still famous

herb in phytomedicine found in ancient civilization of Neanderthals. Evidences

support its probable therapeutic use by this nation who lived 60,000 years ago in

Iraq (62-64).

There are many reviews available enlighten the history of herbal medicine, for

example, Indian-subcontinent is also very rich in historical aspect. One can read

a number of Ayurvedic hymns from India 1000 BC and earlier based on

common use of thousands of local herbs (64-65). Ibn Sina (980-1037 CE),

known as Avicenna in west, is considered as father of early modern medicine.

At times when Asian people depended on herbal remedies, same wave was

observed in other parts of world. Asclepius is considered as a hero and the

Roman god of medicine and healing in 1500 BC (64). Galen, in 200 ad,

summarized the height of medicine in Roman Empire in which he discussed his

achievements in herbal therapeutics. (65). History reveals that all medical

systems were once based on herbal therapies. Though, the west had almost

forgotten its enriched history of phytomdicine, revival of ancient herbal era in

America was observed in the early 19th century.

Random use of plants as remedy ultimately dragged the attention of scientists

towards compound purification. The isolation of first plant compound was

observed almost in the beginning of the era of organic chemistry when analgesic

morphine was isolated from terrestrial plant, Papaver somniferum by Sterturner

in 1805. Furthermore, in nineteenth century, atrophine, codeine, cocaine,

noscapine (narcotine), and papaverine were also purified from same specie and

developed as single chemical drugs (66). In twentieth century digoxin,

ergotomine, ergometrine, resperpine and vincristine were added in herbal

medicine (67). Many bioactive compounds have also been isolated from various

Asian terrestrial plants for example, Forskolin, a highly oxygenated diterpenoid,

isolated from the roots of an Indian medicinal plant, Coleus forskohlii exhibited

very strong antihypertensive, antithrombotic and bronchopasmolytic activities

(68). A series of bioactive tetratriterpenoides was isolated from Swietenia

mahagoni- a plant found in Sumatra region of Indonesia (69). An indole

alkaloid, Reserpine was isolated from an Indian plant Rauwolfia serpertina is a

good example of recent days’ popular plant derived compound having efflux

pump inhibiting activity (70). Recently, some Indian medicine plants including

Acorus calamus, Holarrhena antidysenterica and Delonix regia were found to

have tannins, flavonoids glycoside, phenols, saponin that were active against

extended spectrum Beta-lactamase (ESBL)- producing enteric bacteria. (71).

Though, over the past two decades, herbal medicine has increased the interest of

microbiologists and pharmacologists. An increment of 380% in use of herbal

products all over the world during 1990-1997 provides us a valid ground to

justify their success rate (72). Indeed, general trends towards spending more

money on alternative remedies do not justify their safe use but blind use of

herbs as remedy is quite different than their use as a source of drug discovery

and development-area still waiting to be appreciated. There are at least 5 or 6

drugs launched during 2000 to 2005. They are either directly purified from plant

resources or semi-synthesized in lab from plant derived compounds template.

Apomorphine hydrochloride, a derivative of morphine introduced in market as

short-acting dopamine receptor agonist, used to treat Parkinson’s disease. It is

the first subcutaneous dopamine receptor to treat sudden, unexpected and

refractory state of this disorder (73).

In Pakistan, antibiotics are costly and emergence of MDR pathogens is

increasing. In this situation, we can not blame local people who find remedy of

his ailment on a street shop without given a thought to toxicity issues. On the

other hand, provision of properly screened indigenous drugs, especially in rural

areas, can be helpful to treat diseases. According to the Drugs Act of Pakistan,

1976, “it is mandatory for the manufacturers to contribute a certain percentage

of their profit (1 %) towards a Drug Research Fund. These funds will be spent

for conducting researches on the development of new drugs and encouraging

rational drug therapy”. Our present situation needs the development of herbal

medicine industry and competent R & D organizations that can provide more

effective, cheaper and save alternative resources to poor people of Pakistan.

2.3) Problems and Challenges Despite the increasing popularity of recombinant proteins, peptides and

probiotics, plants still are considered as rich biofactories for future drugs.

Problems and hallenges faced by today’s scientists working in the area of herbal

medicine are the following;

2.3.1) Slow Methodologies

To accelerate the speed of drug development, it is important to take up latest

technology. Slow process of conventional phytotherapy techniques is the major

factor that compels the pharmaceutical industry to take out their investment

from this area. High-throughput screening (HTS) method gives the idea of a

large number of targets available and but still a very long and tiring procedure.

Although there is applaud able development in the procedures of compound

isolation and purification but processes involve in initial screening needs

improvement, room for new ideas is still available.

2.3.2) Limited Availability of Plant Material After the initial screening for biological activity, bulk amount of plant material

is usually required for proper extraction. Various seasonal variations also affect

the chemotaxonomy of the plant. Therefore, it is preferred to collect the plant

from geographical area and in same season. Continuous supply of plant material

is a problem typically faced during the second phase of drug discovery

2.3.3) Low Investment Pharmaceutical companies are loosing their interests to invest capital in herbal

drug development. Cost of analytical procedures and expensive hiring of more

competent staff touch the ceiling of expenditure. Regardless of these efforts,

success rate is going down. Over all down-fall in world economy also drop the

morale of investor in an area with less chances of return. In the developing

countries like Pakistan pharmaceutical companies want to put more efforts

towards the stability of their existing setup especially after WTO regulation

instead of giving attention in R & D.

2.3.4) Decreasing Plant Resources In past 5000 years humans have destroyed 50% forests from the earth's land

surface. Most of medicinal herbs are the part of tropical flora. Extinction of

herbal flora is associated with deforestation- a major threat faced by herbal

medicine research. In one of the annual report WHO notified the presence of 77

out of 389 endangered medicinal herbal species in China. In India, 120 plants fit

that category, 35 of which are said to be medicinally important. The situation is

same in Pakistan. Medicinal plant species in Pakistan generally and specially in

NWFP region face the same threat that will result with the ultimate loss of

biodiversity associated with these areas. In Interim Strategic Plan for 2003-

2005, USAID stated, “Pakistan has some of the world’s rarest plants but these

are now in danger of disappearing forever due to overuse and loss of natural

habitat. Misguided economic policies have widened inequalities and forced

rural people and others to exploit biodiversity at rates that are no longer

sustainable. As a result, processes such as deforestation, overgrazing, soil

erosion, salinity and water logging have become major threats to the remaining

biodiversity in Pakistan” (74).

It is therefore necessary to build a bridge among technical forest experts,

botanists, microbiologists, chemists and local forest communities so that local

people can be educated to adopt safe ways of harvesting. Scientific community

should also be engaged in decision making to work out a sustainable forest

management.

2.4) Methods/ Approaches in Herbal Medicine It is a multi disciplinary approach which needs joint efforts from

microbiologists, pharmacologists, chemists and botanical taxanomists. A

number of stages are involved from the screening of plants to the development

of a refined form of drug in market. Stages may be summarized as follows;

a. Selection of Plant

b. Extraction

c. Biological screening of crude extracts

d. bio-assay guided fractionation

e. purification and chemical characterization of bioactive components

f. screening of biological activities of purified products

g. molecular modeling studies for the development of derivatives

h. clinical trials

a) Selection of Plant Right selection of the plant provides a stable start to pave the way of drug

discovery. Improper selection of the source material not only affects the budget

allocated for the program but also deteriorate the morale of the scientists

involved. Generally there are two approaches popular in the selection of plant

material for drug discovery;

Serendipity

Someone says “Chance does not produce drug”. Although we are entering in

the era of rationale drug design but we have to accept the fact that it is most of

the time serendipity to a greater or lesser extent, behind the success story of

every drug. It is not necessary to have a happy ending of every experiment.

Sometimes some side effects or so called unsuccessful experiments may result

in the discovery of lead components like Livamisole (75) that proves the distinct

role of serendipity.

However, despite its historical role it is important to know about the general

characteristics of candidate plant and track the existing literature of bioactivity

and probable common use.

Ethnobiological Approach

This is another approach about the selection of candidate plant for drug

discovery. This is comprehensive combination of knowledge collected from

literature resources as well as by indigenous people about the reported

compounds, activities, geographical location of plant material. Chemotaxonomy

especially play an important role about the ethnobiological selection of a plant

as it is accepted that taxonomically related plants often synthesize similar type

of secondary metabolites.

There are more chances of success if candidate plant belongs from the area with

diverse flora and has history of stable common or medicinal use by the people

reside in the same location since many generations. Another important aspect is

probably the selection of species which is endemic in the area but previously

not studied or improperly studied (76). These were the key factors behind the

selection of plant species for our study.

b) Extraction It is necessary to macerate or crush the plant material before initial extraction as

it can make the mixing of solvent easier especially in case of dried bark and

fruits. Initial extraction can be done in wide range of solvents from polar to non-

polar. Water can be the solvent of choice from a biologist’s point of view. Use

of medicinal plants with warm water either in the form of suspension (e.g. green

tea) or inhalation via steam is very common, therefore extraction in boiling

water is near to their natural use (61). However, it might be possible that

chemist don’t agree with this approach as organic solvents always considered

being more efficient extractants than water.

Factors which may affect the efficiency of extraction process include extraction

time, temperature, and addition of foreign agents and removal of solvent. For

example, in case of methanolic extraction, prolonged soaking of ground plant

material like 1-2 weeks gives best results. However, extraction can be achieved

by continuous shaking of material for shorter period of time e.g. within 24

hours. It is preferred to do extraction at room temperature. High temperature can

enhance the extraction process but due to possible liability of bioactive

components, it is not desirable. Addition of any substance that can be helpful to

break emulsion or in the evaporation of solvent can hinder the activity of actual

candidate component.

c) Screening of Crude Extracts Biological screening of plant material is a very crucial stage. It is necessary to

have reproducible results. Instability of plant derived compounds is well-

known and a main hindrance in the way of their potential candidacy for drug

development. Crude extracts are screened for antimicrobial activity by disc

diffusion (77) , agar well diffusion (78-79), agar dilution and broth dilution

assays (80). Assays for antimicrobial action include spore germination assay

for antifungal activity, investigation of cytopathic effects and plaque formation

for antiviral activity and microscopic determination of antiparasitic activity with

the help of fluorescence or inverted microscope.

d) Bio-assay guided Fractionation After getting positive results from initial screening, crude extract is further

processed to locate bioactive components. Many chromatographic methods can

be employed with the combination of bioassays. Fractions can be physically

separated by two phases of a liquid–liquid extraction. Continuous elution of

crude extract with a series of polar and non-polar solvents is usually done by

column chromatography that gave various fractions collected by fraction

collector. In herbal medicine science, goal is always making the way easy to

identify bioactive component however, most of the time it depends on the type

of sample. It is important to keep solvent extraction schemes simple in order to

increase reproducibility of results. Fig # 2 illustrates the simple and widely used

scheme of fractionation. Different chemical fractions and sub-fractions can be

further processed for the screening of bioactivity and for chemical analysis by

Thin Layer Chromatography (TLC). It is an easy and inexpensive method that

quickly separates various compounds according to their Rf value thus, gives a

quick idea about the components present in a mixture.

The collection of large number of different fractions where on one hand

improves the probability of success, it is also time and labor consuming.

Moreover, this method may result in the dilution of desired compound in

different fractions so it will be difficult to detect and locate the bioactivity.

Alternatively, if the separation process is cruder, it can deal with vast variety of

compounds at the same time and make the goal more rapidly achievable (81).

Another easier and quick approach to locate bioactive component and/or their

combination is the employment of bioautography. The assay is a wonderful

combination of chemical and biological analysis which helps to localize

bioactivity especially antimicrobial activity on a chromatogram. The method

has been used previously in a number of studies (82-84). In this method, crude

plant extract or fraction is employed on a TLC plate and allowed to separate in

different compounds according to their Rf value. Assay for antimicrobial

activity is then performed over TLC plate by incorporation of agar containing

test strain. The spots containing bioactive compound are visualized using

microbial indicators (tetrazolium salts) (85). This is the approach we applied in

our study.

e) Purification and Chemical Characterization of Bioactive

Components After the successful location of bioactive component or their combination, next

required step is purification. A variety of different techniques can be used for

the isolation and purification of plant derived compounds including, high-

performance liquid chromatography (HPLC), gradient high-performance liquid

chromatography, countercurrent chromatography, droplet countercurrent

chromatography, vacuum column chromatography, desalting, ion exchange

chromatography , size exclusion chromatography, acid–base switching

technology , centrifugal partition chromatography, microwave-assisted

extraction, pressurized solvent extraction etc (81). Other important methods

help in structure elucidation are NMR spectroscopy, infrared (IR) spectroscopy,

X-ray cystallography and MS/MS.

An alternative to common analytical approaches have been introduced in 1987

by Karas et al (86) named Matrix assisted Laser Desorption/ Ionization-Time-

of-Flight mass spectrometry (MALDI-TOF-MS). The technique was originally

developed for large molecules like proteins, lipids, nucleic acids and

carbohydrates now have been proved successful for the characterization of

small molecules in food samples (87-91). This method not only allows the

analysis of macropolymers but also tells about their chain lengths. MALDI-

TOF-MS have several advantages including easy sample preparation, rapid

generation of reliable data, and good tolerance towards additives and

determination of wide range of masses from low to high molecular weight in

complex samples. The details about the principle of this technique are discussed

in Material and Methods-section 2.4.3.

It is the matter of fact that no standard procedure is available for this area. It

varies from laboratory to laboratory but good purity of compound is always

desired in the end. Finally, purified compound required to test for bioactivity

by various methods and then to clinical trials. It depends what type of activity

one is looking for.

2.5) Types of Biological Activities Many kind of biological activities are associated with plants and plant derived

compounds. Activities can be categorized according to disease area as described

previously (92).

A) Anti cancer Activity

B) Nervous System Activation/ Supression

C) Cardiovascular/ Metabolic

D) Antimicrobial Activity

E) Immunomodulating and anti-inflammatory Activity

2.5.1) Anti-cancer Activity Plants behaved very responsibly in the therapy of cancer. Most of efforts put in

herbal medicine are to look for novel anti-cancer agents especially for breast,

colorectal, lung and ovarian cancers. For many years, National Cancer institute

in Bethesda-USA has been the forefront of the anticancer drug development

from plants since many years. They introduced the approach of testing various

tumor cell lines covering a wide range of human cancers in a panel to test each

candidate compound, fraction and extract with a variety of individual assays

(76). The procedures usually undertaken for screening are microculture

tetrazolium assay, DNA topoiomerase I and II, protein kinase C, aromatse ,

tyrosine kinase, tubulin binding and assays for DNA damage (93). A number of

plant derived anti-cancer compounds includes vinblastine, vincristine,

derivatives of camptothecin, paclitaxel, topotecan, irinotecan, etoposide,

epipodophyllotoxin, teniposide, homoharringtonine, taxol etc (94-96) (Table 1).

In Pakistan, indigenous plants have been explored and there is publications

available on anti-cancer properties of Onosma limitaneum, Curcuma longa,

Fagonia cretica and Alpinia galangal (97-99). Other new and promising agents

undergoing clinical trials are enlisted in Table # 2.

2.5.2) Nervous System Suppressing/Activating or Analgesic

Activity Who don’t remember morphine? The historic alkaloid purified from opium

poppy, Papaver somniferum has long been used as an analgesic. The Phase III

clinical trials of M6G, a metabolic end product of morphine showed better

ability to suppress post-operative nausea and vomiting as compared to morphine

The endogenous cannabinoid system is an ubiquitous lipid signalling system

that appeared early in evolution and regulate essential functions of the body like

autonomic nervous system, the immune system and microcirculation (100). The

discovery of this system have drawn the attention of phytochemists and other

related scientists to synthesize cannabinoid receptor agonists and antagonists

and inhibitors of endocannabinoid degradation that leads to the control of pain,

obesity, neurological diseases including multiple sclerosis, emotional

disturbances such as anxiety and other psychiatric disorders including drug

addiction. The two new pharmaceutical products are already in market of

several countries (101).

2.5.3) Cardiovascular/ Metabolic Impact of plant derived compounds on cardiovascular and metabolic disorders

is worth noteworthy. Like for diabetes, according to an observation, there are

approximately 800 to 1200 plants that exhibit hypoglycemic activity. Olea

europeaea (The Olive tree) has been reported for having anti-diabetic and anti-

hypertensive activities (102). Use of peanuts is very universal. One could

never think to have treatment of obesity in peanut shells. Ethanolic extract of

dried shells of Arachis hypogaea L. (peanuts) was evaluated for lipid lowering

activity in rats (103). Terminalia arjuna bark extract exhibited an inhibitory

effect for thyroid hormones which might the explanation of its proven cardio-

protective role (104).

3) Antimicrobial Activity A number of infectious diseases including bacterial, viral, fungal and parasitic

have become serious threat to health care professionals. Increasing global issue

of antibiotic resistance among serious pathogen makes the situation worse. We

have already discussed in detail in section 1. In the battle of infection and

infection eradication, plant products are long running colleague of human and

animal bodies. Historical use of many plants as alternative tools of antibiotics to

combat infectious diseases is an unavoidable fact. Efforts to confirm their use as

putative therapeutic agent are on rise and under strict scrutiny worldwide. We

would like to divide antimicrobial activity into few categories in order to

provide an ease understanding the candidate plants.

3.1) Antibacterial Activity Many plants have shown their power to eradicate bacterial infections. Sato et al

examined the ethanolic extract of Terminalia chebula and two of its purified

products gallic acid and ethyl ester for antibacterial effect against methicillin

resistant strains of Staphylococcus aureus and 12 other gram-negative and

gram-positive bacteria and found them to be active (105). Seven Chinese herbs

include Aloe vera Mill. (Aloaceae), Angelica species (Umbelliferae), Astragalus

Table # 1: List of Anti Cancer Drugs in Clinical Trials S.# Name of Drug Plant Source Mechanism

of action Developmental Status

1 camptothecin Ophiorrhiza prostrata

Anti cancer launched in Korea 2004

2 paclitaxel Taxus species

Anti cancer Luitpold

3 epipodophyllotoxin Podophyllum peltatum

Anti cancer Pierre Fabre

4 vinblastine Catharanthus roseus

Anti cancer Pierre Fabre/Bristol-Myers Squibb

5 combretastatin A4 phosphate

Combretum caffrum

Anti cancer Sanofi Aventis

6 AVE-8062 Combretum caffrum

Anti cancer Sanofi Aventis

7 Homoharringtonine Cephalotaxus harringtonia

Protein Synthesis Inhibitor

ChemGenex

8 Ingenol 3- O - angelate

Euphorbia peplus

Anti cancer Peplin

9 Phenoxodiol Glycine max (Soyabean)

Anti-inflammatory

Marshall Edwards

10 Protopanaxadiol Panax ginseng

Anti cancer against MDR tumors

PanaGin

11 vincristin Vinca minor Anti cancer

embranaceus Bunge. (Leguminosae), Ganoderma lucidum (Fr.) Karst.

(Ganodermataceae), Panax ginseng (Araliaceae), Scutellaria species

(Lamiaceae) and Zingiber officinale Rosc. (Zingiberaceae) were also found to

be effective against many gram positive and gram negative organisms (106).

According to another report, Australian and Maxican plants; Eucalyptus

globolus Labill, Punica granatum L., Artemisia mexicana Willd., and Bocconia

arborea possess strong in vitro inhibitory effects against Staphylococcus

aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans (78).

An interesting story came in microbiology arena about the positive role of wine.

Observation revealed the increased antimicrobial properties in wine sample with

high concentration of polyphenols. Escherichia coli was the most sensitive

bacterium but interestingly wine polyphenols were unable to harm

Flavobacterium sp. (107). Tea tree oil (TTO), the volatile essential oil derived

mainly from the Australian native plant Melaleuca alternifolia is another

classical example of plant active against skin commensals like Staphylococci

and Micrococci and pathogens like Enterococcus faecalis, and Pseudomonas

aeruginosa (108). Studies were also undertaken to see their effect on resistant

organisms like MRSA. Presence of terpenes in TTO and a number of supporting

observations like leakage of potassium ions, inhibition of respiration and gross

morphological changes in cell wall of TTO sensitized Staphylococci even after

the retreatment to sodium chloride forced the microbiologists to assume their

cell wall inhibiting nature (109).

Pakistani herbal flora is also rich in bioactive phytochemicals. In last two years

many publications witnessed it. Derris elliptica, Derris indica and Derris

trifoliate showed broad spectrum antibacterial activity (110). Two more detailed

accounts published recently about the antimicrobial potential of essential oils of

Perovskia atriplicifolia Benth and Ocimum basilicum L. The antimicrobial test

results showed significant potential against Staphylococcus aureus, Escherichia

coli, Bacillus subtilis and Pasteurella multocida (111, 112).

3.2) Strategies for Eradication of Bacterial Infection

In the debate of infection and infection eradication, one need to sort out the

ways yet taken up by plants and plant derived substances to eradicate infection.

Novel targets of bacterial pathogens are not for the antibiotics only but they are

equally charming for plant extracts and their compounds. Quercetin, a widely

distributed bioflavonoid proved to have dual mechanism of bacterial cell

inhibition. It binds with 24 kDa fragment of gyrase B, specific site for binding

ATPase thus jams this energy producing machinery as well as directly interact

with bacterial DNA (113). Antibacterial activity of Radix tinosporae (Tinospora

Root) was also explained by possible mechanism of nucleic acid inhibition

(114).

Different herbs from family Lamiaceae (lemon balm, rosemary RoLA, sage,

oregano, rosemary and chocolate mint) also exhibited good anti- S. aureus

activity due to presence of proline analog that mimic the production of proline

dehydrogenase, important for bacterial plasma membrane (115). Terpene

alcohols were found to be antibacterial due to their effect on initial rate of

leakage of K+ ions, suggesting the cell membrane damage as a possible

mechanism of antibacterial action (116). Same action is also present in three

different monoterpenes purified from essential oil (117).

Hemsleya pengxianensi, a Chinese plant is recently reported as antimicrobial

because of the potential to inhibit bacterial cell wall synthesis (118). Plant

defensins and γ-thionins, small polypeptides present in a variety of plant species

e.g. in cowpea seedlings alter cytoplasmic membrane septum formation, inhibit

cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or

inhibit enzymatic activity (119). The antimicrobial effect of allicin, a purified

product from garlic was characterized against multidrug-resistant

enterotoxicogenic strains of Escherichia coli with the possible interference in

bacterial alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase

(120).

Some plants derived compounds affect different virulence factors of bacteria

associated with their colonization and pathogenesis for example, quorum

sensing. It is cell-to-cell communication among bacterial cells mediated by

small, diffusible signals. In gram negative bacteria, AHLs or N-acyl-homoserine

lactones is the most studied system proved to be associated with production of

extracellular polysaccharide capsule, biofilm formation and other important

virulence factors (121, 122). Teplitski et al. (123) found a mimic in AHL signals

of Pseudomonas aureofacien and Escherichia coli due to methanolic extract and

exudates of Pisum sativum (pea) plant. Later on, an interesting hypothesis about

interplay of signals and signal inhibitors was published that revealed that plants

with defective immune system secrete some chemicals that can modulate

bacterial communication system so that colonization of pathogens can be

prevented. (124).

Efflux pump is inducible protein channel present on the cell membrane of gram

positive and gram negative organisms associated with multiple drug resistance.

Regulation of efflux protein expression can make the bug fully resistant or

super-susceptible respectively (125). Novel mechanism of efflux pump

expression in MDR bacteria is one more target for plant derived compounds.

This new role of plant derived compounds as efflux pump inhibitors (EPIs) is

unequivocally applauded by microbiology community. Reserpine, an alkaloid in

Rauwolfia vomitoria is known to inhibit Bmr efflux pump Bacillus subtilis,

Tet(K) efflux of Staphylococcus aureus conferring tetracycline resistance and

NorA conferring MDR in S. aureus (126). Berberine, isolated from Berberis

fremontii, flavonolignan 50-methoxyhydnocarpin-D (50-MHC-D),

arylbenzofuran aldehyde from Dalea spinosa (smoke tree) and some

oligosaccharides from Mexican Morning Glory Species are also included in the

long list of EPIs of plant origin (127).

3.3) Synergistic Antibacterial Combinations Synergistic antimicrobial combinations have been introduced as more

successful strategy to combat MDR infections. Combinations of amoxicillin/

clavulanic acid, sulfmethoxazole/trimethoprim, piperacline/ tazobactum,

amoxicillin/sulbactum are the classical examples of their success. According to

published data, plants from different geographical locations act synergistically

with common antibiotics and exhibited greater antimicrobial activity against

MDR pathogens. A report from Taiwan stated the synergistic activity of

flavnoids with cefoxitin against ESBL producing Klebseilla pneumoniae (128).

Local Indian medicinal plants; Acorus calamus, Hemidesmus indicus,

Holarrhena antidysenterica and Plumbago zeylanica exhibited synergistic

antibacterial potential with tetracycline and ciprofloxacin against ESBL

producing and MDR Escherichia coli (71). Some daily use spices and plants

like clove, garlic, ginger, lemongrass and guava were also checked and some of

them were found to be inhibitory against Staphylococcus aureus, if were used

with protein synthesis inhibitors like tetracycline (129). Various organic extracts

of root bark of Cordia gilletii De Wild (Boraginaceae), a traditional medicinal

plant in Congo, were tested for antibacterial activity alone and in combination

with different antibiotics against MRSA. Hexane and dichloromethane extracts

decreased the MICs of penicillin and streptomycin 4–64-fold (reversal of

antibiotic resistance) whereas the combination of methanol extract and

tetracycline also showed synergistic nature (130). Ethanolic extracts of propolis

gave same effect with several Beta-lactum antibiotics against Salmonella

enterica serovar Typhi (131).

When co-action of Humulus lupulus derived compounds lupulone and

xanthohumol was tested with polymyxin B sulfate, tobramycin and

ciprofloxacin, interesting observations were noted that there was co-action

against all Gram-positive bacteria tested but in case of gram negative bacteria,

co-action was observed in some species but not in all. Especially in E.coli,

there was no significant synergism observed (132).

3.4) Antimycobacterial Activity It is more difficult to combat intracellular pathogens like Mycobacterium

species. Increase of multi-drug resistant TB (MDR) and emergence of

extremely drug-resistant-TB (XDR) increase the intensity of threat. No new

drug has been was marketed for 30 years. Attempts have been made on

medicinal plants e.g. in India, almost 255 plant species have antimycobacterial

activity (133). Methanolic extract of roots and leaves of Leucophyllum

frutescens and ethyl ether extract of the roots of Chrysanctinia mexicana gave

very capable results against the drug-resistant strain of Mycobacterium

tuberculosis. Both plants are used in northeast Mexican folklore (134).

Xanthium cavanillesii Schouw is a wild herb of Uruguay. Crude extract was

found to be antimicrobial against Mycobacterium smegmatis. Extract was

declared as non-toxic at the dose of 200 mg/kg, in animal toxicity experiments

(135). Glycyrrhiza glabra roots extracts was active against Mycobacterium

tuberculosis H37Ra and H37Rv strainsat MIC 500µg/ml whereas its purified

compound, glabridin at MIC 29.16µg/ml (136). South African plants; Acacia

nilotica and Combretum kraussii gave antimycobacterial activity against

Mycobacterium aurum (137).

Strategy of synergistic combinations was also taken up to fight with

Mycobacterium species. In a report synergistic activity of some Saudi plants

including Plumbago zeylanicawas and others was noticed in combination with

isoniazid against M. tuberculosis H37Rv and four atypical species including

Mycobacterium intracellulare, M. smegmatis, M. xenopei and M. chelonei

(138). Laurel oil, its fractions and two purified sesquiterpene lactones;

costunolide and dehydrocostuslactone, were tested for antimycobacterial

activity against Mycobacterium tuberculosis H37Rv (reference strain) and

clinical drug-resistant M. tuberculosis isolates. Lactones gave better activity

when tested as a mixture than as individual pure compounds (139). Bapela et al

(140) analyzed African plant, Euclea natalensis and a pure compound,

naphthoquinone, 7-methyljuglone (isolated from roots) alone and in

combination with isoniazid and rifampicin against both extracellular and

intracellular M. tuberculosis. Combinations of 7-methyljuglone with anti-TB

drugs resulted in synergism witth FICI 0.2-0.5.

3.5) Anti-parasitic Activity From gastrointestinal tract to blood, genital tract and skin, every area is

vulnerable for parasitic infection. That are more common in developing nations.

Although, increasing number of HIV cases and travel among different nations

also let the developed world think about prevention and cure of parasitic

ailments. It is true that drug resistance is not as common in parasites as in

among bacterial pathogens but reports about their resistance are on the rise

every year. Medicinal plants have been found very rich in case of anti-parasitic

activity and there are many studies confirming their potential in the disease

treatment. Mead and McNair found many plant derived Flavonoids and

polyphenols active against Cryptosporidium parvum and Encephalitozoon

intestinalis(141). Recently anti-parasitic activity was observed in some local

plants of New Caledonia. The parasities used in this study were, Leishmania

donovani, Trypanosoma brucei brucei, Trichomonas vaginalis and

Caenorhabditis elegans (142). In a WHO facilitated project to discover novel

antiparasitic and larvicidal compounds from Panamanian plants, around 150

plants were tested and some of them gave very promsing results (143).

According to an estimate almost 153 plant species from 69 families from

different geographical distributions have been reported active for giardicidal

activity (144). In vitro and in vivo activity of phytoproducts against

Trypanosoma species have also been reported widely (145, 146).

Trichomonas vaginalis is a flagellated protozoan parasite, causing agent of

Trichomaniasis worldwide. The disease is among leading sexually transmitted

infections. Prevalence of infection is extremely high in the developing world.

Available data does not reflect the actual situation of the disease as expertise

and resources of good diagnosis are lacking in this part of the world. A survey

conducted in South Africa revealed 65% pregnant women severely infected

with T. vaginalis (147). Metronidazole with the dose of 250 mg three times a

day for 7 days or single 2-g dose is current recommended regimen. Other

notable drugs are tinidazole and other nitroimidazole derivatives. All have same

mode of action but different in pharmokinectics, toxicity and drug distribution.

Despite of lots of efforts, neither of the drug is as effective as metronidazole.

Unfortunately, there is increasing number of reports about metronidazole

resistance among these flagellates (148). Therefore, it is important to look for

some alternates at least equally effective and cost saving as metronidazole is.

Herbalists and Microbiologists again look at the rich factories of plants for

remedy. If we review the reports apparent since last year, we will find some

interesting and promising reports for example Calzada et al from Mexico

reported about anti-trichomonad activity of Mexican plants; Carica papaya and

Cocos nucifera with IC50 values of 5.6 and 5.8 µg/ml, respectively (149). Ofer

et al worked on Methyl jasmonate (MJ), small lipid molecule widely present in

plants. He found the fragmentation and condensation of the DNA of T. vaginalis

after the treatment with MJ. His findings suggested synergistic role of a

glycolysis blocker with MJ, thus arrest the cell cycle at G2/M phase and cause

death of T. vaginalis cells (150).

4) Immunomodulation The remarkable defense system of host body, immune system was introduced

dates back by Louis Pasteur. Later a number of scientists gave their contribution

for making the concepts clear. We often see immune system playing at various

ends from fighting with foreign agents to lining up against its own particles.

Whatever scenario is, the supreme function is to protect body against infectious

diseases either directly or indirectly. Different external conditions need different

status of immune response for example, infectious disease problem become

aggravated in a host with impaired immune response whereas, in organ

transplantation, it is important to keep it suppressed. Therefore, it is of special

interest how to modulate the immune response according to our need. Being a

microbiologist, we appreciate immunomodulation in a way that help in the

eradication of infection. In modern age immunostimulatory effect of some

antibiotics like clindamycin, erythromycin and chloramphenicol are known,

however, it is generally accepted that most of the antibiotics exert adverse effect

to host immune system that somewhat limit their beneficial effect. Therefore, it

is important to search new antimicrobial substances with immunostimulating

properties.

Immunomodulation achieved by plants and plant derived substances is an old

fact. A number of medicinal plants have successfully interrupted in the normal

mechanism of immune response at various points. Some polysaccharides from

higher plants, mushrooms, lichens and algae bind with macrophage surface

receptors specific for pathogens and induce similar immune response. The

approach can be useful in the discovery of new adjuvants (151). Adjuvant

ability was also observed in green propolis, when it was injected to mice with

inactivated Suid herpesvirus type 1 (SuHV-1) vaccine. The same plant also

exhibited ability to enhance cell-mediated response by increase in IFN-γ

production (152). Plants from Korean folklore have ability to trigger innate

immune response e.g. safflower leaf extract has proved as a stimulant for

lymphocyte proliferation whereas mustard leaf extract induce nitric oxide

production (153). A Thai plant, Aeginetia indica Roxbert ascends T cell

stimulation (154). In our study, we figured out the possible role of some

indigenous plant species in the modulation of innate immune response at three

different levels. Further details about each mechanism are discussed below.

4.1) Immunomodulation and Phagocytosis It is the novel mechanism of innate defense system which involves the

engulfment and clearing of foreign particle like pathogenic microorganisms.

Two main cell lineages are involved in the professional phagocytosis;

1. Polymorphonuclear leukocytes (PMNLs) consist of Neutrophils and

Eosinophils

2. Mononuclear cells (Monocytes and Macrophages)

PMNLs are abundant in number than other lineage and usually present in

circulation and but a considerable amount can be found emarginated with

endothelial lining. On the whole their function is similar but they do differ in

some aspects like antigenic heterogeneity and induction. The overall production

of PMNLs is about 109cells/kg/day but number of circulating cells is markedly

increased during infection and inflammation.

Though, they have short life span but play main role in the innate immune

response against extracellular pathogens and their toxin products. In some cases,

their role to limit intracellular parasites has also been proven. On the other hand,

long-lived mononuclear cells, that are less in number (only 10% of total

circulating leucocytes), are front line soldiers to eradicate obligate intracellular

pathogens.

When a pathogen enters inside the body, it triggers the activity of local

endothelial cells, macrophages and plasma proteins. Microbial products made

some modification in endothelial cell surface receptors to help in slowing down

blood flow and produce chemotactic signals e.g. cytokines, microbial peptides,

and platelet activating factors. In response, circulating PMNLs attract towards

endothelial lining, make changes in their shape via rearrangement in

cytoskeleton and attach on the cell surface by membrane integrins. Attachment

is followed by generalized migration (Diapedesis) of PMNLs in vascular

endothelial linings and finally directed migration towards inflamed area.

Chemotactic signals also activate PMNLs during migration so that they can be

equipped with necessary aid like receptors against C3b and Fc portion of Ig or

lectins to recognize pathogens. Activated cells entrapped the pathogen in a

vacuole (phagosome) and engulf them.

After the engulfment, two main events are held in activated cells to kill

entrapped bugs. First is the release of granular contents in phagosome.

Azurophilic (peroxidase positive) granules are the storage factories of

microbicidal proteins and peptides like defensins, cationic protein37,

permeability-increasing protein etc and whereas cytochrome b558, endotoxin-

binding proteins-cathelicidin and many other receptors are bound to the

membrane of peroxidase negative granules. Second important event occurred in

PMNLs is reassembly of respiratory burst oxidase (constituted by several

membrane and cytosol associated proteins including p47phox, p67phox, Rac-

related guanine nucleotide (GTP)-binding proteins, and membrane-bound

cytochrome b558. The enzyme reduces NADPH and form superoxide anion

(O2-) which is further reduced into H2O2 by myeloperoxidase (present in

azurophilic granules) and finally in singlet oxygen and hydroxy radicals that

help in intracellular killing of pathogen (155). Diagrammatic illustration is in

Fig # 3.

Fig # 3: Function of Phagocytic Cells

Localized inflammation following pathogen invasion activate pahgocytic cells including (cP) that results in strong binding of PMNs to EC (1) and transendothelial migration (2). PMNLs are attracted to the infected area by chemotaxis (3). During chemotaxis, they are primed by various signals (cytokines). they recognize pathogens via membrane receptors for immunoglobulins (Ig) or complement proteins (C3b/iC3b) or via lectins. Engulfment of adherent pathogen in a phagosome occur (4). Oxidative Brust was activated by NADPH oxidase (5). Specific and azurophilic granules are released into the phagosome, which becomes a phagolysosome (degranulation, exocytosis) (6).Intracellular killing of pathogens occur with the help of cellular proteins (7). Digestion of bacterial debris is occurred by PMNL hydrolases (8). In certain cases, degranulation and ROS production takes place (9). (Picture excerpts from Reference # 155)

NADPH + 2O2 NADP+ + H+ + 2O-

Bacterial virulence factors, impairment in metabolic machinery, longer half life,

presence of precursor cells, interrupted interaction with neighbor cells,

impairment in regulatory proteins, defective receptors and disturbed expression

of PMNLs gene profile are the factors effecting phagocytosis. Organisms like

Staphylococcus aureus can escape the intracellular killing. Instead of killing,

phagocytes rather serve as reservoir for this organism and help in the

dissemination to cause recurrent and chronic infections. The reason might be

up-regulation of bacterial genes encoding several virulence factors following

ingestion (156). A recent report describes the role of host cells factors like

Azurophilic granules and Iron-regulated surface determinant (Isd) proteins in

the up-regulation of several virulence genes of MRSA (157).

Modulation of phagocytic activity by antibiotics has been recognized

previously. Antibacterial agents can interfere at different level of phagocytosis.

Possible pathways for the modulation of phagocytosis are illustrated in Fig # 4.

However, it may also possible that phagocytic cells and/or products interact

with drug in synergistic or antagonistic manner (155). In any case results lead to

the modulation of intracellular killing of pathogen. Factors affecting the

immunomodulators’ work include the location, antibacterial activity, cellular

uptake and accumulation of drug, external host factors, cell type, metabolic

status and drug induced structural and functional changes in host cell as well as

sensitivity and virulence of pathogens.

A number of plants and plant origin compounds have altered phagocytosis.

Stimulation of macrophages mediated phagocytosis has been observed in many

plants e.g. Tinospora cordifolia-an Ayurvedic plant (158), Chenopodium

ambrosioides- a Brazalian plant (159) and Acanthopanax senticosus- a Korean

Fig. # 4: Effect of Antibiotics on Phagocytosis

Various antibiotics affects at different steps of phagocytosis including chemotaxis, engulfment, respiratory oxidative burst and intracellular killing or indirectly on cytokines production, thus responsible of modulation of immune cell functions.

species (160), Symphytum asperum, Symphytum caucasicum (161), Magnifera

indica (mango) (162), Capparis zeylanica (163), Astragalus radix and

Scutellaria radix (164) are the few examples of neutrophil function stimulators.

4.2) Immunomodulation and Humoral Immune

Response Humoral immune response is characterized by the production of antibodies

specifically directed to a particular pathogen or antigen. Extremely diverse

antibodies are helpful in the eradication of infection in many ways i.e. direct

binding with pathogens, toxin neutralization, taking a part in complement

activation, opsonization and activation of cell-mediated immunity.

B cells serve as production houses for antibodies. Mature B cells have

approximately 1.5 x 105 antibody molecules/ cell on the surface and every

molecule has identical binding site for antigen. After maturation in bone

marrow, they migrate to peripheral lymphoid organs like spleen, lymph nodes

etc. After encounter with an antigen, clonal selection and cell proliferation of B

cells start that give two populations; plasma cells and memory cells. Clonal

selection occurs in lag phase of primary immune response. After cell

proliferation, log phase starts where peak serum antibody level is achieved. In

case of SRBCs lag phase lasts in 3-4 days, plasma cells reach height of

proliferation between 4-5 days and peak serum antibody level achieve 5-7days.

Primary response lasts for different time period that depend on time of antigen

as well as competence of host immune system.

It is important to give booster dose of antigen to get secondary immune

response where memory cells play lead role (165).

A number of plants and plant derived substances have proved to be stimulatory

for B cell proliferation and antibody production, thus become helpful in

combating infections. Platycodon grandiflorum was found to markedly increase

polyclonal IgM antibody production and B cells proliferation (166). In another

study, abrupt withdrawal of morphine showed > 80% suppression of murine

spleenocyte number and function that confirms its intense effect on B cell

proliferation and antibody production (167). Some plant alkaloids like

Monocrotaline (pyrrolizidine alkaloids) and flavanoides down regulated B cell

function and proliferation (168, 169).

Many assays have been developed to evaluate modulation of immune response

but the system of using spleen cells and SRBCs is generally more useful. In

1963, Jerne and Nordin introduced a simple technique called hemolytic plaque

assay for elucidating individual antibody-forming cells by suspending spleen

cells of an immunized animal in SRBC-agar matrix and observing the formation

of distinct zones of complement-mediated hemolysis. The technique provides a

good opportunity to evaluate in-vivo stimulation of plasma cells especially in

spleen that is mixed platform of cells involved in immune response. Thus, the

use of spleen rules out the possible effect of cell-cell interaction on the action of

immunomodulating drugs. The method with some modification is still in use

successfully (166-169).

4.3) Immunomodulation and Oxidative Challenge

Mitochondria are the ATP generating machinery of the cell that involves the

transmission of hydrogen ions across the mitochondrial membrane via electron

transport chain. A series of cellular proteins, lined up in descending order

according to their redox potential transfer the electron to last player, oxygen.

Reduction of oxygen normally leads to the formation of water with some

byproducts like reactive Oxygen species (ROS) that gives an oxidative

challenge and makes the role of oxygen controversial inside mammalian cell.

ROS is a collective term used for certain free oxygen radicals like superoxide,

hydroxyl, alkoxyl, nitric oxide etc and non-radicals hydrogen peroxide, ozone

and singlet oxygen. These are relatively unstable molecules and require an

electron. Electron donors are usually nearby sources like mitochondrial DNA,

membrane proteins and fatty acids. The most likely electron donor is

mitochondrial DNA (170) which faces severe damage due to the loss of

electron. Consequences of DNA damage starts from strand breaking, mutation

and leads to localized cell injury and organ failure. Association of oxidative

DNA damage with many diseases like Alzheimer's disease, aging (171), cancers

and ischemic heart diseases have been established. Oxidative challenge is also

faced by host body in case of chronic inflammation-a consequence of some

infectious diseases like chronic gastric ulcers caused by Helicobacter pylori,

tuberculosis, ulcerative colitis, chronic cholecystitis caused by E.coli and

Bacteroides fragalis, bronchiectasis seen in pertussis and Staphylococcal

infections and some autoimmune disorders like Crohn’s disease A number of

cellular enzymes are involved ROS formation includes Ubiquinone-cytochrome

C reductase, NADH dehydrogenase. Other factors contributing in ROS

formations are overloading of mitochondrial Ca2+ content that regulate ATP

formation (172), UV irradiation, ionizing irradiation, antibiotics and other

chemotherapeutic agents, microbes and microbial products.

In case of intense cell damage, membrane proteins become activated and

rupture mitochondria. cytochrome C ooze out from mitochondria and combines

with a cytoplasmic protein-apoptotic protease activating factor-1 (Apaf 1) to

form apoptosome. Apoptosome is a multiprotein complex consists of

cytochrome C, Apaf-1, pro-caspase 9 and ATP (Fig # 7). Formation of

apoptosome initiates denaturation of a cascade of proteases namely caspases,

beginning with caspase-9 and then caspase-3. Activation of capase cascade

cleaves cytoskeletal proteins, that leads to the extensive morphological changes

and lead the cell undergoes apoptosis (173).

To terminate the series of oxidation reaction before it damage mitochondrial

DNA and other bio-molecules of the cell, some reducing agents usually called

anti-oxidants play their role. They either remove free radical intermediates or

oxidize themseleves to terminate chain reaction. Thus prevent the cell from

oxidative damage. Anti-oxidants can be found in internal and external

environement. Inside the cell, a number of enzymes like catalases and

peroxidases act like anti-oxidant whereas in external environment, glutathione,

vitamin C, and vitamin E are well-known anti-oxidants.

Anti-oxidants have also been found in many plant species like anthocyanins rich

berries (174), flavanoids of Ginkgo biloba and red wine (175), Hypericum

perforatum (176), Brassica oleracea (the crusiferous vegetable) (177) and a lot

more. The use of anti-oxidants in the treatment of many chronic inflammatory

diseases has been successful. Approach of combinatorial therapy of anti-

oxidants and antibiotics for the treatment of chronic infection is also useful.

LITERATURE REVIEW

THE PLANTS USED IN THIS

1) Camellia sinensis Camellia sinensis is the Tea plant belongs to family Theaceae due to which it

was previously known as Thea sinensis. The plant is widely cultivated in

tropical and subtropical regions all over the world. Leaf buds and young light

green leaves upto 4-15cm are usually trimmed to get tea. After plucking often,

they have been processed for oxidation to get Black Tea. However, ones that

steamed for a very short period of time (preclude oxidation process), soon after

plucking, are the source of Green Tea.

Green Tea is enjoying its popularity since 2727 BC when Chinese Emperor

Shen-Nung used to drink boiling water with very few leaves of Tea plant in his

royal bowl. Now Green Tea is considered as all day drink in most of the

countries. Today large Tea growing countries are China, India, Bangladesh,

Indonesia, Sri Lanka, Kenya, Japan Argentina, Brazil, Peru, Ecuador, Uganda,

Tanzania, Malawi, Rwanda and Mozambique. Before, Pakistan was considered

among the major Tea importers, spending around $300m a year on tea purchase.

In 1982, Pakistan started growing tea in Mansehra Region of NWFP Province

and now is able to export to many other countries.

A number of polyphenols have been isolated from C.sinensis. Of that, catechins

of flavanol group are very important from biological point of view. They

constitute up to 30% of the dry leaf weight but vary according to the age, area

and method of extraction. Maximum concentration may be achieved by

extraction at 95oC for 10 minutes. Degradation of polyphenols may occur at

higher temperature and with prolonged extraction time. The most common

Green tea Catechins are epigallocatechin gallate (EGCG), epigallocatechin

(EGC), epicatechin gallate (ECG) and epicatechin (EC). EGCG accounts for

major ratio among all. According to some estimates, a cup of tea contains about

142 mg EGCG, 65 mg EGC, 28 mg ECG and 17 mg EC. Other polyphenols

found in Green tea include flavanols (myricetin, caempherol, quercetin), flaanol

glycosides like chlorogenic acid, coumarylquinic acid, theogallin (3-

galloylquinic acid), proanthocyanidins, phenolic acids and flavones (178).

Caffeine is another important compound, constitute 3% (around 76mg/ cup of

tea). Other related substances are methylxanthines, theobromine and

theophylline. A number of quinones are also a regular chemical feature of

Green tea. Upon oxidation, they form second chemical series containing

bisflavanols, theaflavins, epitheaflavic acids, and thearubigens that further form

complex with caffeine and gave a variety of taste and color. Usually polyphenol

oxidases catalyze the reaction. Unlike theaflavins, complete characterization of

thearugbins has not yet done. In a study, characterization of black tea by

MALDI-TOF-MS revealed thearugbins as polymers of catechins in which the 3-

OH group is more and less esterified by gallic acid during condensation process

(179). Theanine (5-N-ethylglutamine) is an amino acid uniquely associated with

green tea. Potassium was found in highest amount among all minerals; however

others are Al, Ca, Mg, Mn, P and S (180). In spite of all information available

about its chemical nature, it is important to characterize Green Tea by using

latest methodologies for better understanding about the presence of more

compounds.

Anti-cancer properties of green tea compounds are widely accepted. EGCG

affect on a wide range of human organs for cancer prevention. A study carried

out among Japanese people, who drink green tea daily, revealed its probable

role in cancer prevention among this population (181). Another epidemiological

study shows the preventive trend of breast cancer development and recurrence

among Canadian women consumed 5 cups of green tea a day (182). Role of

EGCG in treatment of adenomatous polyps and chronic atrophic gastritis has

also been demonstrated. However, their exact role in stomach cancer prevention

is sill unclear (183). Induction of apoptotis of cancer cells is a possible reason of

the anti-cancer activity for example in case of colorectal cancer lines, apoptosis

was markedly induced by EGCG suppress the formation of cancer cells (184).

Major Green Tea Catechins

Fig # 6: The most common Catechins isolated from Camellia sinensis (Green

Tea) are epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin

gallate (ECG) and epicatechin (EC).

Camellia sinensis (Green Tea Leaves)

A Glimpse of Tea Garden in Pakistan

Same phenomenon was observed in Theaflavins who seem to be responsible for

misbalancing between pro-and antiapoptotic proteins (Bcl-2, caspases) and

down-regulation of survival pathways of prostatic cancer cells, leading to

apoptosis (185). Another mechanism of anti-cancer activity revealed when

green tea extract showed in-situ up- regulation of annexin-I (an actin binding

protein) that modulates actin remodeling in bladder tumor cell. As a result

adhesion of cell increased that inhibit cell proliferation and motility (186).

Different mechanism and anti-cancer activity in crude extract indicate the

possible presence bioactive compound(s) other than catechins. Some authors

think that interruption in signal transduction pathways is the main cause of

selective anti-proliferative and neuroprotective behavior of green tea towards

tumor cells (187). Chae et al recently prove this claim by observing inhibitory

activity of EGCG in signal transduction pathway of angiotensin II, thus results

in the inhibition of inflammatory cells binding to vascular wall in

atherosclerosis (188, 189).

Many other enrich plants green tea also has potential of antibacterial activity. It

is generally accepted that activity of tea polyphenols are better against gram

positive bacteria than gram negative. In a study carried out in India, green tea

extract was tested against many organisms including Staphylococcus aureus,

Vibrio cholerae, Escherichia coli, Shigella spp., Salmonella spp., Bacillus spp.,

Klebsiella spp. and Pseudomonas aeruginosa. The spectrum of activity was the

same (190). MIC of green tea extract against S. aureus was reported 2.0mg/ml

(191), in some other reports MIC against MRSA is found between 1.3-8.2

mg/ml (192). Antimicrobial behavior of green tea catechins towards

Staphylococcus aureus have also been studied previously but concentrations

required to exert cidal effects is quite high (193). Tea and tea derived

compounds become more effective against MRSA, when tested with well-

known antibiotics i.e. ampicillin, tetracycline (192). Change in oxacillin and

carbepenem resistance among MRSA isolates was observed when they were

used with ECG. The authors claimed the presence of gallate moiety essential for

this action (194). They gave the hypothesis that due to gallate moiety,

glycosylated catechins (ECG) can penetrate deeper into phosphatidylcholine

and phosphatidylethanolamine bilayers which is enough to modulate β-lactum

resistance in MRSA (195). Further investigations proved the synergistic

interaction of galloylated and nongalloylated catechins to enhance binding with

bacterial cell wall (196). Another synergistic combination of green tea and

butylated hydroxyanisole was observed to be effective against Streptococcus

mutans-an organism involved in dental plaque formation (197). Recently, Choi

et al reported the inhibitory effect of green tea extract on artificial dental plaque

formation. Extract was also found non-toxic for human gingival fibroblast

(198). Saraya et al upon their observations suggested the possible addition of

green tea extract in commercial mouth washes to prevent dental caries studied

its combination with commercial mouthwashes (199). Tea catechin, although,

are very active against gram positive bacteria but their antimicrobial potential

against gram negatives is still questionable. Possible explanation may be a

difference in target side i.e. cell wall (200).

Antiparasitic activity was also observed in green tea catechins when

Trypanosoma cruzi was tested at two different developmental stages such as

nonproliferative bloodstream trypomastigotes and intracellular replicative

amastigotes. MBC50 was 0.53 pM and 100 nM respectively (201). Sheep

nematodes; Teladorsagia circumcincta and Trichostrongylus colubriformis

were also found susceptible by green tea extract (202). Antifungal activity

against some fungal human and plant pathogens was also observed (203, 204).

Tichopad et al detected inhibition of reverse transcriptase and slowing down of

DNA polymerase process by green tea (205).

Green tea components also modulate immune cell function like γδ T cell,

macrophages and monocytes and effect on the intracellular survival of some

bacterial species (206-208). Other important functions of green tea are anti-

oxidant (209, 210), antiangiogenic (211), neuroprotective (212) cardioprotective

(213) and inhibition of fatty acid synthase (214). Regardless of the fact that a

number of studies has been carried out, green tea is still not in therapeutic

practice. Less or no in-vivo evidences, pharmodynamic studies and lack of

clinical trials are the hindrances.

2) Juglans regia Walnut is a common temperate forest tree found throughout the world. The

plant belongs to the family Juglandaceae, known as Juglans regia in the world

of botany. It is native of Eastern Europe but easily found in east Himalayas

and China (215). According to their cultivar, the plant is called as Persian

Walnut and English Walnut. They grow best in mild climatic conditions. Due

to their deep roots, needs well-drained and chalky soil. It is a medium to large

sized tree reach to maximum of 100 ft height with short trunk, broad and

round-topped, open crown. The bark is thick, often smooth and light gray in

color. Trunk is soft and coarse grained (216). The dried bark of Juglans regia

is locally available in Pakistan with the name of Dandasa. It is a very famous

traditional teeth brightening and lip decorating substance among the females

of NWFP region of Pakistan. It increase the pH of saliva, therefore, this bark

may improve oral hygiene. It is of special interest that there are very few

reports stating about side effects after their oral use but none is reported any

severe toxicity outcomes (217). The outer husk of the bark, that gives color to

lips, have also been used to make a brown colored natural dye. The plants

have a rich history of use in Italian, Turkish and Indian folklore (218, 219). It

has been used for the treatment of skin diseases such as eczema, scabbing

pruritus, blisters and varicose ulcers, blood cleansing and as laxative (216).

The tree is rich in polyphenols. There are 16 different types that has been found

in this plant including three ellagitannins, two dicarbooxylic acid derivatives,

glansreginin A and B, dimeric hydrolysable tannin i.e. glansrin D, valoneic acid

dilactone (220). Concentration of polyphenols is significantly effected by

cultivar, and season of sampling. Flavanoides found in Juglans regia include

catechins and myricetin (221). Another important compound is a

naphthohydroquinone or called juglone uniquely isolated from the stem bark

(222). Walnuts are also considered to be rich in vitamins. It has been found

years back that ascorbic acid or vitamin C is present in a considerable amount

i.e. 252mg/100g (223). According to some estimation, if Vitamin C and tannins

would extracted from bark, the residue can only be used as fuel (215). VitaminE

or tocopherol are also found in nuts in approximately 267.87 mug/g. γ-

tocopherol is the main tocopherol present. Major fatty acids found in Juglans

regia are linoleic, α-linoleic, palmitic and stearic acid (224).

There is published data available about the antimicrobial potential of this plant.

In 1997, a dose dependent antimicrobial activity of dried bark extract was

reported against Staphylococcus aureus, Streptococcus mutans, Esherichia coli

and Pseudomonas aeruginosa (225). Bark was also found to be useful in

maintaining oral hygiene as it inhibited the growth of cariogenic bacteria,

Streptococcus mutans, Streptococcus salivarius, Lactobacillus casei and

Actinomyces viscosus and found to be non-toxic for oral fibroblast cells (226).

Ethanolic extracts of leaves show antilisterial activity when tested with some

other plans of Turkish folk medicine (227). An interesting study came in 2004

from Iran when Nariman et al revealed anti-Helicobacter pylori potential of this

plant (228). Activity against Propionibacterium acnes and other acne producing

bacteria was observed in leaf extract (229). Some investigators think flavanoids

as responsible of antimicrobial activity of leaves (230) whereas others are in

favor of polyphenols (231). Plant leaves were also tested for anti-fungal

activities and found to be good inhibitor of Microsporum canis and

Trichophyton violaceum, Trichophyton mentagrophytes and Microsporum

gypseum (232, 233). Recently, bark and leaf crude extracts of Juglans regia L

are also found to be antimycobacterial (234). Investigations of antimicrobial

activity in this plant are more focused on leaves than bark. However, in

Pakistan, common use of dried bark in traditional medicine provides an insight

for probable antimicrobial activity of bark against bacterial pathogens.

Secondly, claims of antimicrobial activity in this plant have not been proved yet

in in-vivo studies. Study of the bioactive components and their antimicrobial

activity against multidrug resistant pathogens is another area need to be

explored.

The plant has also been reported as strong anti-oxidant, previously but with the

debate of active ingredient that can be phenolics (235) or gamma tocopherol

(224). Different other bioactivities have also been previously reported including

antiaging, antiproliferative, antimutagenic, anti inflammatory and

antinociceptive activities (236-238 and 239).

3) Hippophae rhamnoides The plant belongs to the family Elaeagnaceae and commonly called as Sea

buckthorn or Sea Berry. It is a native plant of northwestern Europe i.e.

Denmark, Netherlands, Germany, Poland, Finland, Sweden and Norway and

Asia including Pakistan, China, Russia, India, Nepal. There are 6 species and 12

subspecies available throughout the world. In Pakistan it is found abundantly in

Gilgit, Kurram Agency, Chitral, upper Swat, Skardu, Baltistan, Ladak and in

other parts of Northern Areas. The wild variety of sea buckthorn available in

Pakistan is Hippophae rhamnoides subsp. turkestanica. The plant grows

naturally in sandy soil at high altitudes in cold climates preferably but can also

grow in low altitude and temprate zones and in nutritionally poor soil. It cannot

grow in the shade. It is a thorny and hardy shrub of 2-4 meters in height with

soft, juicy, globose shaped, yellow to orange colored berries with 6-9 mm in

diameters and an average weight of 0.2-0.35g. The extensive root system is

capable of fixing nitrogen.

In ancient times, Greek people used Sea buckthorn in horse feed as weight

gaining supplement and shiny coat on feet. Today the plant has many

nutritional and medicinal benefits and growing as major economic crop in

many countries. China is especially on the ceiling of Sea buckthorn products

sale. In 1990, almost US$ 20million was recovered by them. Food industry all

over the world, use sea buckthorn berries for jam, jelly, juices and liquors.

Seeds are useful to get oil that is very famous cosmetic product. Upper two

layers of berry skin are also processed for skin creams. Leaves are believed to

be good for making herbal tea. The medicinal use of sea buckthorn is proved

Indian and Chinese systems.

Hippophae rhamnoides, the source of many food and cosmetic products is

known to rich in Vitamin C and carotinoides. Other important nutrients include

fatty acids, free amino acids, flavonoids, essential oil, carbohydrates, minerals,

organic acids and soluble sugars (240). Berries have a very characteristic aroma

which is tjought to be due to several aliphatic esters such as ethyl, 3-

methylbutyl andcis-3-hexen-1-y1 esters. The important ones are ethyl

hexanoate, 3-methylbutyl 3-methylbutanoate, 3-methylbutanoic acid, 3-

methylbutyl hexanoate, 3-methylbutyl benzoate and 3-methylbutyl octanoate

(241). Seeds and fruits are also rich in pigments and lipoproteins.

Carotenolipoprotein complexes are located particularly in upper membrane of

fruit. The ratio of unsaturated fatty acids is more than their saturated analogues.

The polar lipids included 61% phospholipids and 39% galactolipids (242).

Flavanol glycosides and aglycones were also separated and identified. Quercetin

aglycone, Myricetin aglycone were present in considerable amount (243)

whereas kaempferol 3-O-β-sophoroside-7-O- -rhamnoside was major flavanol

glycoside isolated (244). Other 5 flavanoides were also found in Sea buckthorn

leaves including catechin, rutin, quercetin, kaempferol and isorhamnetin (245).

It is interesting to note that place of origin and environmental conditions

immensely effect the composition. in a study, significant differences were

observed in the vitamin C, total sugar and acidity among different varieties of

Sea buckthorn fruit. Hippophae rhamnoides subsp rhamnoides (from Finland)

was characterized by low vitamin C, total sugar and high acidity while

Hippophae rhamnoides subsp sinensis (Chinese variety) showed high vitamin C

and total sugar. Hippophae rhamnoides subsp Turkestanica (that was a hybrid

of Chinese and Finnish variety) was found to be intermediate (246). Recently,

Chen et al introduced a chemical fingerprinting method by using HPLC to

differentiate the variation of flavonoid content and type. The method is useful to

identify berries from different species (247). In Pakistan, amount of moisture,

fatty acids, proteins, vitamins and sugars varies in two different color varieties

i.e. orange and red (248).

Sea buckthorn is known to have antioxidant activity. Vitamin C takes the major

part of antioxidant activity whereas phenolic compounds including quercetin 3-

O-glycosides, catechins, and hydroxybenzoic acids contribute up to 5% (249).

However, some other group observed the major role of phenolics (250). One

more group reported the cytoprotective role of Sea buckthorn leaves and berries

for lymphocytes. They found the extracts as remedy for chromium-induced

inhibition of lymphocyte proliferation (251). These results give insight for the

possible immunoenhancing role of this plant. Lately, a study was carried out in

Quebec, Canada to test the anticancer activity in Canadian sea buck thorn

berries. They found the extract inhibitory for the growth of stomach, prostate,

intestine and breast carcinoma cell lines. The inhibition of cancer cell

proliferation was the result of cell-cycle arrest (252). Isorhamnetin, a flavonol

aglycone of Sea buckthorn was found inhibitory for human hepatocellular

carcinoma cells (BEL-7402) in dose dependent manner. Cellular accumulation

of the compound leads to permeation of the cell membrane and fragmentation

and condensation of cellular chromatin (253).

Sea buckthorn seed and berry oil showed a significant inhibition in adenosine-

5′-diphosphate-induced platelet aggregation (254). Goel et al demonstrated the

protective nature of variety RH-3, on gama-rays induced spermatogenesis by

enhancing the spermatogonial proliferation, enhancing the stem cell survival

and reducing sperm abnormalities (255).

Explroing antimicrobial activity in this natural treasure has also been a focus for

microbiologists and phytoscientists. Investigation carried out on Finnish variety

of berries revealed least antimicrobial activity against gram negative organisms.

commensale of intestinal tract were inhibited but there was no activity observed

against Salmonella (256). In contrast, indian variety showed strong inhibtion

against same organism (257). Crude seed extract also inhibited Bacillus species,

Listeria monocytogenes and Yersinia enterocolitica (258, 259). In spite of these

few reports, there is no in-vivo experiment reported to verify antimicrobial

potential.

Aims and Objectives

Isolation, Identification and characterization of different

intracellular and extracellular bacterial pathogens.

Investigation of the antimicrobial activity of water and

organic extracts of different indigenous plants and plant

derived substances alone and in combination with well-

known antibiotics, which have lost their efficacy against

bacterial pathogens.

Further investigation of effect of plants on virulence

factors of various bacterial pathogens.

Study of the immunopharmacological properties of plants.

MATERIAL AND

METHODS

2.1 Collection, Isolation and Characterization of Bacterial

Pathogens A total of 378 different clinical bacterial isolates were collected from various

public and private sector laboratories of Karachi-Pakistan. Details regarding

their type, site of infection and others are listed in Table 1a. Various American

Type Culture Collection (ATCC) reference strains were also used (Table 1b).

Mueller Hinton agar (MHA), MacConkey’s agar and Luria-Bertani (LB) were

used to sub culture all gram negative organisms, whereas, gram positives were

grown on MHA and Blood agar. In case of Staphyloccous aureus, 2% NaCl was

added to MHA. Organisms were grown on respective media aerobically at 37oC

for 24 hrs. Mycobacterium tuberculosis H37Rv was subcultured on

Middlebrook 7H10 medium (BBL) and incubated for 15-20 days at 37oC in the

environment enriched with 5% CO2.

2.1.1 Characterization of Bacterial Pathogens by Conventional

Methods

A Identification

All clinical isolates were re-identified at Immunology and Infectious Diseases

Research Laboratory, Department of Microbiology, University of Karachi by

standard biochemical methods (260). Brief identification schemes are given in

Table 2a, b and c. Rapid identification systems like QTS 24(DESTO Labs-

Karachi) and API NE (Biomeurex) were used wherever required. Serotyping of

Salmonella species was also done.

B Antibiotic Susceptibility Pattern Antibiotics susceptibility profiles of all bacterial isolates were determined by

using Kirby-Bauer disc diffusion method as per performance

Table 2a: List of Clinical Bacterial Isolates

S.No. Organisms Specimen collected

Age of Patients (yrs)

No. of Isolates

1 Methicillin Resistant Staphylococcus aureus (MRSA)

Blood, Pus/ wound

any 99

2 Methicillin Sensitive Staphylococcus aureus (MSSA)

Blood, Pus/ wound

any 59

3 Salmonella enterica serovar Typhi (MDR)

Blood <5 16

4 Salmonella enterica serovar Typhi (Non- MDR)

Blood <5 22

5 Salmonella enterica serovar Paratyphi A

Blood <5 8

6 Salmonella enterica serovar Typhi (MDR) (Indian isolates)

Blood unknown 02

7 Salmonella enterica serovar Typhi (MDR) (Tanzanian isolates)

Blood unknown 01

8 Streptococcus pyogenes Throat 15-45 08

9 Enterotoxicgenic Escherichia coli (ETEC)

Diarrheal Stool

10 Enteropathogenic Escherichia coli (EPEC)

Diarrheal Stool

11 Enteroaggregative Escherichia coli (EAggEC)

Diarrheal Stool

12 Escherichia coli (uropathogenic) Urine any 30

13 Shigella species Stool any 35

16 Klebseilla pneumoniae urine adult 01

17 Vibrio cholerae stool adult 01

18 Bacillus subtilis Environmental - 01

19 Neisseria gonorrhoae uretheral swab adult 01

20 Mycobacterium tuberculosis sputum adult 07

21 Mycobacterium bovis - unknown 01

22 Mycobacterium avium - unknown 01

23 Pasteurella multocida serotype B-2 Blood (Buffalo)

24 Micrococcus species Environmental - 01

25 Pseudomonas aeurginosa Pus unknown 01

Total 383

Table 2b: List of Reference Bacterial Strains

S. No. Organism ATCC number

1 Mycobacterium tuberculosis H37Rv 27294

2 Escherichia coli 25922

3 Klebseilla pneumoniae 13883

4 Staphylococcus aureus 25923

5 Salmonella enterica serovar Typhi 13311

6 Shigella flexneri 9199

7 Pseudomonas aeruginosa 27853

8 Vibrio cholerae 9459

9 Mycobacterium smegmatus M2

10 Pseudomonas aeruginosa 27853

11 Salmonella braenderup H9812

Table 3a: Identification Scheme for Gram Positive Cocci S. #

Organisms Biochemical TestsCatalase Coagulase DNAse Hemolysis Oxidase Pigementation Mannitol Bacitracin Lactose

1 Staph. aureus + + + β _ white-Golden + ND + 2 Staph.

epidermidis + - + β - white - ND +

3 Micrococcus + - ND γ + orange + ND - 4 Strept. pyogenes - - - β - none - sensitive +

Table 3b: Identification Scheme for Gram Positive Rods S. # Organisms Biochemical Tests

Spore Catalase Glucose Mannitol Lactose 6.5% NaCl VP Nitrate Lecithinase Motility

1 Bacillus subtilis

+ + + + - + + + - +

2 Bacillus anthracis

+ + + - - + + ND + -

Table 3c: Identification Scheme for Gram Negative Rods S. #

Organisms Biochemical Tests Oxidase Sulfide Indole Motility Citrate Urea TSI Pyocinin

Production Lactose Growth on

MacConkeys 42oC

1 S. Typhi - - - + - - a/ak - - + - 2 S. Paratyphi A - + - + - - a/ak - - + - 3 Escherichia coli - - + + - - a/a - + + - 4 Klebseilla

pneumoniae - - - - + + a/a - + + -

5 Vibrio cholerae + + ND ND ND - + suppressed - 6 Pseudomonas

aeurginosa + - + + + + ak/ak + - + +

7 Shigella dysenteriae

- - - - - - a/ak - - + -

8 Shigella flexneri - - - - - - a/ak - - + -

standards set by Clinical Laboratory Standards Institute (261). The readings

were also interpreted using NCCLS breakpoint criteria Antibiotics discs (Oxoid)

used against gram negative organisms Ampicillin, Amoxicillin/Clavulanic acid,

Ofloxacin, Tetracycline, Amikacin, Gentamicin, Co-trimoxazole/ trimethoprim,

Chloramphenicol and nalidixic acid. In case of gram positive organisms,

susceptibility against Oxacillin, Erythromycin, Amikacin, Ampicillin,

Ofloxacin, Tetracyclin, Vancomycin and Co-trimoxazole/ trimethoprim was

checked.

In case of those strains of Staphylococcus aureus who appeared to be resistant

against oxacillin by disc diffusion method, minimum inhibitory concentration

(MIC) of oxacillin was determined by Etest-strips and agar dilution method by

using Muller Hinton Agar supplemented with 4% NaCl. Strains with

MICs >6µg/ml of oxacillin were defined as Methicillin resistant Staphylococcus

aureus (MRSA).

In case of Salmonella, strains resistant to first line drugs i.e ampicillin,

chloramphenicol and co-trimoxazole with or without resistance to tetracycline

and streptomycin were defined as multidrug resistant (MDR).

2.1.2 Characterization by Molecular Methods A total of 86 isolates of diarrheal E. coli were already characterized

genotypically according to the presence of different virulence factors by one of

my colleagues. The virulence genes which were taken under consideration for

their characterization are listed in Table 3.

Table # 4: Genotypic Characterization of Escherichia Coli Isolates

Strain Target gene

E. coli UID-A

ETEC LT, ST

EPEC eae, bfpA

EAggEC AstA, EAST

36 strains of Salmonella enterica serovar Typhi (S. Typhi) and 8 of Salmonella

enterica serovar Paratyphi A (S. Paratyphi A), isolated from blood cultures of

the patients suffering from enteric fever were grown

overnight on LB agar at 37°C and then subjected to molecular characterization.

A Plasmid Analysis Multi-drug resistant Salmonella strains harbor a plasmid encoding resistance to

all three antibiotics. Usually, a large and conjugative resistant plasmid (R

plasmid) of 98.6 mega-dalton(150 kb) is found to be responsible for the

resistance which belongs to the incompatibility complex group IncH1.

For size determination, Plasmid DNA was extracted from all isolates of

Salmonella by the alkaline lysis method of Kado and Liu with minor

modifications (262). Briefly, a loopful of bacterial culture was suspended in

50µl of resuspending buffer in a microtube, followed by the addition of 150µl

lysis buffer. Suspension was mixed properly and incubated at 56oC for 40

minutes. Then, 150µl Phenol-chloroform was added. Tubes were centrifuged

for 20minutes at 12000 xg. Upper layer which had Plasmid DNA was removed

carefully, mixed with 15µl of loading dye and electrophoresed on horizontal

0.75% agarose gels and stained with 0.05% ethidium bromide. DNA bands

were then visualized using a UV transilluminator (UVP).The Escherichia coli

reference strains V517 and 39R861 were used as molecular standards for the

determination of plasmid sizes.

i) Bacterial DNA Extraction A loopful of log phase culture was added in 500µl of sterile Mili Q water,

placed into screw-capped Eppendorf tubes and boiled for 20 minutes. Samples

were centrifuged for 15 minutes at 12000rpm and 300µl of supernatant was

transferred in another tube and store at –20C.

ii) Incompatibility grouping of plasmids by PCR All strains of S. Typhi and S. Paratyphi A who appeared having plasmid of

150kb by agarose gel electrophoresis, were subjected to PCR to determine

whether they belonged to the IncHI1 incompatibility group. The repHI1A

replicon, present in IncHI plasmids, was amplified via the polymerase chain

reaction using the primers

5' -CGA AAT CGG TCC AAC CCA TTG-3’, 5' -CGA CAA CTC ATC AGA

AGC GTC AAC- 3' as previously reported (263). Primers were used at a final

concentration of 1µM in a reaction mixture containing 1.5 mM MgCl2; 200µM

each) dATP, dCTP, dGTP, and dTTP; DNA polymerase buffer; 2 U of DNA

polymerase (Sigma) and 1 µl of genomic DNA as the template. Amplification

conditions were initial denaturation at 95.0°C for 5 min; 30 cycles of 95.0°C for

30 s, 57.5° for 1 min and 72.0°C for 1 min with a final extension of 72.0°C for 7

min. PCR products were resolved by electrophoresis on 1% gels at 100 V run

for 1 h with positive and negative controls. 100bp DNA ladder (0.5µg/lane) was

used as marker. Gels were stained with buffer containing 0.05% ethidium

bromide for 20 min and then destained with distilled water on rotation for 20

min. Visualization was done by using UV transilluminator (UVP). Amplicons

of 110 bp were considered positive for the RepHI1A.

B Determination of Class 1 Integron Integrons are genetic elements that usually have one or more integrated

antibiotic resistance gene cassettes. Three classes of integrons have been

characterized and among them class 1 often contain antimicrobial resistance

genes in clinical isolates of S. Typhi. All integrons have a 5´ conserved

segment (5´-CS), which has an intI gene encoding integrase and attI

recombination site, but a distinct 3´ conserved segments (3´-CS). attI

recombination site, located next to intI, is recognized by the IntI1 integrase, and

a promoter, Pc, which directs transcription of the cassette-borne genes, lies

within the intI1 gene. The 3´ conserved segment of class 1 integrons includes

qacE∆1, a deletion derivate of the antiseptic resistance gene qacE, and the sul1

gene, which encodes sulfonamide resistance (264, 265).

Fig 1: Schematic diagram of class 1 integron in S. Typhi. Two resistance gene

cassettes were detected—dfrA15 conferring resistance to trimethoprim and

aadA1 conferring resistance to spectinomycin and streptomycin. The

combination of the dfrA15 gene with the sul1 gene (sulfamethoxazole) results

in resistance to co-trimoxazole.

All Strains of S.Typhi and S. Paratyphi A were screened for the presence of

integrons with specific primers for the integrase genes intI1 via the polymerase

chain reaction using the primers L-5'-ACATGTGATGGCGACGCACGA-3 and

R-5'-ATTTCTGTCCTGGCTGGCGA-3'' as previously reported (266). Reaction

mixture consisted of 1.5 mM MgCl2, 200 mM dNTPs, 50 pmol of each primer,

DNA polymerase buffer, 2 U of DNA polymerase (Sigma), and 1 µl of genomic

DNA as the template. Amplification conditions were initial denaturation at

95.0°C for 5 min; 30 cycles of 95.0°C for 1 min, 62° for 1 min and 72.0°C for 1

min with a final extension of 72.0°C for 3 min. positive and negative controls

were run with test samples. PCR products were resolved by electrophoresis on

1.5% gels at 100 V run for 1 h. Amplicons of 569 bp were considered positive.

C Analysis of Conserved Region of Class 1 Integron (CS

5'-3') Analysis of the class 1 integron variable region was performed on intI1-positive

strains by using same reaction mixture with primers 5´ -CS-5'-

GGCATCCAAGCAGCAAG-3' and 3´ -CS-5'-AAAGCAGACTTGACCTGA-

3'(267) Amplification conditions were initial cycle at 94.0°C for 1 min; 30

cycles of 94.0°C for 1 min, 55° for 30sec and 72.0°C for 3 min with a final

extension of 72.0°C for 5 min. PCR products were resolved by electrophoresis

on 1% gels at 100 V run for 2h. Size of variable region was determined by

observing the gel under UV transilluminater.

D PCR for dfrA7

All CS positive strains were subjected to cassette assortment of dfrA7,

conferring resistance to trimethoprim, by PCR. Reaction mixture consisted of

1.5 mM MgCl2, 200 mM dNTPs, 50 pmol of each primer, DNA polymerase

buffer, 2 U of DNA polymerase (Sigma), and 1 µl of genomic DNA as the

template. Primers used were F 5' GTG TCG AGG AAA GGA ATT TCA AGC

TC 3' and 5' TCA CCT TCA ACC TCA ACG TGA ACA G 3'. Amplification

conditions were same as in case of InCH1. Positive and negative controls were

run with test samples. Amplified products were run at 1.5% agarose gel at 150V

for ~2 hrs. Amplicons of 191bp were considered as positive.

Table # 5: Oligonucleotides used for Identification of Resistant Genes in Salmonella enterica serovar Typhi and Salmonella

enterica serovar Paratyphi A

Primer Sequence Length of Product (bp)

incH-F

InCH-R

5'-CGAAATCGGTCCAACCCATTG-3'

5'-CGACAACTCATCAGAAGCGTCAAC-3'

intI1L

intI1R

5'-ACATGTGATGGCGACGCACGA-3'

5'-ATTTCTGTCCTGGCTGGCGA-3'

dfrA7-F

dfrA7-R

5'-GTG TCG AGG AAA GGA ATT

TCAAGCTC-3'

5'- TCA CCT TCA ACC TCAACG TGA ACA G-

5´ -CS

3´-CS

5´ -CS-5'-GGCATCCAAGCAGCAAG-3'

5'-AAAGCAGACTTGACCTGA-3'

Variable

2.1.3 Pulse Field Gel Electrophoresis

I. Introduction PFGE is considered gold standard in molecular typing as it is reliable,

reproducible and highly discriminatory. The technique was first developed in

1983 and describes the separation of large molecular weight DNA fragments in

agarose gels. (268) An array of 24 electrodes is clamped and produces

homogenous electric fields from 90o-120o within the gel to prevent lane

distortion. Due to the alternative pulses of current, DNA fragments move

backward and forward into the gel. Electrodes sense changes in gel thickness,

constituents of buffer and temperature and readjust immediately to maintain

uniform field, thus give high resolution. DNAs from 100bp to 10mb can be

effectively resolved.

II. Method

A. Growth of Bacterial Cells

All strains of Salmonella enterica serover Typhi and Paratyphi A were grown

overnight on LB agar for individual colonies. Sufficient colonies were

suspended directly with cotton swabs in 5ml of CSB. Cell suspensions were

adjusted with CSB to obtain an OD of 0.5-0.55 at 600nm. CSB was used as

blank.

B. Preparation of Bacteria Embedded Agarose Plugs

2% chromosomal grade agarose solution (BioRad) was prepared in TE and

placed into a water bath maintained at 55oC. Aliquots of 500µl of cell

suspensions were transferred to microcentrifuge tubes. 20µl (8 U) of proteinase

K (stock solution= 20mg/ml) was added to each tube and mixed well. 500µl of

molten agarose was added to each microtube. Bacterium-agarose mixture was

mixed well and immediately added to plug molds by avoiding any bubbles. The

plugs were allowed to solidify for 10 min at 4oC. Solidified plugs were

transferred to sterile tubes containing 2ml of CLysis B containing 3 U of

proteinase K and incubated for 2hr at 55oC in water bath for proteolysis. Then

plugs were washed twice with 10ml of sterile, preheated (55oC) distilled water

at 55oC for 10 min in water bath with gentle mixing. Subsequently, three

washes in 5 ml of preheated (55oC) TE was done with same procedure. The

plugs were then cooled at room temperature in TE buffer.

C. Restriction Enzyme Digestion of Agarose Plugs

For restriction enzyme digestion, One-third (3mm) slices of each plug was

incubated at 37oC overnight in 100µl restriction mix containing 50 U of Xba1

enzyme. Tubes were then kept into fridge for 30min to harden the plugs for ease

of handling.

D. Gel Electrophoresis

1% agarose gel was prepared in 0.5 X TBE and poured into gel slab. Plug slices

were loaded with a little low melting point agarose so that the slices remain in

position. Approximately 2 liters of 0.5 x TBE buffer was placed into

electrophoretic tank. PFGE of agarose plug inserts was then performed on a

CHEF-DR III system (Bio-Rad) for 22 hrs at the rate of 6V/cm (200 V), with a

pulse time of 2s to 64s and a 120o linear corner at 14°C. Salmonella braenderup

H9812 was treated same as unknown samples throughout the procedure and run

as reference standard.

E. Staining of Gel

The gels were stained for 20min by immersing in 500ml of sterile distilled

water containing 50µl of ethidium bromide (10mg/ml) and destained in distilled

water only for 30min. gels were photographed on a UV transilluminator. The

restriction endonuclease digest patterns were compared, and their similarities

were scored to determine pulsotypes.

2.2 Collection, Preparation and Characterization of

Plants 2.2.1 Plants Collection Three different plants of indigenous origin were studied during the study. Dried

leaves of Camellia sinensis (Green Tea) was purchased from local market.

Juglans regia (Dandasa) is the dried bark of Persian walnut tree which is very

famous teeth brightening and lip decorating substance among the ladies of

NWFP region of Pakistan. Dried bark was purchased from local market of

Swat-NWFP region of Pakistan. Hippophae rhamnoides (sea buckthorn), berry

fruits commonly found in Gilgit-Pakistan were collected in the form of dried

berries. All dried plants material was ground in a commercial blender and kept

at room temperature until required.

2.2.2 Preparation of Aqueous Extracts A 5% solution of each dried plant material was prepared in sterile distilled

water by heating at 95oC in water bath for two minutes and cooling for two

minutes. Procedure was repeated three times and final extracts were centrifuged.

Supernatants were filtered through 0.2µm membrane, stored at -20oC and

thawed before use. Every time stored aqueous extracts were used for not more

than one week for different bioassays.

2.2.3 Preparation of Organic Extracts Ground and dried material of every plant was seeped into 95% methanol in a

1:10 (w/v) ratio for at least 24hrs on shaking at room temperature separately.

The crude organic extracts were obtained after evaporation of solvent in rotary

evaporator under vacuum, weighed and stored in dried form at room

temperature until required. For different bioassays, stock solutions were

prepared by dissolving concentrated methanolic extracts into DMSO (Merck).

2.2.4 Bioassay-guided Chemical Analysis of Extracts Various techniques were employed to separate chemical components present in

these plants including Thin Layer Chromatography (TLC) and Column

chromatography. Bioautography was performed to locate bioactive components.

Analysis of crude extracts as well as bioactive fractions was performed by

MALDI-TOF-MS.

A Thin Layer Chromatography Thin layer chromatography is a method which allows the separation of

compounds depending on their chemical structure. Different compounds can be

separated according to their affinity with the solvent and the silica particles of

the plate. Commercial Silica gel plates (10x10cmx0.25mm thickness, Fisher

Scientific) were used for the whole study.

100ml of 10% and 30% methanolic extracts were kept in rotavapor to

concentrate. 100µl of each concentrated extract was spotted on TLC plates and

developed in different solvent mixtures which are listed in Table # mm5. Plates

were placed in pre-poured glass tanks, lined with filter paper for sufficient time

period to allow a better solvent distribution in the entire developing chamber.

Plates were then dried at room temperature and visualized under UV light (254

and 365nm) to recognize and mark the separated components. All

chromatograms were developed in duplicate; one plate was used to test the

microbiological activity and the other one to analyze the bands corresponding to

the active ones. Procedure was repeated several times in same condition for the

confirmation. All chemical used in this procedure were of HPLC grade.

Table # 6:

Solvent Mixtures Used in Thin Layer Chromatography

A -------- CHCl3/MeOH = 85:15

B----------Ethyl acetate/n-hexane = 60:40

C--------- CHCl3/MeOH = 70:30

D--------- Ethyl acetate/MeOH = 85:15

E----------CHCl3/MeOH/acetonitrile = 80:10:10

F-----------CHCl3: Ethyl acetate/MeOH= 50:40:10

B Bioautography This technique has long been used to locate antimicrobial component(s) present

in plant extracts (269, 270). This technique is based on the direct killing of test

organism on contact with band having antimicrobial activity present on

chromatogram.

Three different bacterial strains were used for screening which includes

Staphyloccus aureus (ATCC 25923), clinical strain of MRSA (strain # 3443-

isolated from blood specimen) and clinical strain of Salmonella enterica serovar

Typhi (strain # 2877-blood isolate, R-type: AmpCCoTNA). Pure cultures were

grown overnight in LB broth at 37oC in orbital shaker. Small size of inoculum

was added to fresh LB broth and incubated again for 2 hrs in same conditions.

Sterile LB agar was prepared, kept molten at 55oC and seeded with various test

strains separately with final inoculum size of 106 CFU/ml of log phase culture.

Agar was further poured on properly dried TLC plates. Plates were incubated in

humid chamber overnight at 37oC aerobically and then sprayed with a solution

of 2mg/ml of p-iodonitrotetrazolium violet (Sigma). Plates were kept incubated

again for 1hr for complete colour development. Areas of bacterial growth

appeared violet because of reduction of tetrazolium salt into colored product-

formazan by biologically active organisms, while clear zones corresponded to

bacterial inhibition. Inhibition zones were compared with related spots and their

Rf values on reference plates.

The spot corresponding to the biological activity was scrapped off from

reference plates, transferred into a pipette Pasteur equipped with a glass wool

filter and eluted with 2 ml Methanol + 1ml of Chloroform. The solvent was

evaporated and the extract re-suspended in 500ul of methanol. This extract was

used for MALDI-TOF determination, on positive and negative ionization mode.

For each compound was taken also a non-active band to allow a comparison.

C MALDI-TOF-MS

i) Principle Matrix-assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF-MS) is a new analytical approach for the characterization of

Matrix-Assisted Laser Desorption/Ionization- Time of Flight

Mass Spectrometer

Target Gold Chip Array Plate

macromolecules. This technique was first developed for large biomolecules i.e.

proteins, carbohydrates, lipids and nucleic acids in 1987 (86, 271) but has also

been used in food technology to characterize small molecules like flavonoids

and tannins present in green tea, onion bulbs, red wine, and fruit juice (90, 91).

Many factors play role in making this tool basic and popular in biomedicine,

food technology and molecular biology including easy sample preparation,

rapid generation of reliable data, and good tolerance towards additives and

determination of wide range of masses from low to high molecular weight in

complex samples. The technique is based on bombardment of sample with a

laser light to bring about sample ionization. Samples in any appropriate solvent

are co-crystallized on an appropriate chemical substance, usually a weak organic acid

(the matrix) which absorbs energy emitted by the pulsed laser beam (often

nitrogen laser of 337nm). Matrix transforms the laser energy into excitation

energy for the sample and a fraction of sample was ionized. During this process,

the matrix absorbs the laser energy and transfers it to the sample in a very

smooth and safe way to prevent its fragmentation and degradation the energy

transfer is very smooth and safe. Different matrices suit different biomolecules.

Table # 7 shows the list of matrixes used for MALDI-TOF.

Table # 7: List of Matrixes used for MALDI-TOF-MS

Name Structure Formula λ max M.W. Applications

Sinapinico acid (SA)

3 - ( 4 - H y d r o x y - 3 ,5 - d i m e t h o x y - p h e n y l ) - a c r y l i c a c i d

C11H12O5 266nm,

337nm,355nm 225.22 Lipids, peptids, proteins

alfa-ciano-4-idrossi

cinnamic acid (CHCA)

2 - C y a n o - 3 - ( 4 - h y d r o x y - p h e n y l ) - a c r y l i c a c id

C10H7NO3 337nm 355nm 190.18 Lipids, peptids, Peptidi, nucleotides

2',4',6'-triidrossi acetophenon

(THAP)

1 - ( 2 ,4 ,6 - T r ih y d r o x y - p h e n y l ) - e t h a n o n e

C8H8O4 337nm 355nm 186.17 Oligonucleotides

3-idrossi

picolinic acid (HPA)

3 - H y d r o x y -p y r id in e - 2 - c a r b o x y l ic a c id

C6H5NO3 337nm,355nm 140.12 Oligonucleotides

2'-6'-diidrossi acetophenon

1 -( 2 ,6 - D i h y d r o x y - p h e n y l) - e t h a n o n e

C8H8O3 337nm,355nm 153.16

Proteines e oligonucleotides

6-aza-2-thiothymine

6 -M e th y l - 3 - t h i o x o - 3 ,4 - d i h y d r o - 2 H - [ 1 ,2 , 4 ] t r i a z in -5 - o n e

C4H5N3OS 266nm, 337nm

355nm 143.17 Oligonucleotides,

lipids

Schematic Diagram of the Principle of MALDI-TOF-MS

Fig # 7 illustrates principle of MALDI-TOF-MS that mainly a

combination of sample ionization through Laser Beam. The ions enter in

a field-free region (mass analyzer) where time required for travel and

velocity of particles is calculated. Ions pass ion mirror and hit detector

that calculate m/z.

MALDI is a pulsed ion source and is usually coupled with discontinuous mass

analyzer such as TOF. The laser pulses vaporize the matrix compound and

produce a plume that carries the protonated molecules into the gas phase. The

gas-phase ions are directed into the mass analyzer by appropriate electric field.

Following acceleration, the ion enters a field-free region where it travels at a

velocity that is linearly dependent to the kinetic energy (reflection voltage) and

inversely proportional to its m/z. The time required for the ion to travel the

length of the field-free region, is measured and used to calculate the velocity

and ultimately the m/z of the ion. Low mass ions travel faster than heavier ions.

The instrument used for this work was equipped with a delay extraction system

(a disposal altering the ion optics of the TOF m/z analyzer), to minimize the

effect of the initial kinetic energy. It was also equipped with a reflectron system,

an ion mirror that reverses the flight path of the ions in a manner that corrects

for the differences in the kinetic energy of ions with the same m/z and increases

the ion flight path. The ions hit the detector and are counted by a micro channel

plate (MCP).

A crucial factor that affects the quality of MALDI-TOF spectra is the

crystallization of the analyte during sample preparation and the behavior of the

matrix after laser irradiation. Therefore three types of matrices were tried in the

beginning i.e. a-cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid (SA),

and dihydroxybenzoic acid (DHB).

Table # 8: Parameters for MALDI-TOF Acquisitions

Sample Period (ns) 0.5

Signal Sensitivity (mV) 100.0

AltReflectronFieldLength 1.25300

AltReflectronLength 0.33500

Polarity LDI+

Reflectron Voltage (v) 5200.00

Source Voltage (v) 12000.00

Pulse Voltage (v) 1950.00

MCP Detector Voltage (v) 2350.00

Laser Energy % 195.0

Matrix Suppression (amu) 400.0

TLF Delay (ns) 500.0

Mass Range from 400 to 3000 AMU

Matrix Suppression 400 AMU

ii) Method 1. The extract of bioactive spots from TLC plate was suspended in 100 µl

methanol and was split according to the ionization modality.

2. 0.1 % of TFA (trifluoro acetic acid) was added to 200 µl of the TLC

methanol extract solution for the samples to be acquired on positive

ionization mode whereas NH4OH was added to 200 µl (final

concentration 0.1 M) of the TLC methanol extract solution for the

samples to be acquired on negative ionization mode.

3. For each analysis, the samples were mixed with the matrix (Alfa-cyano-

4-hydroxycinnamic acid (CHCA) with a ratio of 1:1 (v/v).

4. An aliquot of 2µl of this mixture was spotted on a target gold chip array

plate and let dried at room temperature.

5. The target plate was loaded onto the MALDI-TOF analyzer of MALDI

MICRO-MX (WATERS).

6. Samples were desorbed and ionized by a nitrogen laser (wavelength 337

nm; 4ns pulse width) and extracted 1950 V pulse voltage with time-

delayed extraction of 500 ns before entering the time-of-flight mass

spectrometer and accelerated under 12 kV. MCP detector voltage to

record spectra was 2350 V.

7. The instrument was operated in the positive and negative ion mode.

8. For the spectra interpretation, peaks with m/z ratio lower than 400, were

not considered because of matrix and reagent interferences. Other

parameters used for aquisitions (positive ionization mode) are listed in

Table 6.

2.2.5 Isolation of newly purified compound from

Camellia sinensis

Aqueous boiled extract of Camellia sinensis (Green Tea) was concentrated to

one fourth of volume and divided into portions i.e. filtrate and residue. Residue

was further extracted in methanol whereupon methanol soluble and insoluble

fractions were separated. Methanol soluble fraction was further fractionated

with different solvents of increasing polarity to separate non polar components

from polar and semi polar. The solvents used were n-hexane, chloroform and

ethyl acetate. Thin layer chromatography of each extract was monitored.

Chloroform extract showed promising result and revealed the presence of 2

major components. The mixture was separated by PLC. The white compound

was purified by re-crystalization. The melting point of compound was 235oC.

Using IR-spectrum and 13C-NMR techniques structural analysis of the

compound was carried out. The code # FA-CS II was used for this compound.

This work was carried out in collaboration with Dr. Rehana Afzal’s research

group at Department of Chemistry, University of Karachi.

2.3 Antimicrobial Activity of Plants and Plant derived

Substances Aqueous and organic plant extracts, their various organic fractions and newly

isolated compound(s) were screened for in-vitro antimicrobial activity against a

total 417 different extracellular and intracellular clinical bacterial isolates and

standard ATCC reference strains (listed in table 1a & b). Log phase bacterial

cultures were used for all assays performed to determine antimicrobial activity

of plants and plant derived substances.

2.3.1 Agar Well Diffusion Method Aqueous and methanolic extracts of Camellia sinensis, Juglans regia and

Hippophae rhamnoides and organic fractions of Juglans regia were screened

for antimicrobial activity by this method. All methanolic extracts and other

organic fractions were first dried in vacuum evaporator and then suspended in

sterile DMSO to get stock solution. Their working solutions were made in

sterile normal saline prior to their use for screening. 24 hrs old pure culture was

sub cultured in Muller Hinton Broth and incubated for 3 hrs at 37oC aerobically

on shaker to achieve log phase active culture. Turbidity was then adjusted in

3ml phosphate buffer saline to 0.5McFarland index. With the help of sterile

cotton swab, bacterial lawn was spread on MHA plates. Wells were dug in agar

plates with the help of sterile metallic borer (diameter=6mm). 20 µl of various

dilutions of aqueous extracts and organic fractions were poured into well which

were marked respectively. Plates were incubated (upside up) at 37oC aerobically

for 18hrs. Size of the zone of inhibition was recorded in mm. extracts or

fractions giving zone of inhibition >15mm were considered of possessing

antimicrobial activity.

2.3.2 Determination of MIC of Plants and Plant derived

Substances by Agar Dilution Method Minimum inhibitory concentrations (MICs) of aqueous and methanolic crude

extracts of Camellia sinensis, Juglans regia, Hippophae rhamnoides and

organic i.e. hexane, methanol, ethylacetate, chloroform and water fractions of

Juglans regia were determined by agar dilution method. Experiments were

carried out in cation adjusted Muller Hinton Agar (oxoid) with the

supplementation of 2% NaCl if test strains were Staphylococcus aureus and

with the supplementation of 4% NaCl if test strains were Methicillin resistant

Staphylococcus aureus. While in case of other gram negative and gram positive

organisms MHA was used without NaCl. Various extracts were added to agar

with a ratio of maximum 1:20 to avoid any hindrance in solidification of agar.

Concentration of aqueous crude extracts tested ranged from 5000 µg/ ml to 50

µg/ml. concentrations of organic fractions tested ranged from 2500µg/ ml to

19.53µg/ ml. 24 hrs old culture was refreshed for the log phase growth and

turbidity was achieved to 0.5McFarland’s nephlometer index as described above.

10 µl from the tube of each strain was inoculated on properly dried MHA plates

with and without extracts. Plates without any plant extracts were served as

positive control. Maximum 20 strains were tested to check their susceptibility

on one plate. After inoculation, plates were left on the bench for 30min for

proper absorption and then incubated at 37oC aerobically for 18hrs. Least

concentration of extract or fraction inhibited bacterial growth was considered as

MIC against that particular strain.

Substances by Microbroth Dilution Method This method was used to determine MICs of FACS II B-new purified

compound from Camellia sinensis and to reconfirm the MICs of aqueous and

methanolic crude extracts of Camellia sinensis, Juglans regia, Hippophae

rhamnoides as well as organic fractions and sub fractions of Juglans regia.

Concentration tested ranged from 5000 µg/ ml to 50 µg/ml whereas the

concentration of FACS II B was ranged from 500 µg/ ml to 0.5 µg/ ml. In

sterile flat bottomed 96-well plates, two fold serial dilutions of each extract or

compound was made in Mueller Hinton Broth. The starting inoculum was 5x105

CFU/ml of log phase culture. Final volume of broth achieved in each well was

100 µl. wells containing no extract but inoculated with test strains were

considered as positive control. Negative control wells consisted of serial

dilution of extracts or compounds only. Plates were incubated at 37oC

aerobically for 18hrs. Highest dilution of plant extracts or compounds showing

no turbidity were recorded as MIC. In every case experiment was carried out in

quadruplicate at two different occasions.

Substances by Tube Dilution Method Minimum inhibitory concentrations (MICs) of aqueous and methanolic crude

extracts of Camellia sinensis, Juglans regia, Hippophae rhamnoides and

organic i.e. hexane, methanol, ethylacetate, chloroform and n-butanol fractions

of Juglans regia were also reconfirmed by tube dilution method. Experiments

were carried out in 2 ml of MHB dispensed in sugar tubes as per standard

guidelines (260) with the starting inoculum of 5x105 CFU/ml. Tubes containing

no extract but inoculated with test strains were considered as positive control.

Negative control tubes consisted of serial dilution of extracts or compounds

only. MICs were determined in duplicate by this method.

2.3.5 Determination of Minimum Bactericidal Concentration

(MBC) of Plants and Plant derived Substances After the determination of MICs by microbroth and tube dilution methods,

organisms were preceded to check cidal activity present in plants and plant

derived substances. Briefly, 100 µl of MHB from the wells and tubes containing

plant extracts, fractions or compounds (32-2 fold higher concentrations than

MICs or 4 tubes/wells before MIC point) was taken and washed with PBS to

remove any residual plant material in it. Sediment was then resuspended in PBS

and inoculated on extract or antibiotic free MHA plates. The plates were then

incubated for 18hrs at 37oC aerobically. The MBC was taken as the lowest

concentration of each drug that resulted in no bacterial growth following

removal of the drug.

2.3.6 Effect of Plants and Plant derived Substances on Time Kill

Kinetics of Bacterial Pathogens

In order to determine antimicrobial effect of aqueous and methanolic extracts of

Camellia sinensis, Juglans regia, Hippophae rhamnoides, various organic

fractions of Juglans regia and new purified compounds on the growth of

various microorganisms, time kill kinetic assays were performed. 10 strains of

MRSA and MSSA each, Enterotoxigenic E. coli, Enteropathogenic E.coli,

Enteroaggregative E. coli, Uropathogenic E. coli, MDR Salmonella enterica

serovar Typhi (3 strains each) and ATCC reference strains of same species were

used for this study. Tests were performed in 50 ml of cation adjusted Muller

Hinton Broth (MHB). Plant extracts or compounds were added to the flask with

the final concentrations of 0.2, 0.5, 1, 2 and 4 x MIC separately. Actively

growing log phase culture of the test organisms with the starting inoculum of 1

x 105 CFU/ml was then added to the flasks. In vitro killing of the organisms

was monitored over 24h at 37oC. 100µl aliquots were collected at different time

intervals including 0, 1, 2, 3, 4, 6, 8 and 24 hrs and cultured on MHA plates for

the determination of CFU/ ml. Curves were constructed by plotting the log10 of

CFU/ ml verses time. All the experiments were performed in duplicate on three

different occasions.

2.3.7 Antimicrobial activity of Plant Extracts in

combination with Antibiotics Increasing number of multidrug resistant organisms, not only compel the

scientists to search new remedies in plants and plant derived substances but to

carry out their interaction studies with already established antibiotics. Seeking

synergistic drug combinations is another reasonable approach that might help in

exploring new treatment regimens. In our study, different aqueous and

methanolic plant extracts who gave very promising antimicrobial activity

especially against multidrug resistant organisms were tested for their interaction

with various those well known antibiotics that have lost their efficacy against

multidrug resistant extracellular and intracellular pathogens i.e. MRSA and

MDR Salmonella Typhi. Following different methods were used for interaction

studies.

A Checkerboard Titration Method for Synergistic Studies

Interaction of aqueous extract of Juglans regia with different antibiotics i.e.

oxacillin, chloramphenicol and tetracycline was determined against 5 different

clinical strains of MRSA, 5 different strains of MSSA and Staphylococcus

aureus ATCC 29213 by standard checkerboard titration method as described

previously (272).

Briefly, assays were performed in cation adjusted MHB dispensed in flat-

bottomed sterile 96 well plates. 2 fold serial dilution of testing antibiotic was

made in all rows (from 1 to 11) of microtitre plate whereas 2 fold dilution of

extract was prepared in separate tubes first. Then diluted extract was dispensed

in all columns (from A to G) in the same quantity. Every well of microtitre plate

had combination of different concentrations of both. Plant extract was dispensed

alone in Row # 12 while column H had testing antibiotic only. Final volume

achieved in each well was 100 µl. 1x 104 CFU of active log phase culture was

added to each well. Highest concentration of Juglan regia extract used in these

experiments was 5000µg/ml. In case of oxacillin highest concentration was

20µg/ml, for chloramphenicol 128 µg/ml and for tetracycline was 64µg/ml.

Plates were incubated at 37oC for 18 hours. Interaction between extract and

antibiotics was interpreted as synergy, additive effect, no effect and antagonism

based on Fractional inhibitory concentration index (FICI), which is the sum of

FICs of both agents. The FIC of each agent is calculated as the MIC of the agent

in combination, divided by the MIC of the agent alone. The FICI results were

interpreted as follows: < 0.5, synergy; 0.5 to 1, additive effect; > 1 to 2, no

effect and >2, antagonism.

Interaction of aqueous and methanolic extracts of Camellia sinensis with

different other antibiotics i.e. nalidixic acid, tetracycline, chloramphenicol were

also determined against 5 different clinical strains of MDR Salmonella enterica

serovar Typhi (R type: AmpCCoTNA) by the above mentioned method.

Highest concentration of Camellia sinensis extract used in these experiments

was 5000µg/ml. 4096µg/ml, 1024 µg/ml and 2048 µg/ml were the highest

concentrations of tetracycline, chloramphenicol and nalidixic acid respectively.

B Disc Diffusion/ Agar incorporation Method for Synergistic

Studies

Two different combinations, who gave synergistic activity by checker board

titration method, were tested to reconfirm their activity by disc diffusion/ agar

incorporation method. These combinations are as follows,

1. Aqueous crude extract of Juglans regia with oxacillin against MRSA

2. Aqueous or methanolic extract of Camellia sinensis with nalidixic acid

against MDR Salmonella enterica serovar Typhi

Plant extracts were incorporated into MHA plates with a ratio of 1:20. Final

concentrations achieved in each plate were 0.5 and 0.2 x MIC. This set of plates

was termed as test. One plate with MHA only was run as control. 24 hrs old

pure culture was sub cultured in Muller Hinton Broth and incubated for 3 hrs at

37oC aerobically on shaker to achieve log phase active culture. Turbidity was

then adjusted in 3ml phosphate buffer saline to 0.5McFarland index. With the

help of sterile cotton swab, bacterial lawn was spread on MHA plates and

antibiotic discs were placed with a proper distance. Plates were then incubated

at 37oC for 18hrs. Size of zone of inhibition was measured. On test plate, if

zone of inhibition around antibiotic disc was 3-5mm than the zone of inhibition

on control plate, the result was considered as synergistic.

C Etest strip/ Agar incorporation Method for Synergistic

Studies

This method was used to confirm the exact effect of Juglans regia on MIC of

oxacillin against MRSA strains. Plates were prepared as described in above

method. Bacterial lawn was spread and Etest strip of oxacillin was placed and

incubated at 37oC for 18hrs. Difference in MIC values of oxacillin in the

presence and absence of Juglans regia was noticed.

D Effect of Synergistic Antimicrobial Combinations on Time

Kill Kinetics of Bacterial Pathogens

Time kill kinetic assays were performed. Plant extracts, antibiotics and

antibiotics + plant extracts were added to the flask containing 50 ml of MHB

separately with the final concentrations of their MIC values. Starting inoculum

size was 1 x 105 CFU/ml. Effect of synergistic combination as compare to their

individual effects on growth cycle of testing organisms was monitored over 24h

at 37oC. 100µl aliquots were collected at different time intervals including 0, 1,

2, 4, 6, 8 and 24 hrs and cultured on MHA plates for the determination of CFU/

ml. Curves were constructed by plotting the log10 of CFU/ ml verses time. 2

log10 decreases in CFU of organisms treated with synergistic combination than

their components alone was considered as synergistic (273).

2.3.8 Effect of Plant Extracts on Bacterial cell

Morphology

To investigate the effect of plant extracts on bacterial cell morphology,

Methicillin resistant Staphylococcus aureus was observed under transmission

electron microscope after getting treatment with sub-inhibitory concentrations

of Camellia sinensis and Juglans regia. Bacteria were grown in MHB with

plant extracts at 0.5 x MIC for 18hours. Organisms were centrifuged at 5000 x g

and pellet was washed twice with normal saline.

Formvar coated 300-mesh copper grids were used for sample coating. 5 µl of

bacterial samples were placed onto a sheet of parafilm. With the help of sterile

tweezers grids were soaked in upside down position into the drop for 10 min.

excessive fluid was drained off by touching sterile filter paper. Grid was rinsed

once with sterile distilled water to remove excessive non-coated material.

Negative staining was done by immersing coated grids in 1% uranyl acetate for

20seconds. Destaining was achieved by one quick dip of grids in sterile distilled

water. Excess fluid was wicked away with filter paper and placed in sample tray

(specimen side up) for air dry. Copper grids were loaded one by one and

observed directly with a JOEL CO-Japan’s JEM 100 transmission electron

microscope operating at 80 kV

2.3.9 Effect of Plant Extracts on Protein Profiles of

Bacterial Pathogens Pathogenicity of an organism depends on its virulence factors i.e. extracellular

and cell associated proteins. The synthesis of these products can be suppressed

by antimicrobial substances at sub-inhibitory concentrations which have little

effect on overall bacterial growth. Change in protein profile of an organism

ultimately leads to the disturbance in the establishment of an infection. To

determine the effect of aqueous and methanolic extracts of Camellia sinensis

and Juglans regia, different organisms were treated with subinhibitory

concentrations of plant extracts, their exoproteins and cell associated proteins

were prepared and run by single dimensional SDS-PAGE. Change in protein

profiles of following organisms was observed,

• Enterotoxigenic Escherichia coli (clinical isolate)

• Methicillin Resistant Staphylococcus aureus (clinical isolate)

Stock cultures were cultivated overnight on MHA. Isolated colonies were

inoculated in MHB carefully and incubated for 3 hrs at 37oC aerobically on

shaker. Plant extracts were added to the flasks containing 200ml of MHB

separately with the final concentration of 0.2 x MIC and termed as Test flasks.

Whereas, flasks having MHB without plant extracts was considered as control.

Actively growing bacteria were then inoculated in each flask with the starting

inoculum size 1 x 105 CFU/ml and incubated with shaking at 37oC for 18hrs.

Broth culture was centrifuged at 10000 rpm at room temperature.

A Preparation of Extracellular Bacterial Proteins 1. Filtrates, obtained by centrifugation, were removed and filtered by

membrane (0.2µm).

2. Chilled absolute ethanol (-20oC) was then added to cell free supernatants

till final concentration reached upto 70%.

3. Flasks were mixed properly and kept at -20oC overnight for precipitation

of exoproteins.

4. Suspensions were centrifuged (10,000g) for 30 min at 4°C.

5. The resulting pellets were dissolved in 2 ml of chilled deionized water.

6. Samples were analyzed for protein concentrations using BioRad Protein

assay reagent and run on sodium dodecyl sulfate (SDS)-polyacrylamide

gel electrophoresis.

7. Proteins were stained with Coomassie blue stain.

B Preparation of Lysate of Cell associated Bacterial Proteins

Principle: Sonication is a popular method of physical disruption of bacterial cells and has

been reported to use as a key procedure in cell fractionation (274, 275). The

method uses pulsed, high frequency sound waves to agitate and lyse cells,

bacteria, spores and finely diced tissues. Sound waves are delivered using an

apparatus with a vibrating probe that is immersed in chilled liquid cell

suspension. Mechanical energy from the probe initiates the formation of

microscopic vapor bubbles that form transiently, causing shock waves to radiate

through a sample. To prevent excessive heating, ultrasonic treatment is applied

in multiple short cycles to a sample immersed in an ice bath.

Method: 1. After centrifugation, sediment containing bacterial cells was washed

twice with10mM Tris HCl (pH 7.5) and resuspended in 2 ml of same

buffer.

2. Sonicator was turned on.

3. Cells were kept on ice bath and disrupted by sonication with the cycles

of bursts for 4 minutes in case of gram negative and 6 min in case of

gram positive organisms. Each cycle was of 15 s, separated by cooling

periods of 15 s.

4. Crude lysate of cell associated proteins were obtained after

centrifugation at 15000 g for 45 min at 4 °C.

5. Lysate was preceded for estimation of proteins concentration,

6. Further analysis was done by sodium dodecyl sulfate (SDS)-

polyacrylamide gel electrophoresis. If not used immediately, were stored

at -20°C.

C Protein Estimation The total protein concentrations in samples were determined using BioRad

Protein assay. It is a dye-binding assay in which Coomassie blue dye binds to

primarily to basic and aromatic amino acid residues that shifts absorbance from

465 nm to 595 nm.

Working solution of the dye was prepared by diluting 1 part dye reagent

(BioRad) Concentrate with 4 parts distilled/ deionized water. Solution was

filtered through Whatman #1 filter to remove particulates. Bovine Serum

Albumin solution was used as standard. The range of concentration was from

0.1 to 1mg/ml. 100 µl of each sample and standard was taken into a clean, dry

test tube. 5.0 ml of diluted dye reagent was added to each tube and mixed

properly by pippeting in and out. Tubes were then left on bench for 5 min at

room temperature. OD was measured at 595 nm. Standard curve was plotted

between concentrations of protein (mg/ml) in standard verses OD.

D SDS-PAGE

1. Polyacrylamide gels were prepared according to Laemmli (276). Briefly,

stacking gel contained 5% and resolving gels contained 10% or 15%

acrylamide. Resolving gels were also contained 1% SDS.

2. Properly polymerized gels were immersed in reservoir buffer.

3. Samples were prepared in 1:1 ratio of sample diluting buffer by heating

them at 100oC for 5 min.

4. 20µl of each sample or standard was loaded into wells.

5. Electrophoresis was carried out in vertical gels (BioRad Minigel

Apparatus) at 100 V for 1 hr or until bromophenol dye (Sigma) front

reached the bottom of resolving gel.

6. Discontinuous buffer system was used in which reservoir buffer has

different pH and ions concentration than buffer used in resolving gel

which makes the resolution of samples more clear.

7. Standard high and low molecular weight proteins markers (Sigma) were

run with each gel for reference.

8. Gels were immersed in 5 volumes of staining solution containing

Coomassie Brilliant Blue R250 dye (Sigma) and placed on slow rotating

platform for 2 hrs.

9. Alternatively gels immersed in staining solution were kept under heat in

microwave oven for 30s.

10. After the removal of staining solution, gels were soaked in destaining

solution to destain false staining of background and leave stained protein

band visual for 24hrs on slow rocking platform with the cycles of

change of destaining solution every 4 hrs.

11. Alternatively gels immersed in destaining solution were kept on heat in

microwave oven for 1 min first and then again for 1 min after the change

of destaining solution. Gels were stored in saran wrap until they were

photographed.

E Development of Antisera against Bacterial Proteins

i) Vaccine Preparation

• Over night bacterial culture was grown in 100 ml MHB with and

without plant extracts

• Formaline solution was added to broth culture with the final

concentration 3%.

• Flasks were kept at 37oC for 24 hours.

• Broth was centrifuged at 5000 x g and washed twice with 0.3%

formalized saline and once with normal saline.

• Remaining pellet was resuspended in 1 ml normal saline.

• Turbidity was further matched with McFarland Nephlometer

Index # 3.

• Sterility was checked on Blood agar, MHA and thioglycolate

ii) Immunization of Animals Three types of polyclonal antisera were developed against whole cell vaccine of

All immunization experiments were performed healthy adult rabbits. For each

immunization experiment rabbits were immunized subcutaneously with 500µl

of diluted whole cell lysate (total protein concentration = 50µg) suspended in

PBS. Each animal received five immunizations for 20 days at 4-day intervals,

and sera were collected by auricular artery of animal. Blood was collected

before immunization (preimmune) and 4 days after the last dose. Control

animals received an identical course of immunization with PBS with alum. All

experimental and control groups contained 3 animals.

F Immunoblotting Suppression of specific bacterial proteins after the treatment of plant extracts

was further confirmed by western blotting. Two different sets of experiments

were run; in first experiment only vaccine of untreated bacteria was preceded

for western blotting and immunoblots were analyzed with three different types

of antisera. In second set of experiment, treated and untreated vaccine samples

were blotted and analyzed by antisera raised against untreated cells of ETEC.

Experiments were run in separate three sets.

• Briefly, gel subjected to electrophoresis was placed in cold blotting

buffer.

• Six pieces of Whatman’s filter paper # 3 and one piece of nitrocellulose

membrane (Millipore), cut according to the size of gel were immersed in

a tray containing blotting buffer.

• Three pieces of filter paper were placed on one top of other on black

(Anode) side of blotting cassette of minigel apparatus (BioRad).

• Nitrocellulose membrane was placed followed by gel and remaining

three filter paper pieces carefully to avoid any air bubbles.

• Roller was then rolled on that sandwich to remove air bubbles, if any.

• Cathode or red side of cassette was placed and closed properly.

• Cassette was then immersed in tank of BioRad Minigel apparatus.

• Electro transfer of bacterial proteins and standards were done at 150 V

for 2 hrs at 4oC.

• Membrane was stained with temporary stain of Ponceau S,

• Membranes were then subjected to immunodetection.

• An initial saturating step with 5% skimmed milk (Oxoid) solution in

TBS-Tween 20 was carried out overnight at 4 °C.

• Nitrocellulose membranes were then incubated with three different

polyclonal antisera separately (1:100 diluted in TBS-Tween20) for 1 hr

at room temperature.

• After four washings in the same buffer, the detection was then

performed with 1: 25000 diluted alkaline phosphatase-labeled goat anti-

rabbit IgG antibodies (directed against whole IgG molecule, purchased

from Sigma).

• Immunoblots were developed with BCIP/NBT Alkaline Phosphatase

Substrate Tablets (Sigma) after the incubation of 20min at room

temperature in dark. Each tablet contains Nitro Blue tetrazolium (NBT)

75 mg/ml in 70% dimethyl formamide; 5-Bromo-4-chloro-3-indolyl

phosphate (BCIP) 50 mg/ml in formamide (100%).

• The suppression of bacterial protein products after the treatment of plant

extracts was confirmed by the comparison of blots developed by 3

different types of sera.

2.3.10 Antimycobacterial Activity of Plant Extracts

Aqueous and methanolic extracts of Camellia sinensis, Juglans regia,

Hippophae rhamnoides were also screened for antimycobacterial activity by

agar dilution method as per standard protocols (277). Different concentrations

of extracts were added to Middlebrook 7H11 agar plates containing 10% OADC

supplement (BD). Different concentrations of each extract ranged from 0.75 to

5 mg/ml were tested against reference strain of Mycobacterium tuberculosis

H37Rv (ATCC 27294) and clinical strains of Mycobacterium tuberculosis,

Mycobacterium avium and Mycobacterium bovis. culture were grown to late log

phase in Middlebrook 7H9 broth supplemented with 0.2% v/v glycerol, 0.05%

Tween 80 and 10% v/v OADC. Culture was diluted in PBS and matched to a

McFarland no. 1 standard. The resulting suspension was carefully vortexed for

optimal homogenization. Standard dilutions were prepared in PBS. Plates were

inoculated with 10µl bacterial culture. Activity was checked with two different

inoculum concentrations. Size of inoculum was 104 and 106 CFU/spot. Agar

without supplementation of extracts was served as growth control. Middlebrook

7H11 plates were also prepared with 0.1µg/ml of isoniazid (INH) to confirm the

susceptibility of standard strains against first line drugs and also used as

positive control. After inoculation, plates were left on the bench for 30min for

proper absorption and then incubated at 37oC in 5% CO2 for two or three weeks.

The MIC was defined as the lowest drug concentration which inhibited the

visible growth of bacteria whereas growth was observed on the extract-free

plate. Experiments were performed in duplicate at three different occasions.

2.3.11 Anti-Trichomonas Activity of Plant Extracts Assays were performed to see anti-trichomonas activity of aqueous and

methanolic plant extracts as per protocol described previously (278).

• Seven different isolates of Trichomnas vaginalis isolated in Italy,

Angola and Mozambique from the cases of vaginal trichomoniasis.

• ome isolates were associated with Mycoplasma homonis and some were

Mycoplasma-free.

• Isolates were retrieved from stock culture vials frozen in liquid nitrogen.

They were grown in Diamond’s medium supplemented with 10% heat

inactivated fetal calf serum (FCS) at 37oC in humid atmosphere

containing 5% CO2.

• For antitrichomonas assay, 100µl of Diamond’s medium containing 10%

FCS was dispensed to each well of 96-well microtitre plate.

• Serial 2 fold dilutions of extracts were made.

• Active log phase protozoa were washed twice with PBS and

• suspended in the same medium.

• For the determination of Protozoal count and viability, Thoma

BLAUBRAND counting chamber was charged. Cells were counted in

WBCs squares.

• Appropriate dilutions of protozoal cultures were made in Diamond’s

medium to achieve density of 2 x 105 cells/ml.

• 100 µl of the calibrate suspension was added to each well.

• Final concentrations of crude extracts tested were ranged from 12.5-

0.312mg/ml. Wells containing only Diamond’s medium served as

growth control.

• To rule out the possibility of interference of water in activity (in case of

aqueous extracts) 100µl of water was added to each well instead of plant

extract. Some wells had only medium or plant extracts and were not

inoculated with protozoa, to check sterility of material used.

• Plates were incubated at 37oC in humid atmosphere containing 5% CO2

for 24hrs.

• The susceptibility of T. vaginalis was determined by the assessment of

growth and motility of flagellates exposed to different concentrations of

drugs under inverted microscope (OLIMPUS CK).

• The lowest concentration of extract in which no motile trophozoites

were seen was considered endpoint and defined as MIC100, according to

Meingassner and Thurner (279).

• The inhibitory effects on the morphology and motility of the

trophozoites were also recorded by Trypan Blue assay at different time

intervals (280).

• Well contents were properly mixed by pippetting in and out with the

help of micropipette.

Table # 9

List of Isolates of Mycobacterium species

S. # Strain R-type

1 Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis RIF

2 Mycobacterium tuberculosis RIF

3 Mycobacterium tuberculosis RIF, SM

4 Mycobacterium tuberculosis RIF, INH 5 Mycobacterium tuberculosis SM, INH 6 Mycobacterium tuberculosis SM, INH, ETH

7 Mycobacterium tuberculosis SM, INH, ETH + CIP (XDR)

8 Mycobacterium bovis - 9 Mycobacterium avium -

10 Mycobacterium smegmatis -

RIF Rifampicin SM Streptomycin INH Isoniazid ETH Ethambutol CIP Ciprofloxacin XDR Extremely Drug Resistant

• 20µl of well content were removed from each well after 30min, 1hr, 2hr,

3hr, 4hr, 6hr, 8hr and 24hr.

• 1:2 dilution was made with 4% Trypan Blue in microtubes.

• Living and dead cells, as revealed by Trypan Blue staining, were

counted in a Thoma BLAUBRAND counting chamber.

2.4 In-Vitro Toxicity Studies of Plants Toxicity studies play a pivotal role in herbal medicine. Toxic effects of any drug

on host cells and in animal model may result the study abandoned. The

objective of toxicological studies is to establish a dose-reaction relationship.

Instead of searching the mechanism of toxicity, it is important to know the

amount of drug which is effective for target organism as well as save for

mammalian cells or body. Toxicity can be studied by different physiological,

morphological and biochemical examination.

Although the plants, we worked on, are already very famous food or cosmetic

items in Pakistan, we decided to evaluate their effects on some mammalian cells

and laboratory animals. During this study, three different type of experiments

were performed to study toxic effects (if any) of plant extracts and isolated

compound.

2.4.1 Hemolytic Activity of Plants and Plant derived Substances To determine the potential of plant extracts cause injury on mammalian cell

membrane, we examined their ability to lyse human erythrocytes as described

previously with some modification (281).

The hemolytic activities of the aqueous and methanolic crude extracts of

Camellia sinensis, Juglans regia, Hippophae rhamnoides and FACS-II (newly

isolated compound from Camellia sinensis) were assayed with heparinized

human RBCs. Blood was collected from a normal volunteer in heparinized

tubes and washed three times in PBS. 1 ml of 10% RBCs suspension was

dispensed in dried, clean glass tubes. Equal volume of crude plant extracts and

FACS-II were added to each tube. Five different concentrations ranged from

1000-1mg/ml for crude extracts and 1000-1µg/ml for FACS-II was tested. 5%

SDS and PBS were served as positive and negative control respectively. After 1

hr incubation, cell suspensions were centrifuged for 10 min at 1500 x g and

supernatants were transferred to a flat bottomed 96-well plate. The absorbance

(A) was read at 492 nm by ELISA reader (Statfax).Since the plant extracts were

colored, so every concentration of extracts has separate blank. For preparation

of blanks, plant extracts were taken in separate tubes without addition of blood,

treated in same manner like tests. The percent hemolysis was calculated using

the formula,

Hemolysis (% of control) = (A of sample – A of blank) x 100

(A of positive control –A of blank)

2.4.2 Cytotoxicity Plant Extracts against Human Vascular

Endothelial cells a) Principle

This is very popular assay to evaluate the effects of plant extracts on various

mammalian cells (282, 283) MTS solution contains a novel, tetrazolium

compound [3-(4, 5-dimethylythiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

Sulfophenyl)-2H-tetrazolium] and an electron coupling reagent (phenazine

ethosulfate or PES). PES is a stable chemical and its combination with MTS

enhances the chemical stability of solution. Succinate dehydrogenase is the

enzyme produced by the mitochondria of metabolically active cells. MTS got

reduced by accepting electrons from oxidized substances like NADPH or

NADH, produced during succinate dehydrogenase activity. After the

bioreduction of MTS, a colored product- formazon is produced and released

from mitochondria by rendering the membrane permeable. Color intensity can

be measured by spectrophotometer.

b) Method

• ECV304 (Human Vascular Endothelial cells lines Modified) were

maintained in 75mm tissue culture flask containing Medium 199

supplemented with 10% FBS and antibiotics.

• Cells were washed with PBS once and then were preceded for

tyrpsinization. 3ml of 1 X trypsin-EDTA for endothelial cells (sigma)

was added to flask for 5 min.

• 1:10 dilution of cells with Nigrosine 0.25% in PBS was made. Counting

chamber was charged with cell suspension to check cell viability and

count.

No. of cells/ml= average # of cells per large square x 800 x dilution factor

• 80,000 cells/ml were added to each well of 96-well polystyrene plate in

final volume of 200µl.

• Plate was incubated for 24h at 37oC in humidified environment with

95% O2 and 5% CO2.

• Tissue culture medium was taken out from each well and 200µl of

different concentrations of aqueous and methanolic plant extracts were

added to wells marked respectively.

• Plates were again incubated for 24h under same atmospheric conditions

and temperature.

• Medium and the extracts were gently aspirated off from each well.

• MTS reagent (Promega) was diluted 1:5 (v/v) in tissue culture medium.

200µl of diluted MTS reagent was added to each well and incubated

again for 1h.

• To avoid interference in absorbance due to background color of extracts,

absorbance was taken immediately at 595nm instead of 490 nm.

• Negative control wells were treated same like tests except the addition

of any plant extract.

2.4.3 Free Radical Scavenging Activity of Plant

Extracts Reactive oxygen species (ROS), which include oxygen ions, free radicals and

peroxides, produced inside the cell under unfavorable conditions, play main role

in cell demage during inflammatory process. Plants and plant derived

susbtances can help the cells in preventing ROS demage by scavenging free

radicals.

Aqueous and methanolic extracts of Camellia sinensis, Juglans regia,

Hippophae rhamnoides were screened for effect on ROS production in Human

Endothelial cell line by using a fluorescent probe, DCFH-DA (2', 7'-

dichlorofluorescin diacetate). This is well-known method for assessment of free

radical scavenging potential in plant extracts (284, 285) and based on the

incubation of cells with DCFH-DA which enters into the cell membrane by

passive diffusion. Upon entry, acetate group of DCFH-DA is cleaved by

intracellular esterases and converted into 2', 7'-dichlorofluorescin (DCFH)

which ultimately oxidized by ROS to a fluorescent dichlorofluorescein (DCF).

Therefore, the fluorescent produced in this assay is directly proportional to the

H2O2 and/or hydroxyl radical concentration.

• ECV304 (Human Vascular Endothelial Modified) cell lines were

maintained in 25mm tissue culture flask containing Medium 199 with L-

glutamine (Gibco) supplemented with 10% FBS, 50 mg/L streptomycin

and 1000 U/L penicillin.

• Cells were grown as monolayers in a humidified atmosphere at 37°C in

5% CO2 enriched environment. Experiments were performed with cells

in a log phase of growth.

• Cell counting and viability was checked as described above.

• 110m M stock solution of probe, DCFH-DA (2', 7'-dichlorofluorescin

diacetate), purchased from Molecular Probes was prepared in DMSO.

• 1mM working solution was prepared in PBS Plus immediately.

• Cells, suspended in pre-warmed Medium 199 with L-glutamine (Gibco)

and 10% FBS, were added to black colored 96-well plate with a density

of 20,000 cells/ well with a final volume of 100µl.

• After overnight incubation in humidified environment with 95% O2 and

5% CO2, cells were washed with PBS and incubated with 200µl of

DCFH-DA for 30min.

• Wells designated to serve as blank were added with PBS Plus instead of

probe.

• Probe was taken out from each well and cells were washed with PBS

Plus once.

• 200µl of different concentrations (ranged from 0.625-2.5mg/ml) of

aqueous and methanolic plant extracts were added in triplicate to their

respective wells. Control wells were added with PBS Plus instead of

plant extracts.

• DCF fluorescence was measured at 37o C for 80 min by TECAN

GENios plus Spectrofluorimeter at excitation 485nm and emission

535nm. Each extract was tested in triplicate and in two different

experiments.

2.4.4 Effect of Plant Extracts on Cell Proliferation

by 3H Thymidine Incorporation Production of ROS inside the cell causes DNA damage which ultimately leads

to detrimental effects on cell proliferation. Therefore, results of free radical

scavenging activity were further confirmed by cell proliferation assay (286).

The following procedure was performed,

• 80,000 cells in Medium 199 with L-glutamine (Gibco) and 10% FBS

were seeded to each well of 24-well plates and incubated overnight for

attachment.

• Medium was aspirated and cells were treated with different

concentrations of plant extracts ranged from 0.625-2.5 mg/ml for 3hrs

and 24 hrs.

• After completion of treatment, cells were washed with PBS to remove

plant extract in medium and pulsed with 1µCi/ ml of (3H) Thymidine.

• After 24hrs, cells were washed with PBS and treated with 500µl of 5%

trichloroacetic acid (TCA) for 10min.

• Washing was again done with 500µl Methanol once.

• 500µl of 25M formic acid was added for 5min.

• Lysate was then transferred to scintillation vials having 450µl

scintillation fluid and processed for liquid scintillation counting.

• Cells processed without extract treatment were served as control.

• The results were expressed as the average cpm value for test vials

compared with control.

2.5 Immunopharmacological Studies of Plants

2.5.1 Animal Toxicity Studies of Plant Extracts

Determination of toxic effects of plant products in laboratory animals is

considered very significant in herbal medicine. In a number of studies,

experiments were done in BALB/C mice (287-289).

Experiments were carried out with 8 weeks old, healthy BALB/C mice of either

sex, weighing 20-25g, to determine the acute and subacute toxic effects of plant

extracts. Animals were divided into different groups. Each group had 6 mice.

They were housed in standard environmental conditions of temperature,

humidity and under clear and dark cycles of 12 h. The mice were fed on diet

and water ad libitum. For acute toxicity studies, plant extracts were

administered by intraperitoneal (i.p) route in doses of 100, 300, 500 and 1000

mg/kg of the body weight. Same volume of normal saline was injected to

control group. The general behavior of mice was observed continuously for 1 h

after the treatment and then intermittently for 4 hrs. Animals were further

monitored for any physical, behavioral change, mortality and morbidity over 1

week of follow up period. To find out acute toxic effects of plant extracts, blood

samples were collected on 8th day and examined for serum chemistry profile

and blood count. Mice were then sacrificed, organs including spleen, kidneys,

liver and lungs were removed and sent to the laboratory in 10% formalin for

histopathological examination. The results obtained were compared with those

for the control. To investigate the subacute toxicity, mice were administered the

same concentrations of extract on every alternate day over the period of 2 weeks

and then sacrificed at day 28. Biochemical, hematological and histopathological

parameters were evaluated.

2.5.2 In-Vivo Antimicrobial Activity

On the basis of observations of in-vitro antimicrobial activity experiments and

non-toxic nature of plant extracts, antimicrobial activity was further tested in

animals. Different experiments were carried out separately to find out in-vivo

efficacy of each plant.

A) In Vivo Antimicrobial Activity of Camellia sinensis against

MRSA in Experimental Murine Septicemia Murine MRSA septicemia model has been used to study the efficacy and

pharmokinectics of antimicrobial compounds in a number of studies (290, 291).

10-12 week old healthy, female BALB/C mice were used in this experiment.

Animals were divided into 3 groups; test group, control group, infected group.

Each group has 16 animals and housed in separate cages. Neutropenia was

developed by giving cyclophosphamide i.p with the dose of 150mg/kg of the

body weight intraperitoneally a day before infection (292). Infection was

established into 2 groups (test group and infected group) with 107 CFU of

methicillin resistant Staphylococus aureus by using tail vein.

Aqueous crude extract of Camellia sinensis was prepared fresh. After 2 hrs of

infection single i.p injection (156mg/kg of body weight) of aqueous crude

extract of camellia sinensis was given to test group and control group. Infected

group of animals left untreated. At 0, 2, 4 and 6 hrs after the administration of

extract, blood samples were collected aseptically by tail bleeding method.

Animals were then sacrificed by cervical dislocation and different organs i.e.

lungs, liver, spleen and heart were aseptically removed. Organ tissue

homogenates were prepared in 3 ml of chilled sterile saline. Homogenates and

blood were suitably diluted in sterile saline and 100µl was plated on MHA

plates to enumerate the bacterial load of each organ. Plating was done in

duplicate in each case. Bacterial eradication was evaluated by comparing the

reduction of bacterial counts in each organ and blood in the test group with the

bacterial loads of the infected animals at different time intervals.

B) In Vivo Antimicrobial Activity of Hippophae rhamnoides

against Pasteurella multocida Pasteurella multocida is the causative agents of hemorrhagic septicemia in

cattles and water buffalos. Hemorrhagic septicemia (HS) is the main cause of

mortality among large and small ruminants in Pakistan. Use of antibiotics in

animal feed and prophylactic antibiotics shots to animals are very common to

prevent this disease that results in emergence of antibiotic resistance in

Pasteurella (293) and their subsequent spread to environment n human beings.

Interestingly, Hippophae rhamnoides exhibited very promising in-vitro

antimicrobial activity. Its frequent use in the form of jam and pulp by common

people of Pakistan suggest its non-toxic nature which was further proved during

toxicity studies. Keeping this in mind, we tested the effect of aqueous extract of

Hippophae rhamnoides against Pasteurella multocida in septicemia mice model.

Susceptibility of mice by Pateurella multocida is already known. Experimental

septicemia by this organism manifests the disease similar to HS in large and

small ruminants (294) and has been extensively used previously (295). Two

sets of experiments were carried out.

i) Determination of LD50

5-6 weeks old healthy, female BALB/c mice, weighing 20-22g, were used for

the first set of experiments. Animals were divided into eight different groups.

Each group had 6 mice. They were housed in standard environmental conditions

of temperature, humidity and under clear and dark cycles of 12 h. The mice

were fed on diet and water ad libitum. A virulent strain of Pateurella multocida

serotype B2, isolated from blood sample of buffalo, suffering from hemorrhagic

septicemia, was used. 24 hrs old culture was inoculated into BHI broth and

incubated at 37oC for 2 hrs in shaking water bath. Culture was adjusted with

0.5Mcfarland’s standard. Appropriate 10 fold serial dilution of exponential

phase culture was made in phosphate-buffered saline (PBS). Seven groups were

injected with 100µl of respective dilution of exponential phase culture by

intraperitoneal route, whereas sixth group was left uninfected which served as

Control. The dilution and their corresponding number of organisms are given in

Table # new

Animals were kept under observation for 48hrs post-challenge. Numbers of

survivors were recorded to determine LD50 of this clinical strain of Pasteurella

multocida serotype B2.

ii) Effect of Different Concentrations of Hippophae

rhamnoides on LD50 5-6 weeks old healthy, female BALB/c mice, weighing 20-22g, were divided

into seven different groups. Each group had 6 mice. The same strain of

Pateurella multocida serotype B2 was used. 24 hrs old culture was inoculated

into BHI broth and incubated at 37oC for 2 hrs in shaking water bath.

Appropriate dilution of exponential phase culture was made in phosphate-

buffered saline (PBS). Six groups were injected with 100µl of exponential

phase culture containing 2 x 104 CFU (100 x LD50) by intraperitoneal route,

whereas last group was left uninfected which served as Healthy Control. After

4hrs of infection, five infected groups were administered i.p. different

concentrations of freshly prepared aqueous extract of Hippophae rhamnoides

(Sea Buckthorn). Concentrations of extract given were 100mg/kg, 80mg/kg

50mg/kg, 10mg/kg and 5mg/kg of the body weight. Mice were observed for

any sign of symptoms and mortality up to 7 days.

Table # 10: Preparation of Challege Dose of

Pasteurella multocida for LD50 Determination

Group # dilution no. of CFU/ml 1 0.5 McFarland’s original 2 x 108

2 10-1 2 x 107

3 10-2 2 x 106

4 10-3 2 x 105

5 10-4 2 x 104

6 10-5 2 x 103

7 10-6 2 x 102

iii) Effect of Different Concentrations of Hippophae

rhamnoides on Organ Dislocation To determine of effect of extract on organ dislocation of organisms, within 1 hrs

of the onset of symptoms, animal was sacrificed and different organs like lungs,

liver, heart, kidneys, and spleen were removed aseptically. Peritoneal fluid was

also aspirated immediately after death. Organs were homogenized in 10ml

portions of PBS, and 10fold serial dilutions (1:10 and 1:100) were plated on

MHA plates and incubated aerobically overnight at 37°C to determine number

of viable organisms.

2.5.3 Intracellular Killing in Phagocytic Cells in

the Presence of Plants Phagocytic cells include Polymorphonuclear leukocytes (PMNLs) and

Monocytes serve as the primary host defense against bacterial infections.

Intracellular killing of the infecting organism with Reactive oxygen species and

microbicidal proteins followed by their ingestion is a vital phenomenon of

phagocytosis. In case of impaired host defense, it is difficult for phagocytic

cells to effectively kill pathogens. Plants and plant derived substances can help

immune system to combat infectious diseases by modulating phagocytic process.

Aqueous extracts of Camellia sinensis, Juglans regia and FACS II (new

purified compound from Camellia sinensis) were tested for their effect on

intracellular killing of MRSA by human PMNLs by following method.

A Isolation of PMNLs 5ml blood collected from healthy donors in EDTA was added to the tube

containing 5ml of Hank’s balance Salt Solution (HBSS) and mixed gently.

• 5ml of diluted blood was then transferred carefully to the tube

containing 7ml of Ficol-hypaque (Pharmacia) without disturbing ficol

layer.

• Tube was centrifuged at 400 x g for 30 minutes at 18-200C.

• After drawing off first three layers, last layer of the tube containing

RBCs and PMNLs was taken in a fresh tube containing 16ml of ice-

cold PMNLs separating solution (see Appendix-1)and incubated at 4oC

for 15min.

• Tube was centrifuged at 55 x g for 10min at 4oC.

• Sediment was suspended in 5 ml of PMNLs separating solution.

• Procedure was repeated until the redness goes.

• Final sediment containing PMNLs was resuspended in HBSS.

B Adjustment of Cell Density and Viability Cells were diluted in 0.4% Trypan Blue dye with ratio of 1:5 and 1:10. cell

density and viability was checked by counting stained (dead) and unstained

(viable) cells in hemocytometer (WBC squares). >95% cell viability is required

to precede the assay. Cell density was adjusted to 1 x 107 cells/ ml by adding

appropriate volume of HBSS. Following formulae were used,

For Cell Viability:

% viability = Number of viable cells x 100

Total number of cells

For Cell Density:

Viable Cells/ ml = N x B x 104

where, N = Average number of viable cells per square

B = Dilution factor

C Assay

• Overnight growing culture of MRSA (strain # 3443) was inoculated 5ml

MHB and incubated for 2 hrs at 37oC under rotation.

• 0.5ml of 10% of pooled human serum was then added to the tube and

incubated for another 30min.

• Opsonized bacteria were harvested, washed and resuspended in HBSS to

match turbidity with 0.5 McFarland’s index. Cell density was adjusted

to 1 x 107 CFU/ml.

• Assay was initiated by adding pre-warmed opsonized bacteria and

PMNLs to microtubes with the ratio of 10:1 respectively.

• Tubes were shaken vigorously for 20 min to allow rapid engulfment of

bacteria by PMNLs and kept on crushed ice to stop phagocytosis.

• Gentamicin was added to each tube for 5 min at 4oC to kill non-engulfed

bacteria.

• Cells were washed with HBSS to remove gentamicin.

• 1 ml Plant extracts/ compound with concentrations 2 x MIC were added

• Tube, added with 1 ml of HBSS in place of plant extracts was served as

control.

• The incubation tubes were slowly rotated for 180 min at 37°C to

facilitate intracellular killing.

• At the beginning of the incubation and every 30 min thereafter, aliquots

of 100µl were removed and washed with 900µl ice-cold HBSS.

• Cells were lysed by adding 1 ml of sterile distilled water.

• Samples were spread on MHA plates for count of surviving bacteria.

Effect of plant extracts on intracellular killing was analyzed by the

comparison of number of surviving organisms in test as compare to

control tubes.

2.5.4 Effect of Plants on Humoral Immune

Response

Immunomodulating properties of aqueous extracts of Camellia sinensis and

Juglans regia were also studied by evaluating their effect on antibody secreting

plasma cells by Hemolytic plaque assay.

A Immunization

5ml of sheep blood was taken in 20 ml of Alsever’s solution. 10% of washed

SRBCs suspension was made in normal saline.

Experiment was carried out with 8 weeks old, healthy female BALB/C mice,

weighing 20-22g. Animals were divided into four groups (n = 5). On day 1,

group 1 and 2 were given 10 mg/kg of aqueous extracts of Camellia sinensis,

Juglans regia and group 3 was given 5mg/kg of FA-CS II by intraperitoneal (i.p)

route. Group # 4 was given normal saline (served as control). On Day2, 0.5ml

of 10% SRBCs was given to all groups with same treatment of day1. On day3,

treatment of day 1 was repeated.

B Assay Animals were scarified on day 5 by cervical dislocation and spleen was

removed aseptically, dipped in 5ml cold RPMI 1640 cell culture medium

(Sigma) and macerated. Content was filtered by glass wool to get single cell

suspension. Filterate was collected in sterile propylene tube and volume was

made upto 10 ml with cold cell culture medium. Centrifugation was done at

1000 x g for 10min. sediment was resuspended in 1ml of cell culture medium

and cell density was adjusted upto 1 x 105 cells/ ml. 100µl of spleen cells were

mixed with 50µl SRBCs in a tube containing 500µl of 1% agarose solution

which was kept on water bath at 50oC. Suspension was mixed properly and

immediately poured on glass slides (pre-coated with 0.1% agarose). At least 5

slides were prepared for each animal. Slides were incubated at 37oC for 1hr.

slides were dipped (upside down) in 1:10 diluted guinea pig serum in veronal

buffer and incubated for another an hr at 37oC. Number of plaques per 105 cells

were counted by inverted microscope.

RESULTS

3.1 Collection, Isolation and Characterization of

Bacterial Pathogens 3.1.1 Characterization of Bacterial strains by Conventional

Methods A total of 377 different gram positive and gram negative clinical bacterial

strains, isolated from various public and private sector laboratories of Karachi-

Pakistan were used in this study (shown in Table # 2a). Bacterial strains were

characterized phenotypically on the basis of standard morphological, cultural,

biochemical characteristics and serotyping (schemes shown in Tables # 3a, b

and c). Out of 377, 289 (76%) were extracellular bacterial pathogens including

Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebseilla

pneumoniae, Vibrio cholera, Pasteuralle multocida serotype B-2, Micrococcus

species and Bacillus subtilis. Rests of 88 (24%) strains were intracellular

bacterial pathogens that include Mycobacterium tuberculosis, Mycobacterium

bovis, Mycobacterium avium, Mycobacterium smegmatus, Neisseria

gonorrhoae, Salmonella enterica serovar Typhi, Salmonella enterica serovar

Paratyphi A and Shigella species. Nine ATCC reference strains were also used

(shown in Table # 2b).

Antibiotics Susceptibility pattern of Staphylococcus aureus isolates against

commonly prescribed antibiotics was determined. Out of 158, 99 isolates

(62.65%) were found to be Methicillin resistant (MRSA) and 59 (37.34%)

methicillin sensitive (MSSA). Isolates with MICs >6µg/ml of oxacillin were

confirmed as MRSA. As shown in Fig # 8, majority of MRSA strains

exhibited high level of resistance to structurally unrelated groups of antibiotics

while MSSA were found to be susceptible to most of the antibiotics tested. No

vancomycin resistant isolates was included in the study. Table # 11

CLSI criteria for reference interpretive standards and MIC breakpoints of

Staph. aureus against antibiotics. Fig # 9 shows the susceptibility pattern

several genotypes of Diarrheal Escherichia coli isolates including

Enteroaggregative Escherichia coli (EAggEC), Enterotoxigenic Escherichia

coli (ETEC) and Enteropathogenic Escherichia coli (EPEC). Generally,

Antimicrobial Susceptibility Pattern of

Staphylococcus aureus

020406080

E CN AK MET AMP OFX TE VA SXT

ANTIBIOTICS

RESISTANT SUSCEPTIBLE

0102030405060708090

E CN AK MET AMP OFX TE VA SXT

ANTIBIOTICS

RESISTANT SUSCEPTIBLE

Fig # 8: Antibiotics Susceptibility Pattern of (a) clinical isolates of Methicillin Resistant Staphylococcus aureus (n = 99) (b) Methicillin Sensitive Staphylococcus aureus (n = 59) included in the study. Key: E = Erythromycin, CN = Gentamicin, AK = Amikacin, Met = Methicillin, AMP = Ampicillin, OFX = Ofloxacin, TE = Tetracycline, VA = Vancomycin, SXT = Co-trimoxazole/Trimethoprim.

Table # 11: Reference Interpretive Standards and MIC

Breakpoints of Antibiotics against Staphylococcus aureus

S. # Antibiotics Disc

Zone Diameter (mm) MIC Breakpoints

(µg/ml)

R I S R S

1 Amikacin 30 <14 15-16 >17 >32 <16

2 Gentamicin 10 <12 13-14 >15 >8 <4

3 Ampicillin 10 <28 - >29 β-

lactamas

4 Methicillin

(oxacillin)

1 <10 11-12 >13 >4 <2

5 Erythromycin 15 <13 14-22 >23 >8 <0.5

6 Vancomycin 30 - - >15 - <4

7 Tetracycline 30 <14 15-18 >19 >16 <4

8 Ofloxacin 5 <14 15-17 >18 >4 <1

9 Co-trimoxazole/

Trimethoprim

<10 11-15 >16 >8/152 <2/38

Standard values are given as per CLSI criteria.

amikacin, gentamicin, fosfomycin, ciprofloxacin and chloramphenicol found to

be useful whereas most of the isolates were resistant to amoxicillin and co-

trimoxazole/trimethoprim. Fig # 10 illustrates the pattern of antibiotics

resistance among uropathogenic Escherichia coli. Trend of higher resistance

ratio among all antibiotics are very evident. .

As shown in Fig # 11, antibiotics resistance among Salmonella enterica serovar

Paratyphi A is quite high. Out of 8, 3 (37%) isolates were found to be MDR (R

type-AmpCSxtT). Resistance against first line therapy was also observed in

Salmonella enterica serovar Typhi where out of 38, 16 (42%) were found to be

multi drug resistant (MDR) with R type-AmpCSxtStrT) i.e. resistant against

ampicillin, chloramphenicol, co-trimoxazole/ trimethoprim, Streptomycin and

tetracycline. Rest of 22 (57%) were either sensitive to all antibiotics or resistant

to single or a couple of first line drugs. Among MDR isolates 2 were found to

be resistant against Nalidixic acid (Fig # 12). Table# 12 shows CLSI criteria for

reference interpretive standards and MIC breakpoints of Enterobacteriacae

against antibiotics.

3.1.2 Characterization of Salmonella enterica serovar Typhi and

Salmonella enterica serovar Paratyphi A by Molecular

Methods A total of 36 clinical blood isolates of Salmonella enterica serovar Typhi (S.

Typhi) and 8 of Salmonella enterica serovar Paratyphi A (S. Paratyphi A) were

processed for determination of plasmid incompatibility grouping, integron

analysis, presence of resistant cassettes and DNA fingerprinting (Table # 13a

and b shows complete characterization of Salmonella isolates included in this

study).

A Plasmid Analysis and incompatibility grouping Out of 38, a total of 16 (42%) S. Typhi and out of 8, 3 (37%) S. Paratyphi A

isolates with full resistance against Ampicillin, Cholramphenicol, Co-

trimoxazole/Trimethoprim and Tetracycline phenotypically were found to have

a single 150 kb (98 Mda) plasmid. However, strains with resistance against

either one or two first line drugs did not have any plasmid. No plasmids were

Fig # 9: Antimicrobial Susceptibility Pattern of

Diarrheal Isolates of Escherichia coli

AK CN AML AMC C TOB CEP CXM CRO ATM FOS NA SXT CIP ER

Antibiotics

susceptible intermediate resistant

AK CN AML AMC C TOB CEP CXM CRO ATM FOS NA SXT CIP ERAntibiotics

Fig # 9: Antibiotics Susceptibility Pattern of (a) diarrheal isolates of

Enteroaggregative Escherichia coli (n = 63) (b) Enterotoxigenic Escherichia

coli (n = 16) (c) Enteropathogenic Escherichia coli (n = 7) included in the

study. Key: AK = Amikacin, CN = Gentamicin, AML = Amoxicllin, AMC =

Amoxicillin/ Clavulanic acid, C = Chloramphenicol, TOB = Tobramycin, CEP

= Cefipime, CXM = Cefuroime, ATM = Aztroenem, FOS = Fosfomycin, NA =

Nalidixic acid, SXT = Co-trimoxazole/Trimethoprim, CIP = Ciprofloxacin and

E = Erythromycin.

AK CN AML C TOB CEP CXM CRO ATM NA SXT CIP ER

Antibioticssusceptible intermediate resistant

Antibiotic Susceptibility Pattern of Uropathogenic Escherchia coli

AMP AMC ATM CN CRO CXM F NA OFX PIP SXT CPM

Antibiotics

Fig # 10: Antibiotics Susceptibility Pattern of urinary isolates of Escherichia

coli (n = 30). Key: AMP = Ampicillin, AMC = Amoxicillin/ Clavulanic acid,

ATM = Aztroenem, CN = Gentamicin, CRO = Ceftriaxone, CXM = Cefuroime,

F = Nitrofurantoin, NA = Nalidixic acid, OFX = Ofloxacin, PIP = Piperacillin,

SXT = Co-trimoxazole/Trimethoprim and CPM = Cefipime.

Fig # 11: Antibiotics Susceptibility Pattern of Salmonella enterica serovar Paratyphi A

AMP AMC C CIP CRO NA SXT T

AntibioticsSensitive Intermediate Resistant

Antibiotics Susceptibility Pattern of clinical isolates of Salmonella enterica

serovar Paratyphi A. Key: AMP = Ampicillin, AMC = Amoxicillin/ Clavulanic

acid, C = Chloramphenicol, CIP = Ciprofloxacin, CRO = Ceftriaxone, NA =

Nalidixic acid, SXT = Co-trimoxazole/Trimethoprim and T = Tetracycline.

Fig # 12: Antibiotics Susceptibility Pattern of Salmonella enterica serovar Typhi

AMP AMC C CIP CRO NA SXT T STR

Antibiotics

Sensitive Intermediate Resistant

Antibiotics Susceptibility Pattern of cilincal isolates of Salmonella enterica

serovar Typhi. Key: AMP = Ampicillin, AMC = Amoxicillin/ Clavulanic acid,

C = Chloramphenicol, CIP = Ciprofloxacin, CRO = Ceftriaxone, NA =

Nalidixic acid, SXT = Co-trimoxazole/Trimethoprim and T = Tetracycline

Table # 12: Reference Interpretive Standards and MIC

Breakpoints of Antibiotics against Enterobacteriacae

S. # Antibiotics Disc

Zone Diameter (mm) MIC

Breakpoints

(µg/ml)

R I S R S

1 Amikacin 30 <14 15-16 >17 >32 <16

2 Gentamicin 10 <12 13-14 >15 >8 <4

3 Amoxicllin 10 <13 14-16 >17 >32 <8

4 Amoxicillin/

Clavulanic acid

20/10 <13 14-17 >18 >32/16 <8/4

5 Chloramphenicol 30 <12 13-17 >18 >32 <8

6 Tobramycin 10 <12 13-14 >15 >8 <4

7 Cefipime 30 <14 15-17 >18 >32 <8

8 Cefuroxime 30 <14 15-17 >18 >32 <8

9 Fosfomycin 200 <12 13-15 >16 >256 <64

10 Nalidixic acid 30 <13 14-18 >19 >32 <8

11 Co-trimoxazole/

Trimethoprim

<10 11-15 >16 >8/152 <2/38

12 Aztroenem 30 <15 16-21 >22 >32 <8

13 Ciprofloxacin 5 <15 16-20 >21 >4 <1

14 Ceftriaxone 30 <13 14-20 >21 >64 <8

15 Nitrofurantoin 300 <14 15-16 >17 >128 <32

16 Piperacillin 75 <17 18-20 >21 >128 <16

17 Cefpiridine. 30 <14 15-17 >18 >32 <8

18 Tetracycline 30 <14 15-18 >19 >16 <4

19 Ofloxacin 5 <12 13-15 >16 >8 <2

isolated from fully sensitive isolates except a single strain of S.Typhi that had a

121 kb plasmid (Fig # 13). To determine the association of plasmids with

antibiotic resistance, transmissibility was tested in some separate conjugation

experiments where recipient strain was Escherichia coli (AmpsTsKr).

Transconjugants (AmprTrKr) were isolated on LB agar supplemented with

ampicillin, Tetracycline and Kanamycin. Conjugation experiments confirmed

the transmissible nature of 150 kb plasmid and its association with antibiotics

resistance. Tranconjugates were also showed same resistant pattern when tested

by Kirby-Bauer disc diffusion method.

Plasmid incompatibility grouping was done by using bacterial DNA as

template, along with rep HI1A-specific primers, in order to amplify a 110-bp

region specific for RepHI1A, a region present in IncHI1 incompatibility group

plasmids. All isolates with 150kb plasmids and with MDR phenotype were

found to be positive for RepHI1A (Fig # 14). Fig # 15 shows 100bp DNA

ladder.

B Analysis of Integron and Antibiotic Resistance Cassettes All Strains of S.Typhi (n = 38) and S. Paratyphi A (n = 8) were screened for the

presence of class I integron by using specific primers for the integrase genes

intI1. Amplicons of 569 bp were considered as positive. The intI1 gene was

detected in

all MDR strains of S. Typhi (n = 16) and S. Paratyphi A (n = 3) (as shown in Fig

# 16). Further characterization of 3´ conserved variable segment (CS 5´3´) was

performed in isolates positive for intI1 gene. All isolates containing class 1

integron possessed a variable region of 750 bp (Fig # 17). All CS5´3´ positive

strains also carried dfrA7 gene, conferring resistance to trimethoprim, when

subjected to PCR for cassette assortment. Amplified product of 191 bp was

considered positive (Fig # 18). Positive control was positive and negative

control appeared negative in all assays.

Table #13a: Molecular Charaterization of Salmonella enterica serovar Typhi

SSM Serotype R-type (SS lab) Plasmid incH1 intI1 CS5'3' dfrA7 Pulso-

type 2264 Typhi - - - - - - STX6 2266 Typhi - - - - - - STX7 2267 Typhi - 121 kb - - - - STX1 2271 Typhi - - - - - - STX9 2272 Typhi - - - - - - STX1 2273 Typhi - - - - - - STX10 2275 Typhi - - - - - - STX1 2276 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2277 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2278 Typhi - - - - - - STX10 2279 Typhi - - - - - - STX1 2280 Typhi - - - - - - STX3 2282 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX4 2283 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX4 2284 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX5 2285 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX5 2286 Typhi Sxt - - - - - STX3 2287 Typhi - - - - - - STX3 2288 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2289 Typhi - - - - - - STX8 2290 Typhi - - - - - - STX3 2291 Typhi - - - - - - STX3 2292 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2864 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2865 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2866 Typhi - 50 kb - - - - ? 2867 Typhi - 50 kb - - - - ? 2868 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2869 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2870 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2 2871 Typhi - - - - - - STX11 2872 Typhi Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2

2873 Typhi NA Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2

2874 Typhi - - - - - - STX9 2875 Typhi - 121 kb - - - - STX11 2876 Typhi - 121 kb - - - - STX11

2877 Typhi NA Amp C Sxt T 150 kb pos pos 750 bp dfrA7 STX2

2878 Typhi - - - - - - STX9

Table # 13b: Molecular Characterization of Salmonella enterica serovar Paratyphi A

SSM Serotype R-type (SS

lab) Plasmid incH1 intI1 CS5'3' dfrA7

Pulso-

2262 Paratyphi A Amp T i - - SPAX1

2263 Paratyphi A Amp Sxt i - - SPAX3

2268 Paratyphi A

Amp C Sxt

T 150 kb pos pos 750 bp dfrA7 SPAX2

2269 Paratyphi A

Amp C Sxt

2270 Paratyphi A

Amp C Sxt

2293 Paratyphi A

Amp i Sxt i

T i - - - SPAX3

2879 Paratyphi A - - - SPAX1

2880 Paratyphi A - - - SPAX4

Fig # 14: Plasmid Incompatibility Grouping of Salmonella enterica

Plasmid incompatibility grouping of clinical isolates of Salmonella

enterica serovar Typhi and Paratyphi A was done by amplifying

RepHI1A region. A 110 bp amplified product was considered as positive

3.1.2 DNA fingerprinting by Pulse Field Gel

Electrophoresis Genetic diversity of all isolates of Salmonella species (S. Typhi {n = 38} and S.

Paratyphi A {n = 8}) was analyzed by Pulse Field Gel Electrophoresis as shown

in Fig # 19. Salmonella braenderup H9812 was run as reference standard strain.

PFGE patterns were assessed visually. Total concordance in the DNA fragment

profiles (after XbaI restriction endonuclease digestion) was considered as single

pattern or clonal strain. Difference in one or more bands represents genetic

diversity, however, we can say that single band difference also represent closely

related strains.

Using digestion with XbaI, a total of 11 PFGE patterns were identified in S.

enterica serovar Typhi isolates (n = 38) i.e. STX1, STX2, STX3, STX4, STX5,

STX6, STX7, STX8, STX9, STX10 and STX11 suggesting that multiple clones

are circulating in Pakistan (see Fig. #20). In particular there was great

variability among the PFGE patterns of strains of S.Typhi susceptible to

antibiotics where eight different patterns were observed. The largest PFGE

pattern was STX2 that was shown by 12 isolates. Exluding two strains which

were additionally resistant to

nalidixic acid, all of the strains in this pattern were MDR exhibiting resistance

to ampicillin, chloramphenicol, cotrimoxazole, streptomycin and tetracycline

carried by an IncHI plasmid.PFGE patterns STX4 and STX5 were also

identified in plasmid mediated MDR strains, suggesting the diffusion of three

different MDR strains. However, STX2 differed by STX1 only for the presence

of plasmid. Table # 13a showed the detailed characteristics of each S. Typhi

strain, included in our study. Among eight strains of S.Paratyphi MDR were

predominant and showed a unique PFGE pattern i.e. SPAX2. However, despite

the small number of samples other 3 PFGE patterns were identified among

sensitive strains. Fig # 21 shows representative patterns of S. Paratyphi A. Table

# 13b illustrates the picture of complete characterization of S. Paratyphi A

isolates included in our study.

Fig # 16 Analysis of Class I Integron in Salmonella enteric

Clinical strains of Salmonella enterica serovar Typhi and Paratyphi A

were subjected to PCR for intI1 gene to check the presence of class I

integron. A 569 bp amplified product was considered as positive.

Fig # 17 Analysis of 3´ conserved variable segment (CS 5´3´) of

intI1 gene

Characterization of 3´ conserved variable segment (CS 5´3´) was performed in

isolates of Salmonella enterica serovar Typhi and Paratyphi A found to be

positive for intI1 gene. 750 bp variable region was observed.

Fig # 18

Analysis of the Presence of Trimethoprim Resistance Cassette (dfrA7)

Fig # 18: All CS5´3´ positive strains of Salmonella enterica serovar Typhi and

Paratyphi A were subjected to PCR for dfrA7 gene, conferring resistance to

trimethoprim. 191 bp amplified product was considered as positive.

To conclude, we can say significant genetic diversity was observed among

Pakistani strains of S. Typhi and S. Paratyphi A especially those sensitive to

first line drugs. In contrast, MDR S. Typhi strains showed significant

homogeneity. Our data suggests that multiple clones of Salmonellae are

circulating in Pakistan.

3.2 Bioassay-guided Chemical Characterization of

Plants A total 3 different plants, used throughout the study period were subjected to

chemical characterization. Table # 14 shows the list of plants used.

3.2.1 Camellia sinensis (Green Tea) a) Bioassay guided MALDI-TOF-MS Analysis Chemical analysis of Camellia sinensis was carried out to detect bioactive

component(s) present in Pakistani green tea by undertaking bioassay guided

approach. Six different solvent systems were used to run thin layer

chromatography

as listed in Table #6. Solvent mixtures E (CHCl3/MeOH/acetonitrile =

80:10:10) and F (CHCl3: Ethyl acetate/MeOH = 50:40:10) separated

components in a wide range of Rf value according to their polarity, whereas

extract remained partially separated in other four solvent mixtures. Fig # 21

shows separated components of plants extracts on TLC run in solvent mixture

F, observed under UV lamp. Chromatograms from both E and F mixtures were

preceded for bioautography to locate bioactive spot(s). Salmonella Typhi (strain

# 2877, R-type: AmpCSxtSTNA) was used as reference strain. Chromatogram

run with solvent mixture F showed more promising results than mixture E.

Antimicrobial activity was observed in spot # 4 and 5 in case of solvent E and

only spot # 4 in solvent F. Bioactive spots are clearly seen in Fig # 22. TLC spot

# 4 from solvent mixture F was then scrapped off for MALDI-TOF-MS analysis

(Table # 15). Samples were analyzed in positive and negative ionization modes

with a matrix of Alfa-cyano-4-hydroxycinnamic acid (CHCA). Negative

ionization did not display significant signals therefore, spectra observed under

Chemical Characterization of Plant Extracts by

Thin Layer Chromatography

Fig # 21: Plant extracts were characterized by Thin layer chromatography by

using solvent system F. this figure shows separated components of plants

extracts, observed under UV lamp.

Table # 14: List of Plants

S. # Botanical

Local Name Part used Solvent/

Extract

1 Camellia

sinensis

Green Tea Dried

Leaves

Aqueous

Methanol

FA-CS II

2 Juglans regia Dandasa Dried Bark Aqueous

Methanol

n-hexane

chloroform

ethyl acetate

n-butanol

3 Hippophae

rhamnoides

buckthorn

Berries

Aqueous

Methanol

positive ionization were considered as results. A total of 37 individual peaks

with masses range from 476da to 998da, as listed in Table # 15, were detected

in the bioactive spot # 4. The mass spectra are illustrated in Fig # 23.

b) Isolation of Newly Purified compound by IR-spectrum

and 13C-NMR IR spectrum λmax cm-1 (CHCl3) showed a small peak at 3300 cm-1 (CH stretch),

2850(N CH3 group), a strong absorption band at 1702 due to C=O group. Signal

at 1660 confirmed the presence of C=C and 1230, covering a range of 1230-

1020 could be the indicative of C-N stretching (12).

Its mass spectrum showed molecular ion peak M+ at m/z 194 and high

resolution mass appeared at 194.0739 corresponding to molecular formula

C8H10N4O2. Other mass fragments appeared at m/e (% intensity) 165(20),

137(30), 109(60), 82(12), 67(35) and 55(42). 1H-NMR (CDCl3, 400 MHz)

showed four resonating signals at δ 3.35 (s), 3.55 (s), 3.95(s) and 7.60(s) (13). 13C-NMR (C 5 D 5 N, 25 MHz) showed eight signals, signifying three methyl at

δ 33.5, 30.0 and 27.9, one methyne at δ 141.3, two olifinic carbon (δ 155.4) and

195, due to C=O carbons). The spectral data signifies the compound belongs to

the alkaloid (purine class). Structure is shown in Fig #24. The code # FA-CS II

was used for this compound.

(This work was carried out in collaboration with the Department of Chemistry,

University of Karachi).

3.2.2 Juglans regia (Dandasa) a) Bioassay guided MALDI-TOF-MS Analysis Juglans regia was also subjected for the detection of bioactive component(s) by

undertaking MALDI-TOF-MS analysis coupled with bioautography-a bioassay

guided approach. Solvent systems were same as for green tea to run thin layer

chromatography (see Table # 6). Solvent mixtures E (CHCl3/MeOH/acetonitrile

= 80:10:10) and F (CHCl3: Ethyl acetate/MeOH = 50:40:10) separated

components in a wide range of Rf value. Fig # 21 shows component separation

achieved by TLC by using Solvent F system. Spot # 4 and 5 in either of solvent

system, were found to have antimicrobial activity, when observed by

bioautography (See Fig # 22, 25). Bioactive spots from each plate were

analyzed by MALDI-TOF-MS (Table # 16). Samples were analyzed in

positive and negative ionization modes with a matrix of Alfa-cyano-4-

hydroxycinnamic acid (CHCA). Positive ionization mode showed significant

data. A wide range of masses were observed in both solvents,however, peaks

with m/z 416, 444, 655, 860, 861, 862 were found with high intensity signals in

all bioactive spots suggesting the compounds responsible for antimicrobial

activity. Spot # 4, in solvent mixture E showed significant peaks of m/z 550.

551, 552 and 788. The mass spectra are illustrated in Fig. # 26-29.

b) Bioassay guided Chemical Fractionation In a separate set of experiments methanolic extract of Juglans regia was

subjected to column chromatography using a range of non-polar to polar

solvents. Chemical components were eluted according to their polarity in

various solvents. A total of 4 fractions were collected including hexane,

chloroform, ethyl acetate, methanol and aqueous and subjected for antimicrobial

activity. Following the results of antimicrobial activity, compounds eluted in n-

hexane were found to be bioactive. MRSA (strain # 3443) was used as reference

strain here. Bioactive spot was scrapped off and subjected for further MIC

determination against MRSA (strain # 3443).

In order to purify bioactive component(s), sub-fractionation of n-hexane

fraction was carried out and a total of 5 sub fractions were subjected for

antimicrobial screening by different methods.

3.2.3 Hippophae rhamnoides (Sea buckthorn) Similar approach was undertaken to analyze bioactive components present in

Hippophae rhamnoides. 3 spots of different Rf values (spot # 1, 5 and 6),

separated by Solvent mixture E, were found to be antimicrobial. Furthermore,

spot # 4 and 6 separated by Solvent system F, showed better zone of inhibition

on bioautography against Salmonella Typhi as shown in Fig # 25. Fig # 21

demonstrates the separation of components according to their Rf value in solvent

F. all bioactive spots were scrapped from reference plates and analyzed by

MALDI-TOF-MS (Table # 17). Significant signals were observed in positive

Table # 15: Summary of Bioassay-guided Chemical Analysis of Bioactive Compound(s) of Camellia sinensis

Bioautography Spot(s) scrapped off

MALDI-TOF-MS

m/z (P) of

significant peaks

Solvent

Bioactive

Bioactiv

e Spot

active

1 4 - -

2 4 4 - 416*, 438*, 451,

522, 550*, 551, 643,

649, 650, 708, 854*,

Fig # 23: MALDI-TOF-MS Analysis of Camellia sinensis

The mass spectra of Camellia sinensis observed by MALDI-TOF-MS showing 37 individual peaks with masses range from 476da to 998da.

Fig # 24: Structure of FA-CS II

Structure of FA-CS II, newly purified compound from Camellia sinensis (Green

Table # 16: Summary of Bioassay-guided Chemical Analysis of Bioactive Compound(s) of Juglans regia

* = peaks with high signal intensity

Bioactive Spot

on Bioautography

Spot(s) scrapped off for

MALDI-TOF-MS

m/z (P) of significant peaks

m/z (P) of

common peaks

Solvent

Bioactive Non-active

1 4 4 - 402, 444, 494, 522, 550*,

551*, 552*, 655, 760, 788*,

789, 790, 860

416, 444,

451, 522,

655, 860,

861, 862

2 5 5 416, 440, 444, 464, 481*,

490*, 643*, 649*, 655, 861,

862, 863

3 4 4 - 416, 444, 451*, 543, 655,

656, 658, 860, 861, 862

4 5 5 400, 416, 440*, 444, 655,

656, 860, 861, 862

Fig # 26: MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. E) of Juglans regia

The MALDI-TOF-MS mass spectra of bioactive spot # 4 of Juglans regia. TLC was run by using Solvent system E.

Fig # 27: MALDI-TOF-MS Analysis of Bioactive Spot # 5 (Sol. E) of Juglans regia

The MALDI-TOF-MS mass spectra of bioactive spot # 5 of Juglans regia. TLC was run by using Solvent system E.

Fig # 28: MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. F) of Juglans regia

The MALDI-TOF-MS mass spectra of bioactive spot # 4 of Juglans regia. TLC was run by using Solvent system F.

Fig # 29: MALDI-TOF-MS Analysis of Bioactive Spot # 5 (Sol. F) of Juglans regia

The MALDI-TOF-MS mass spectra of bioactive spot # 5 of Juglans regia. TLC was run by using Solvent system F.

Table # 17: Summary of Bioassay-guided Chemical Analysis of

Bioactive Compound(s) of Hippophae rhamnoides

S. # Bioactive Spot

on Bioautography

Spot(s) scrapped off for

MALDI-TOF-MS

m/z (P) of significant peaks m/z (P) of common

Solvent

Bioactiv

active

1 1 1 - 440, 494*, 496, 522, 532, 714, 741*, 757, 767*,

860, 875

452, 522,

550, 551,

655, 860,

2 5 5 416, 440, 444*, 451, 452, 855*, 856,860,861, 862

3 6 6 400, 438*, 550*, 551*, 649*, 650*, 655, 706,

708, 709, 854*, 860*, 861*

4 4 4 2 404*, 452, 467, 478*, 493*, 494, 495, 550*, 551,

788*, 789, 790

5 5 6 435*, 437*, 464, 465, 490*, 524, 542*, 543,

558*, 569, 625*, 626, 655*, 658, 860, 861, 862

ionization mode. Compounds with m/z ranged from 400 dal to 800 dal were

observed in both solvents as illustrated by Fig. # 30-34. Common peaks found in

both solvent systems were 452, 550, 551, 655, 860 and 861. It is interesting to note

that m/z (P) 860 and 861, probably isomers, were observed in all 5 bioactive spots

with intense signals. Although, signal intensity is not directly related to the

quantification of components, but the presence of these compounds especially in

bioactive spots with high intensity increase their candidacy as major antimicrobial

components present in Sea buckthorn. It is interesting to note that compounds with

mass: 451, 550, 522 were common in bioactive spots of all three plants. Mass; 416,

440 were observed in solvent system F.

3.3 Antimicrobial Activity of Plants and Plant derived

Substances Three different indigenous plants; Camellia sinensis, Juglans regia and Hippophae

rhamnoides were screened for antimicrobial activity against a total of 377 clinical

bacterial isolates and standard strains representing 15 different gram positive and

gram negative bacterial species. Detailed antimicrobial activity present in every

plant and plant derived substances is given below;

3.3.1 Camellia sinensis (Green Tea) a) Susceptibility Profile Camellia sinensis was screened for antimicrobial activity by agar well diffusion,

agar dilution, microbroth dilution and tube dilution methods against a wide range of

intracellular and extracellular bacterial pathogens. Figure # 35 displays the

percentage of pathogens found susceptible against Camellia sinensis. Aqueous

crude extract of green tea was found to be most effective against Methicillin

Resistant Staphylococcus aureus (n = 99) and gave a zone of 17-18mm against

them. 99% of MRSA strains were found to be sensitive with an average MIC of

0.19 mg/ml (190µg/ ml). 91% of MSSA were also found susceptible with relatively

higher MIC values i.e. 0.78 mg/ml. MIC against reference strain Staphylococcus

aureus was also same. Extract exhibited cidal

Fig # 30: MALDI-TOF-MS Analysis of Bioactive Spot # 1 (Sol. E) of Hippophae rhamnoides

The MALDI-TOF-MS mass spectra of bioactive spot # 1 of Hippophae rhamnoides. TLC was run by using Solvent system E.

Fig # 33: MALDI-TOF-MS Analysis of Bioactive Spot # 4 (Sol. F) of Hippophae rhamnoides

Fi The MALDI-TOF-MS mass spectra of bioactive spot # 4 of Hippophae rhamnoides. TLC was run by using Solvent system F.

Fig # 34: MALDI-TOF-MS Analysis of Bioactive Spot # 6 (Sol. F) of Hippophae rhamnoides

The MALDI-TOF-MS mass spectra of bioactive spot # 6 of Hippophae rhamnoides. TLC was run by using Solvent system F.

activity at concentration 2 x MIC, most of the time. Table # 18 shows a

comprehensive view of antimicrobial susceptibility profile of Camellia sinensis.

Among gram negative isolates 100% MDR Salmonella enterica serovar Typhi,

100% Salmonella enterica serovar Paratyphi A, 71% EPEC, 75% EAggEC and

67% Shigella species were found to be susceptible with average MICs of

3.12mg/ml. Among all MDR Salmonella Typhi found sensitive to green tea, 2

were also resistant to Nalidixic acid. 87% of total ETEC tested were inhibited at

1.56mg/ml of green tea extract. Interestingly MICs against reference strain of

Shigella flexneri ATCC 9199 was comparable to MIC against MRSA, however,

MIC against clinical strains of Shigella species was not too low. Among all

clinical gram negative isolates, MDR strains of Salmonella enterica serovar

Typhi showed the most sensitive pattern (100% susceptibility, as shown in Fig #

36 with MICs 1.56mg/ml. Same trend was observed in methanolic extract with

relatively lower MIC values. The lowest MIC was 0.39mg/ml, observed against

Staphylococcus aureus irrespective of methicillin resistance.

b) Effect on Bacterial Growth Kinetics Time-kill kinetic studies of different bacterial strains were carried out to analyze

the effect of green tea on bacterial growth cycle. A concentration dependent

killing pattern was observed in all organisms irrespective of species. Among

gram positive isolates, time kill curves of MRSA (n = 10), MSSA (n = 10) and

Staphylococcus aureus ATCC 29213 demonstrated the concentration dependent

bactericidal killing.

4 x MIC of Camellia sinensis inhibited the organisms the earliest in case of all

isolates tested. Among all cases concentrations at MIC and above appeared

bactericidal whereas 0.2 and 0.5 x MICs were found to be static. However, it is

interesting to note that MSSA were killed at 6 hours whereas it took longer for

Camellia sinensis to kill MRSA that supports the observations of susceptibility

data where MIC/ MBC of MRSA was 0.25 and MIC/ MBC of MSSA was 0.5

(Table # 18). Fig # 37 demonstrates the inhibition of MRSA (representative

strain # 3443) in the presence of 4 and 2 x MIC of green tea from the beginning

with the sharp fall in CFU after 6 hours of incubation. Organisms were

Fig # 35: Susceptibility Profile of Camellia sinensis

Shigella

Organisms

Sensitive Resistant Susceptibility of Camellia sinensis against different intracellular and

extracellular bacterial pathogens.

Key: MRSA = Methicillin Resistant Staphylococcus aureus

MSSA = Methicillin Sensitive Staphylococcus aureus S. pyo = Streptococcus pyogenes ETEC = Enterotoxigenic Escherichia coli EPEC = Enteropathogenic Escherichia coli EAggEC = Enteroaggregative Escherichia coli MDR-ST = Multidrug Resistant Salmonella enterica serovar Typhi ST = MDR Salmonella enterica serovar Typhi (sensitive strains) SPA = MDR Salmonella enterica serovar Paratyphi A Shigella = Shigella species

Table # 18: Antimicrobial Activity of camellia sinensis against a wide range of intracellular and extracellular bacterial pathogens

Organisms

Aqueous Methanolic FA-CS II

µg/ml

Staphylococcus aureus ATCC 25923 01 17 0.78 1.56 20 0.39 1.56 125 250

Methicillin Resistant Staphylococcus aureus 99 17-18 0.19 0.76 20 0.39 1.56 125 250

Staphylococcus aureus (MSSA-clinical isolates) 59 17 0.78 1.56 20 0.39 1.56 125 250

Streptococcus pyogenes 08 17 0.78 1.56 ND ND ND ND ND

Escherichia coli ATCC 25922 01 11 3.12 >5 ND 3.12 >5 250 >500

Enterotoxigenic Escherichia coli 16 13 1.56 5.0 ND ND ND 250 >500

Enteropathogenic Escherichia coli 07 10 3.12 >5 ND ND ND 250 >500

Enteroaggregative Escherichia coli 63 10 3.12 >5 ND ND ND 250 >500

Uropathogenic Escherichia coli 30 10 3.12 >5 ND ND ND 250 >500

Salmonella enterica serovar Typhi ATCC 13311 01 11 3.12 3.12 ND 2.5 2.5 125 500

MDR Salmonella enterica serovar Typhi 16 11 1.56 1.56 ND 1.25 2.5 62.5 250

Salmonella enterica serovar Typhi (senstive strains) 22 11 3.12 3.12 ND 2.5 2.5 62.5 250

Salmonella enterica serovar Paratyphi A 08 ND 1.56 3.12 ND 1.25 2.5 ND ND

Shigella flexneri ATCC 9199 01 ND 0.78 3.12 ND ND ND 250 >500

Shigella flexneri (clinical strains) 35 ND 3.12 6.25 ND ND ND 250 >500

Pasteurella multocida 02 0 0.39 0.78 ND ND ND ND ND

Note: Results mentioned in average values

completely killed at 24hours. At 1 x MIC, organisms remained in lag phase till

6 hours and become completely killed at 24 hours. at concentrations below MIC

level, organisms started multiplying in first 6 hours at much slower rate than

growth control (more than 2 x log10 difference in CFU). Differences in CFU

become more significant between 8-24 hours of incubation period. Oxacillin

(32µg/ ml) was also tested against MRSA strains for comparison purpose.

Fig # 38 and 39 shows the effect of various concentrations of green tea on

clinical strain of MSSA and Staphylococcus aureus ATCC 29213 respectively.

Graphs illustrate that Camellia sinensis at MIC level and more started effecting

CFU in first two hours, and completely inhibited the growth at 6 hours of

incubation. Effect remained cidal at 24hours. the concentrations at lower than

MIC were found to be static till 8 hours and organisms started multiplying

again, though the rate of multiplication is slower than untreated organisms.

Time-kill curve analysis of the ETEC, EPEC, EAggEC and Uropathogenic E.

coli also demonstrated concentration-dependent antimicrobial effect, with 4 x

MIC of green tea showing the most active inhibitory trend. In contrast to gram

positive strains, green tea behaved bacteriostatic against most of gram negatives

except ETEC. As demonstrated in Fig # 40, Bactericidal killing was observed at

8 hours when green tea was tested at 4 and 2 x MIC against ETEC (n = 3). At 1

x MIC, the organisms remained in lag phase till 4 hours then started dieing,

however complete cidal activity was not observed.

With EPEC and EAggEC, concentration dependent response was observed.

With 4 and 2 x MIC, lag phase up to 2 hours was followed by the significant

reduction in CFU between 2-8 hours time points (Fig # 41, 42). By 8 hours,

CFU was reduced >2 x log10 from the original inoculum size. Organisms treated

with green tea at 1 x MIC remained in lag phase of growth till 8 hours of

incubation period. Green tea inhibited uropathogenic E. coli in a different

manner. Reduction in CFU at 4, 2 and 1 MIC was very distinct at time point 4

hours where bacteria were killed from an average of 6 log10 to 2 log10 CFU/ml

(Fig # 43).

Fig # 37: Effect of Camellia sinensis on Time Kill Kinetics of MRSA

0 2 3 4 5 6 8 24

Time (hrs)

Growth Control Camellia sinensis 4 x MICCamellia sinensis 2 x MIC Camellia sinensis 1 x MICCamellia sinensis 0.5 x MIC Camellia sinensis 0.2 x MICOxacillin (32µg/ml)

Time-kill Curve of a representative isolate of Methicillin Resistant Staphylococcus aureus (strain # 3443) in the presence of five different concentrations of aqueous extract of Camellia sinensis (Green Tea). Data of Oxacillin effect on growth cycle is presented for comparison purpose and growth control of untreated organism is also shown. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Concentrations at MIC level and more started effecting CFU in first few hours, however, at concentrations 0.2 x MIC and 0.5 x MIC CFU started dropping after 8 hours of incubation. Results are presented as an average standard deviation for three experiments.

Fig # 38: Effect of Camellia sinensis on Time Kill Kinetics of MSSA

0 2 3 4 5 6 8 24

Time (hrs)

Growth Control Camellia sinensis 4 x MICCamellia sinensis 2 x MIC Camellia sinensis 1 x MICCamellia sinensis 0.5 x MIC Camellia sinensis 0.2 x MIC

Time-kill Curve of a representative isolate of Methicillin Sensitive Staphylococcus aureus (strain # 3438) in the presence of five different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Concentrations at MIC level and more started effecting CFU in first two hours, and completely inhibited the growth at 6 hours of incubation. Effect remained cidal at 24hours. Results are presented as an average standard deviation for three experiments.

Fig # 39: Effect of Camellia sinensis on Time Kill Kinetics of Staphylococcus aureus ATCC 29213

0 2 3 4 5 6 8 24

Time (hrs)

Growth Control Camellia sinensis 4 x MICCamellia sinensis 2 x MIC Camellia sinensis 1 x MICCamellia sinensis 0.5 x MIC

Time-kill Curve of Staphylococcus aureus ATCC 29213 in the presence of four different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Concentrations at MIC level and more started effecting CFU in first two hours with a sharp fall in CFU between 5 -6 hours. Organisms were completely inhibited at 6 hours of incubation and remained inhibited at 24hours. Results are presented as an average standard deviation for three experiments.

3.3.2 FA-CS II, a New Purified Compound from Green

Tea FA-CS II, a newly purified compound from green tea was found to be very

active against MDR Salmonella Typhi (n = 8) and sensitive clinical strains of

Salmonella Typhi (n = 4) with MIC 62.5 µg/ml. MIC against reference S. Typhi

strain was 125 µg/ml. rest of all gram negative isolates were found to be

susceptible at an average of MICs 250 µg/ml, however compound did not

behave bactericidal >500 µg/ml. Antimicrobial activity of FA-CS II was better

against gram positive isolates including MRSA, with MIC 125 µg/ml and MBC

250 µg/ml (Table # 18).

3.3.3 Juglans regia (Dandasa) a) Susceptibility Profile In order to determine antimicrobial potential, aqueous crude extract and organic

fractions of Juglans regia bark were subjected to screening by agar well

diffusion method and for MIC determination. A number of gram positive and

gram negative bacteria were found to be susceptible as shown in Fig # 44.

aqueous extract of Juglans regia inhibited 95% Methicillin Resistant

Staphylococcus aureus (n = 99) isolates with an average 23 mm zone of

inhibition and MIC 0.31mg/ ml. Interestingly, there was a significant difference

in the MIC values of Juglans regia against MRSA and MSSA strains (P ≤ 0.05)

as shown in Table # 19, however, value of MIC/ MBC is 0.5 in either case,

suggesting its bactericidal activity against Staphylococcus aureus. The activity

of aqueous crude extract against other gram positive isolates was not very

promising except against Streptococcus pyogenes who became susceptible at

MIC 1.25mg/ ml but with relatively very high MBC. Among all gram negative

species, ETEC were found to be most susceptible; i.e. 72% of them were

inhibited at 2.5mg/ ml of Juglans regia.

Fig # 40: Effect of Camellia sinensis on Time Kill

Kinetics of ETEC

0 2 4 6 8Time (hrs)

Time-kill Curve of Enterotoxigenic Escherichia coli in the presence of four different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Concentrations more than MIC significantly decrease the bacterial count from 2 hours of growth with a sharp fall between 6 -8 hours. Results are presented as an average standard deviation for three experiments.

Fig # 41: Effect of Camellia sinensis on Time Kill Kinetics of EPEC

0 2 4 6 8Time (hrs)

Time-kill Curve of Enteropathogenic Escherichia coli in the presence of four different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Results are presented as an average standard deviation for three experiments.

Fig # 42: Effect of Camellia sinensis on Time Kill Kinetics of EAggEC

0 2 4 6 8

Time (hrs)

Time-kill Curve of Enteroaggregative Escherichia coli in the presence of four different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Results are presented as an average standard± deviation for three experiments.

Fig # 43: Effect of Camellia sinensis on Time Kill Kinetics of Uropathogenic E. coli

0 2 4 6 8Time (hrs)

Growth Control Camellia sinensis 4 x MICCamellia sinensis 2 x MIC Camellia sinensis 1 x MIC

Time-kill Curve of Uropathogenic Escherichia coli in the presence of three different concentrations of aqueous extract of Camellia sinensis (Green Tea). Untreated organisms served as Growth Control. 2 x Log10 decrease in the CFU was considered as significant. Camellia sinensis inhibited the organism in dose dependent manner. Results are presented as an average ± standard deviation for three experiments.

Antimicrobial activity of Juglans regia organic fractions was found to be better

than aqueous crude extract. MICs were lesser for gram positive bacteria than

gram negative; similar trend as observed in aqueous extract. Interestingly, there

was a significant difference in the MIC values of all fractions against MRSA

and MSSA strains (P ≤ 0.05). The fair activity in terms of zone inhibition size

and MIC was demonstrated in all fractions of Juglans regia but relative high

activity was observed in compounds eluted in n-hexane especially against

MRSA (MIC 0.032mg/ml). MIC of chloroform fraction against Staphylococcus

aureus ATCC 29213 (0.19mg/ml) was lesser than hexane (0.25) but MBCs

were vise versa. The results of antimicrobial activity were also confirmed by

bioautography as discussed earlier.

On the basis of promising results against MRSA, n-hexane fraction was further

subjected to column chromatography in order to purify bioactive component(s).

Out of 5 different sub-fractions, only one fraction (code # PP-1) showed

significant antibacterial effect (Table # 19) with MICs 25µg/ ml and 50 µg/ml

against MRSA (n = 10) and MSSA (n = 6) respectively, though, it lost its

activity within 5-6 hours after dissolving in DMSO. Due to its unstable nature,

we were unable to identify the components.

Chloroform and ethyl-acetate fractions showed better activity against 85%

MDR Salmonella enterica serovar Typhi isolates (R-type: AmpCSxtT) with

MIC/ MBC 0.5 whereas methanol fraction effected on same strains at MIC

1.06mg/ml. Despite of higher MIC levels (5 mg/ml), crude methanolic extract

of Juglans regia appeared to have some strong antibacterial components against

Salmonella, when tested by bioautography. In Fig # 22, 25, spot number 4 and 5

in lane # 3 demonstrate very strong antibacterial effect on MDR Salmonella

Typhi strain (R-type: AmpCSxtTNa). For other gram negative organisms like

ETEC and EPEC, aqueous and organic fractions showed antibacterial effect at

higher MIC levels. n-hexane fraction inhibited the organisms at lower MIC than

other fractions.

Fig # 44: Susceptibility Profile of Juglans regia on Intracellular and Extracellular Pathogens

Organisms

Sensitive Resistant Susceptibility of Juglans regia against various clinical bacterial isolates. Key: ETEC = Enterotoxigenic Escherichia coli EPEC = Enteropathogenic Escherichia coli EAggEC = Enteroaggregative Escherichia coli

MRSA = Methicillin Resistant Staphylococcus aureus MSSA = Methicillin Sensitive Staphylococcus aureus MDR-ST = Multidrug Resistant Salmonella enterica serovar Typhi Shigella = Shigella species

S. pyo = Streptococcus pyogenes

Table # 19: Antimicrobial Activity of Juglans regia against Intracellular and Extracellular Bacterial Pathogens

Note: results are given in average. Key: 1 = Methicillin Resistant Staphylococcus aureus, 2 = Methicillin Sensitive Staphylococcus aureus, 3 = Bacillus subtilis, 4 = Escherichia coli ATCC 25922, 5 = MDR Salmonella enterica serovar Typhi, 6 = Pseudomonas aeruginosa ATCC 27853, 7 = Enterotoxigenic Escherichia coli, 8 = Enteropathogenic Escherichia coli, 9 = Staphylococcus aureus ATCC 25923, 10 = Streptococcus pyogenes, 11 = Streptococcus pneumoniae, 12 = Shigella dysentriae, 13 = Enterobacter cloacae and 14 = Pasteurella multocida.

n Aqueous Crude Methanol Ethyleacetate CHCl3 Hexane Hexane Sub-fraction (PP-1)

MIC mg/ml

MBC mg/ml

MIC mg/ml

MBC mg/ml

MIC mg/ml

MBC mg/ml

Zone mm

MIC mg/ml

MBC mg/ml

MIC µg/ml

MBC µg/ml

1 99 23 0.31 0.6 28 0.31 1.06 26 0.06 0.97 34 0.04 >2 37 0.032 0.5 19 25 100 2 59 >20 1.25 2.5 18 0.53 1.06 16 0.48 >3 27 0.04 >3 28 0.064 >2 15 50 100 3 01 00 2.5 >5 19 1.06 - 17 0.97 0.97 30 0.09 0.39 33 0.25 0.5 15 50 250 4 01 00 >5 >5 00 1.06 2.1 00 0.97 0.97 17 0.39 1.57 19 0.5 1 00 100 >500 5 15 13 >5 >5 00 1.06 4.2 11 0.48 0.97 18 0.39 0.78 20 0.25 >2 00 100 >500 6 1 00 >5 >5 00 ND ND 00 ND ND 00 ND ND 00 >5 ND 00 >100 >500 7 16 00 2.5 >5 00 4.2 4.2 10 0.97 0.97 15 0.78 >5 18 0.5 1 00 100 500 8 07 00 5 >5 00 2.1 4.2 10 0.97 0.97 17 0.78 1.57 17 0.5 1.03 00 100 500 9 01 >20 1.25 2.5 17 1.06 1.06 19 0.24 >3 30 0.19 1.57 32 0.25 0.5 18 50 10010 03 15 1.25 5 ND ND ND ND ND ND ND ND ND ND ND ND 12 >500 >500 11 01 00 >5 >5 ND ND ND ND ND ND ND ND ND ND ND ND 12 >500 >500 12 15 00 2.5 >5 ND ND ND ND ND ND ND ND ND ND ND ND 15 250 >500 13 01 00 5 >5 ND ND ND ND ND ND ND ND ND ND ND ND 11 >500 >500 14 02 15 1.25 5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND

b) Effect on Bacterial Growth Kinetics Fig # 45 shows bactericidal activity of Juglans regia extract at different

concentrations against Methicillin Resistant Staphylococcus aureus. Organisms

were completely inhibited between 9-24 hours. Concentrations at MIC and

lower levels are appeared to bacteriostatic till 8 hours. When same extract

concentrations were tested against clinical isolates of Methicillin Sensitive

Staphylococcus aureus (n = 10), a concentration dependent killing pattern was

observed. Extract at 4 x MIC and 2 x MIC was successfully killed the

organisms within 8 hours of incubation but took 24 hours in case of 1 x MIC.

Rapid decrease in bacterial count was seen at 8 hours (Fig # 46). Oxacillin

(32µg/ ml) was also tested against MRSA strains for comparison purpose. It is

also very clear in Fig # 47 that shows the effect of various concentrations on

Staphylococcus aureus ATCC 29213. It was observed that Juglans regia at MIC

level and more started effecting CFU in first two hours (P<0.01), and

completely inhibited the growth at 8th hour of incubation. Extract at 0.2 x MIC

kept the organisms static till 6 hours with a significant fall between 6-8 hours,

however organisms started multiplying again after 8 hours though, the rate of

multiplication is slower than untreated organisms. Results of time kill kinetics

were in complete accordance of MIC/ MBC results.

Among gram negative isolates, extract was tested against ETEC at three

different concentrations. 2 x Log10 decrease at any time point from original

CFU was considered as significant. It is worthwhile to note that Juglans regia at

concentrations 2 x MIC, completely inhibited the growth of ETEC from 6 to 8

hours of incubation. After 8 hours organisms started multiplying again. Extract

at lower concentrations were found to be static till 5 hours. a quick fall in

bacterial count was observed at 6 hours time point (> 2 log10 difference from

original count) that recovered in the same manner as discussed above (Fig # 48).

A total of four different fractions i.e. hexane, chloroform, ethyl-acetate and

methanol were tested for the effect on MRSA (n = 10). Results of organic

fractions of Juglans regia were very encouraging. All fractions completely

inhibited the organisms at 24 hours. Rate of CFU/ml decline was faster in

hexane than other fractions (see Fig # 49).

Fig # 45: Effect of Juglans regia on Time Kill Kinetics of

0 2 3 4 5 6 8 9 24

Time (hrs)

MRSA Control MRSA Juglans regia 4 x MICMRSA Juglans regia 2 x MIC MRSA Juglans regia 1 x MICMRSA Juglans regia 0.5 x MIC Oxacillin (32µg/ml)

Time-kill Curve of a representative isolate of Methicillin Resistant Staphylococcus aureus (strain # 3443) in the presence of four different concentrations of aqueous extract of Juglans regia (Dandasa). Data of Oxacillin effect on growth cycle is presented for comparison purpose and growth control of untreated organism is also shown. 2 x Log10 decrease at any time point from original CFU was considered as significant. Results are presented as an average standard deviation for three experiments.

0 2 3 4 5 6 8 24

Time (hrs)

MSSA Control MSSA Dan 4 x MICMSSA Dan 2 x MIC MSSA Dan 1 x MICMSSA Dan 0.2 x MIC

Time-kill Curve of a representative isolate of Methicillin Sensitive Staphylococcus aureus (strain # 3438) in the presence of four different concentrations of aqueous extract of Juglans regia (Dandasa). Growth cycle of untreated organisms was served as Growth Control. 2 x Log10 decrease at any time point from original CFU was considered as significant. Results are presented as an average ±standard deviation for three experiments.

Staphylococcus aureus ATCC 29213

0 2 3 4 5 6 8 24

Time (hours)

Staphylococcus aureus ATCC 29213 ControlJuglans regia 4 x MICJuglans regia 2 x MICJuglans regia 1 x MICJuglans regia 0.2 x MIC

Time-kill Curve of a representative isolate of Staphylococcus aureus ATCC 29213 in the presence of four different concentrations of aqueous extract of Juglans regia (Dandasa). Growth cycle of untreated organisms was served as Growth Control. 2 x Log10 decrease at any time point from original CFU was considered as significant. CFU started declining after 6 hour of incubation in case of organisms treated with Juglans regia at MIC and above. Growth was completely inhibited at 8th hour. Significant fall in bacterial count was also observed between 6-8 hours in organisms exposed to Juglans regia 0.2 x MIC. Results are presented as an average standard deviation for three experiments.

0 2 3 4 5 6 8 24

Time (hrs)

ETEC Growth Control Juglans regia 2 x MICJuglans regia 1 x MIC Juglans regia 0.5 x MIC

Time-kill Curve of a representative isolate of Enterotoxigenic Escherichia coli (ETEC) in the presence of three different concentrations of aqueous extract of Juglans regia (Dandasa). Untreated organisms were served as Growth Control. 2 x Log10 decrease at any time point from original CFU was considered as significant. CFU was towards decline from the beginning of curve with rapid fall between 5-6 hours incubation. Growth was inhibited from 6 to 8 hours in case of ETEC treated with 2 x MIC of Juglaans regia extract. In case of extract at 1 x MIC and 0.5 x MIC, after 6 hours organisms again started multiplying, however, the rate of multiplication was slower than growth control. Results are presented as an average standard deviation for three experiments.

3.3.4 Hippophae rhamnoides (Sea buckthorn) a) Susceptibility Profile Aqueous and methanolic extracts of Hippophae rhamnoides (Sea buckthorn)

was tested for a variety of human and animal pathogens. Screening was done

by agar well diffusion method and MIC by microbroth, agar dilution and tube

dilution methods. Fig # 50 A shows the spectrum of antimicrobial activity

present in Sea buckthorn. It was very interesting to note that among all gram

positive and gram negative isolates, aqueous extract of Sea buckthorn berries

selectively inhibited Pasteurella multocida serotype B2 (n =2) with MIC

50µg/ml and MBC 100µg/ml (Table # 20). Results were reconfirmed many

times by three different methods. These two strains of Pasteurella multocida

serotype B2 were isolated from blood samples of buffalos, suffering from

hemorrhagic septicemia in a dairy farm situated in the province of Punjab. Our

finding further stimulated our interest to explore effect of various concentrations

of Sea buckthorn berries on growth kinetics of Pasteurella multocida. Among

others, Staphylococcus aureus (including MRSA) were also found susceptible

by this extract at low MIC level i.e. 390µg/ml.

b) Effect on Bacterial Growth Kinetics Time-kill kinetic studies of Pasteurella multocida serotype B2 were carried out

to analyze the effect of different concentrations of Sea buckthorn on bacterial

growth cycle. Fig # R20 illustrates the bactericidal activity of Hippophae

rhamnoides (Sea buckthorn) at 10 x MIC and 4 x MIC levels. Organisms treated

with these concentrations of extract were started dieing from stationary phase of

growth cycle. Complete growth inhibition was observed at 6th hour of incubation

and remain dead till 24 hours. Hippophae rhamnoides at MIC showed bacteriostatic

effect till 6 hours. Organisms started multiplying after 6 hours; however, the rate of

multiplication was slower than growth control. Extract was observed ineffective at

ceoncentration less than MIC. Results of time kill kinetics were in complete

accordance of MIC/ MBC results (Fig # 50 B).

Fig # 49: Effect of Organic Fractions of Juglans regia on

Time Kill Kinetics of MRSA

0 2 4 6 8 24

Time (hours)

MRSA Control Ethyl-acetate ChloroformHexane Methanol

Time-kill Curve of a representative isolate of Methicillin Resistant Staphylococcus aureus (strain # 3443) in the presence of different organic fractions of Juglans regia (Dandasa). Concentrations of fractions was 1 x MIC in all cases. Untreated organisms were served as Growth Control. 2 x Log10 decrease at any time point from original CFU was considered as significant. In all cases, growth was inhibited between 8 to 24 hours. Results are given as an average ± standard deviation for three experiments.

Table # 20

Antimicrobial Activity of Hippophae rhamnoides

Organisms

Hippophae rhamnoides

Extract

Staphylococcus aureus ATCC 25923 12 0.39 0.78

Methicillin Resistant Staphylococcus aureus 12-15 0.39 0.78

Staphylococcus aureus (MSSA-clinical isolates) 13 0.39 0.78

Escherichia coli ATCC 25922 00 2.5 5

MDR Salmonella enterica serovar Typhi 15 >5 >5

Salmonella enterica serovar Typhi (senstive) 15 >5 3.12

Salmonella enterica serovar Paratyphi A 13 3.12 >5

Pasteurella multocida serotype B2 25 0.05 0.1

Pseudomonas aeruginosa 00 2.5 >5

3.3.5 Synergistic Antimicrobial Activity of New

Combinations In order to develop an effective and promising antimicrobial candidate against

multidrug resistant (MDR) pathogen, plant extracts were also tested for

synergistic activity with some commonly prescribed and well known antibiotics

that have lost their efficacy against MDR pathogens. On the basis of

antimicrobial activity of extracts alone, different complicated organisms were

chosen to test different synergistic combinations.

Juglans regia extract was tested for synergistic antimicrobial activity against

MRSA (n = 5), MSSA (n = 5) and Staphylococcus aureus ATCC 29213 with

oxacillin, chloramphenicol and tetracycline, the commonly prescribed

antibiotics for Staphylococcal infections. Camellia sinensis extract was tested

for synergistic activity against MDR Salmonella enterica serovar Typhi (n = 2)

with nalidixic acid, tetracycline and chloramphenicol, a commonly given

treatment regime in case of typhoid. Both plant extracts showed indifferent

activity with most of the antibiotics tested (data not shown). Two following

successful synergistic combinations were explored.

a) Juglans regia with Oxacillin

Synergistic antimicrobial activity of Juglans regia Extract with oxacillin was

determined against different clinical isolates of MRSA (n = 5), MSSA (n = 5)

and Staphylococcus aureus ATCC 29213 by four different methods. On MHA

incorporated with 0.2 x MIC of Juglans regia extract, zone of inhibition around

oxacillin (5µg) disc was 10 mm whereas there was no zone of inhibition

observed on control MHA plates.

MIC of oxacillin alone was observed >256 – 132 µg/ ml for MRSA strains by

Etest strip placed on MHA. MIC of oxacillin was 8-16µg/ ml on the plates

incorporated with 0.2 x MIC of Juglans regia extract. Results of checkerboard

titration method revealed significant reduction of MICs of oxacillin and Juglans

regia among all tested isolates of MRSA. As shown in Table # 21, MIC of

Juglans regia was reduced from 312µg/ml to 39µg/ml. All isolates were

resistant to highest concentration of oxacillin tested i.e. 20µg/ml, therefore,

MIC was considered as ≥20µg/ml. MIC of oxacillin in combination with

Juglans regia was appeared 0.312µg/ml which is 1: 64 times lower than MIC

alone. An average FIC index (FICI) of 0.193 was observed that strongly suggest

synergism between both partners of combination. The combination was also

found susceptible against MSSA isolates as well (data not shown). The FIC

index results were interpreted on the following criteria < 0.5, synergy; 0.5 to 1,

additive effect; > 1 to 2, no effect.

Effect of synergistic combination on growth kinetics of MRSA strains was also

studied. Juglans regia extract and Oxacillin, separately, at concentrations

39µg/ml and 0.312µg/ml respectively did not exert significant effects on growth

cycle of MRSA strains. However, their combination (at same concentration)

was found to be inhibitory for MRSA growth cycle. The significant fall in

bacterial count was observed in peak log phase (8th hour) with complete

inhibition in 9th hour (Fig # 51) that confirmed the results of checker board

titration method.

b) Camellia sinensis with Nalidixic acid Synergistic antimicrobial activity of Camellia sinensis Extract with Nalidixic

acid was determined against five Pakistani and one Tanzanian isolates of

Salmonella enterica serovar Typhi by two different methods. On MHA

incorporated with 0.5 x MIC of Camellia sinensis extract, zone of inhibition

around Nalidixic acid (30µg) disc was bigger than zone size around Nalidixic

acid disc placed on MHA control plates. A difference of 4-6 mm in zone of

inhibition was observed. Table # 22 showed the results of Disc diffusion/ agar

incorporation assay. In case of two strains with R-type: AmpCSxtTNa, no zone

of inhibition was observed in either of the plate.

To investigate synergisitic activity of this combination against S.Typhi (R-type:

AmpCSxtTNa), checkerboard titration method was employed against two

Pakistani isolates. A very strong synergistic activity between green tea and

Nalidixic acid was observed. MIC of Nalidixic acid alone was 256µg/ ml for

both strains and MIC of Camellia sinensis alone was 2.5mg/ml. significant

reduction in the MICs of both drugs was observed. As shown in Table # 23,

MIC of Camellia sinensis was reduced up to 0.62mg/ ml. MIC of Nalidixic acid

in combination was 32µg/ml that is 8 times reduced from original MIC value.

Despite of the significant reduction, MIC of Nal was unable to reach breakpoint

level (8µg/ml) that explained the lack of zone of inhibition around Nal disc in

previously discussed method. However, FIC index (FICI) of 0.37 was observed

that strongly suggest synergism between both partners of combination. The FIC

index results were interpreted on the following criteria < 0.5, synergy; 0.5 to 1,

additive effect; > 1 to 2, no effect.

Table # 21: Synergistic Antimicrobial Activity of

Juglans regia with Oxacillin against MRSA

strains

MICs of Oxacillin

(mean values in µg/

MICs of Juglans regia

(mean values in µg/ml)

Fractional

Inhibitory

Concentration

Index (FICI) alone In

combination

alone In

combination

3443 ≥20 0.312 312.5 39 0.1404

501 ≥20 0.312 312.5 39 0.1404

493 ≥20 0.625 312.5 78 0.28

MR1 ≥20 0.312 156 39 0.265

MR2 ≥20 0.312 312.5 39 0.1404

average FICI 0.193 ± 0.72

Summarized results of synergistic activity of Juglans regia Extract with

Oxacillin against 5 different clinical isolates of Methicillin Resistant

Staphylococcus aureus (MRSA) by Checkerboard titration method. FICI is

expressed as average ± SD (n = 5).

Fig # 51: Synergistic Antimicrobial Activity of Juglans regia

with Oxacillin against MRSA

0 2 4 6 8 9 18Time (hours)

Juglans regia (39µg/ml)Oxacillin(0.312µg/ml)Juglans regia (39µg/ml) + Oxacillin (0.312µg/ml)MRSA Grow th Control

Synergistic antimicrobial activity of Juglans regia with oxacillin was observed by Time-kill kinetics of Methicillin Resistant Staphylococcus aureus (n = 5). Organisms were tested in the presence of Juglans regia extract (39µg/ml), Oxacillin (0.312µg/ml) and a combination of Juglans regia extract (39µg/ml) + Oxacillin (0.312µg/ml) at different time intervals. Untreated organisms were served as Growth Control. 2 x Log10 decrease at any time point from original CFU was considered as significant. Results are given as average standard deviation for five experiments.

Table # 22: Synergistic Antimicrobial Activity of Camellia

sinensis with Nalidixic acid against Salmonella enterica serovar

Typhi by Disc Diffusion/ Agar Incorporation Method culture code R-type Zone of Inhibition around Nalidixic acid (30µg)

with Camellia sinensis without Camellia

sinensis

2276 Amp C Sxt T 27 23

2277 Amp C Sxt T 30 24

2279 sensitive to all 26 23

2873 NA Amp C Sxt

2877 NA Amp C Sxt

2899 Amp C Sxt T 25 23

Table # 23: Synergistic Antimicrobial Activity of Camellia sinensis with Nalidixic acid against Salmonella enterica

serovar Typhi by Checkerboard Titration Method

Culture (n = 2) MIC of Nalidixic acid

(µg/ ml)

MIC of Camellia sinensis

(mg/ml)

alone In

combination

alone In

combination

Salmonella enterica

serovar Typhi (Rtype: AmpCSxtTNa)

256 32 2.5 0.62 0.37

3.3.6 Effect of Plant Extracts on Bacterial Cell

Morphology

In order to see the change in bacterial ultrastructure, MRSA was observed with/

without treatment of Camellia sinensis and Juglans regia by Electron

microscope. Fig. # 52 shows characteristic morphological changes in MRSA

after getting treatment with Juglans regia extract for 18 hours. Presence of thick

intercellular masses/ cell walls was observed in almost all bacteria (Fig # 52b).

The most significant change observed was the coating of thread like material on

some swollen and de-shaped bacterial cells (Fig # 52 c and d) indicating the

presence of some unknown material on bacterial surface, even in few cases

completely hollow and deformed cells were observed (Fig # 52d). Prominent

changes in bacterial cell morphology without the complete disappearance of cell

indicate the cell wall as possible target of action for Juglans regia extract.

3.3.7 Effect of Plant Extracts on Protein Profiles of

Bacterial Pathogens It is known that antimicrobial substances affect the microorganisms in many

ways. They can directly inhibit bacterial growth or may interfere in the

synthesis of virulence factors at sub-inhibitory concentrations. In order to see

the effect of Camellia sinensis and Juglans regia protein pattern of the

following organisms was observed by SDS-PAGE in the presence / absence of

plant extracts;

A Methicillin Resistant Staphylococcus aureus (MRSA) Fig # 53A shows a comparison in extracellular protein profile of MRSA in the

presence 0.5 x MIC (lane # 2) and absence (lane #1) of Camellia sinensis after

18hours. In order to avoid the effect of decreased bacterial densities, equal

amount of protein (10µg) was loaded in each lane of 10% PAGE and stained by

Coomassie Brilliant Blue R250 dye (Sigma). There was a very clear difference

in the exoprotein profile of MRSA in both lanes. A dramatic inhibition of high

molecular weight proteins can be easily observed by the above mentioned

figure. In Control sample (lane #1), there are 17 protein bands ranging from

>200-33 kda whereas very few can be observed in lane #2. Due to the limitation

of procedure it was not possible to identify the individual band, however,

according to the literature extracellular protein bands of MRSA, seems down

regulated by Camellia sinensis in our study, could be autolysin (97kda), lipase/

glycerol ester hydrolase (90 kda), Protein A (60kda) and α-hemolysin (33kda).

A new protein band between 45-55kda was expressed after Camellia sinensis

treatment.

In case of cell-associated proteins, we were unable to find any band in both

samples by Coomassie straining. Only 5µg samples were loaded to each lane,

therefore, gels were stained by silver staining. As shown in Fig # 53B a

difference in cell-associated protein pattern of MRSA in the presence of

Camellia sinensis (lane 1), Juglans regia (lane2) and absence (lane3) is very

clear. A total of seven bands can be observed in control (lane3), whereas three

bands with molecular weight >66kda are missing in lane1 and 2 that according

to the literature can be assumed as bifunctional autolysin (145kda), autolysin

(97kda) and protein ORFID (80kda).

B Enterotoxigenic Escherichia coli (ETEC) Although, Camellia sinensis showed antibacterial activity against ETEC at

higher levels of MIC but a very interesting dose dependent inhibition of

extracellular and intracellular protein profiles was observed. ETEC strain used

in this study was genotypically positive for LT and ST. Fig # 54A showed a

dramatic change in high molecular weight exoproteins of ETEC after getting

treatment with graded doses of Camellia sinensis extract. In lanes1-4, equal

amount of ETEC exoprotein samples in the presence of 2 x MIC, 1 x MIC, 0.5 x

MIC and 0.2 x MIC of Camellia sinensis were loaded respectively. Lane 5

received exoproteins of untreated/ control cells. There are no band observed in

test samples (lane 2-4) except a pair, appeared immediately after 36kda that due

to their molecular weight may be assumed as cell envelope proteins; omp F,C

(35.2 kda) and omp A (37.2kda). In case of treatment with higher doses of

extract i.e. 2 x MIC, no band was appeared (lane1). It is important to note that

Labile Toxin (LT) with molecular weight 85kda, the main virulence factor

secreted by ETEC is also among high molecular weight proteins suppressed by

Camellia sinensis.

Effect of graded doses of Camellia sinensis on low molecular weight

exoproteins was also observed on 14% gel. Fig # 54B gives a very good

difference between low molecular weight exoprotein profile of Camellia

sinensis treated (lane 1-4) and untreated bacteria (lane5). Untreated bacterial

exoprotein profile shows the expression of a number of low molecular weight

proteins including a band of 5kda. The results at 14% PAGE also verified the

persistence of 37 kda protein band in all camellia sinensis treated samples

except ones with 2 x MIC.

In order to verify the disappearance of 37kda band (probably omp A) after the

treatment with 2 x MIC of Camellia sinensis, immunoblot analysis was carried

out using anti-ETEC (whole cell) antisera. Immunoblot analysis showed the

same expression pattern of proteins as seen by SDS-PAGE (Fig # 55A). Effect

of the immunogenic nature of 37kda protein band was further confirmed in a

separate set of experiments where exoproteins of untreated organisms were

analyzed using three different types of antisera was rose in the presence or

absence of Camellia sinensis and Juglans regia. No difference was observed in

any case (Fig # 55B).

Fig # 54A also shows effect of Camellia sinensis on cell associated bacterial

proteins. In lane 6-9, cell associated ETEC protein samples were loaded with

the similar treatment whereas lane 10 served as control. A dose dependent

down-regulation in band expression was observed. Lane 6, contained ETEC

with 2 x MIC Camellia sinensis showed only few (4-5) proteins, however lane 7

and 8 showed 10 and 12 bands respectively. Proteins between 36-45 kda

appeared in all cases. Fig # 54B indicates disappearance of several low

molecular cell associated proteins after Camellia sinensis exposure (lane 6-9)

but effect was not in dose dependent nature.

Fig # 55: Immunoblot Analysis

A: Persistance of 37kda protein band was confirmed by western blot analysis. Protein samples of bacteria grown in presence of lane # 1: Camellia sinensis, lane # 2: Juglans regia lane #3: or absence was treated with anti-whole cell vaccine antisera.

B: This figure illustrates the effect of Plant extracts on the mmunogenic nature of 37kda protein band. Antisera against ETEC whole cell vaccine was raised in the presence of lane # 1: Camellia sinensis, lane# 2: Juglans regia and lane # 3: without any plant extract. No difference was observed in any case.

In case of Juglans regia, it was difficult to find out the difference in protein

profiles due to dark color of Juglans regia extract, however inhibition of a

number of exo and cell associated proteins were not visible (data not shown).

3.3.8 Antimycobacterial Activity of Plant Extracts Aqueous and methanolic crude extracts of all three plants namely Camellia

sinensis, Juglans regia and Hippophae rhamnoides were screened for

antimycobacterial against reference strain of Mycobacterium tuberculosis

H37Rv and seven clinical strains of Mycobacterium tuberculosis including

MDR-TB and XDR-TB, Mycobacterium avium, Mycobacterium smegmatis and

Mycobacterium bovis. It was very interesting to note that all three plants

showed very effective antimycobacterial activity against Mycobacterium

tuberculosis H37Rv with MIC <0.75mg/ml (the least concentration tested).

Selective activity of all three aqueous extracts against reference and clinical

strains of MTB was also observed (Table # 24). Furthermore, aqueous extracts

did not show antibacterial potential against M. bovis and M. avium. Methanolic

extract of Camellia sinensis was found to be active against all species of

Mycobacterium whereas the activity of its aqueous extract was restricted to

Mycobacterium tuberculosis. In particular we found that three strains have a

MIC of 2.5 mg/ml, two strains a MIC of 5 mg/ml, and the XDR strains has a

MIC of 1.25 mg/ml.

Methanolic extract of Juglans regia was inactive against M. bovis, whereas was

found to be active against M. avium (MIC 5 mg/ml). In case of M. tuberculosis

strains, it showed inhibition at MIC 0.75 mg/ml against H37Rv and 6 clinical

strains including XDR-TB. Only one strains had a MIC of 2.5 mg/ml. In

contrast, aqueous extract of Juglans regia had no activity against M. bovis and

M. avium and had higher MIC against all clinical strains too. In particular: two

strains were resistant, three had a MIC 5 mg/ml, two strains had a MIC 2.5

mg/ml and only H37Rv had a MIC 0.75 mg/ml.

Table # 24: Antimycobacterial Activity of Plant Extracts

S. # Strain R-type Camellia sinensis MIC (mg/ml)

Juglans regia MIC (mg/ml)

MIC (mg/ml) Aqueous MeOH Aqueous MeOH Aqueous MeOH

1 Mycobacterium tuberculosis H37Rv

- 0.75 0.75 0.75 0.75 0.75 0.75

Mycobacterium tuberculosis RIF 2.5 2.5 >5 0.75 2.5 >5

2 Mycobacterium tuberculosis RIF 2.5 2.5 >5 0.75 2.5 0.75

3 Mycobacterium tuberculosis RIF, SM 2.5 5 5 2.5 >5 0.75

4 Mycobacterium tuberculosis RIF, INH 1.25 5 5 0.75 >5 0.75

5 Mycobacterium tuberculosis SM, INH 2.5 2.5 5 0.75 2.5 0.75

6 Mycobacterium tuberculosis SM, INH, ETH

2.5 2.5 2.5 0.75 >5 0.75

7 Mycobacterium tuberculosis XDR 2.5 1.25 2.5 0.75 >5 0.75

8 Mycobacterium bovis - >5 2.5 >5 5 >5 2.5

9 Mycobacterium avium - >5 2.5 >5 5 >5 <0.75

10 Mycobacterium smegmatis 1.25 ND 0.75 ND ND ND

Antimycobacterial activity of aqueous and methanolic extracts of indigenous plants against clinical and reference

isolates of Mycobacterium tuberculosis and other species. Results are given as an average of three experiments.

Methanolic extract of Hippophae rhamnoides had a good activity against all the

strain tested, with the exception of one clinical strain resistant. The results are

the: six clinical strains, H37Rv and M. avium had a MIC 0.75 mg/ml, whereas

M. bovis had MIC 2.5 mg/ml. Aqueous extract of Hippophae rhamnoides had

the worst results: M. avium, M. bovis and four clinical strains were resistant,

three clinical strains had a MIC 2.5 mg/ml and only H37Rv had a MIC 0.75

mg/ml.

It is interesting to note that aqueous crude extracts of all plants exhibited

inhibitory activity against reference and clinical strains of MTB but no activity

against other species. In contrast, methanolic extracts of Juglans regia and

hippophae rhamnoides inhibited all species of Mycobacterium tested.

3.3.9 Anti-Trichomonas Activity of Plant Extracts Anti-protozoal activity of aqueous and methanolic plant extracts was assessed

against seven different clinical isolates of Trichomonas vaginalis, collected in

different geographic areas (Italy, Angola, and Mozambique) by endpoint

method. Some isolates were associated with Mycoplasma homonis and some

were Mycoplasma-free. One strain of Trichomonas, isolated from Angola-

Africa was resistant to metronidazole. Among three indigenous plants only

Juglans regia showed strong anti-trichomonas activity. All isolates were found

to be susceptible by aqueous and methanolic extracts of Juglans regia with

MIC100 3125 µg/ml and 125µg/ml respectively. Activity was irrespective of

their association with Mycoplasma homonis. Among all cases, 90% trophozoites

became rounded up, non-motile and ultimately dead within 30m of the exposure

of plant extracts as shown in Fig # 56. Effect of Juglans regia extracts on the

morphology and the motility of Trichomonas vaginalis was also confirmed by

Trypan Blue exclusion assay at different time intervals. 100% inhibition was

observed within 3 hours of incubation that excludes the possibility of anti-

protozoal action in time dependent manner.

3.4 In-Vitro Toxicity Studies of Plants All plants used in this study are in common community use but to fulfill this

basic criterion, it was important to carry out toxicity studies. Three different

assays were performed to analyze toxic effects of plants and their purified

compounds on mammalian cells and to establish a dose-reaction relationship.

3.4.1 Hemolytic Activity of Plants and Plant derived

Substances

A possible limitation of plant extracts to be used in therapy is their potential to

cause injury to mammalian cell membranes. In order to assess this potential

shortcoming, we examined the ability of Camellia sinensis, Juglans regia and

FA-CS II to lyse human RBCs. A normal rate of hemolysis in 1 hour was

17.33%, therefore extracts showed hemolysis double than normal rate of

hemolysis were considered as significant.

Among all plants extracts and compounds tested, only 2 highest concentrations

of Juglans regia (Dandasa) i.e. 1000mg/ml and 500mg/ml were appeared to be

toxic (>30% hemolysis) as shown in Fig # 57b. Rest of three concentrations

tested was non-toxic. The lowest hemolytic activity was observed in 10mg/ml

and 1 mg/ml Camellia sinensis (Fig # 57a) and concentrations ≤100µg/ml of

FA-CS II (Fig # 57c). Our results confirm that Juglans regia, Camellia sinensis

and FA-CS II do not contain broad spectrum cytolytic activity at concentrations

show antibacterial potential. Our results do not exclude the possibility of

cytotoxicity against other cell types.

3.4.2 Cytotoxicity of Plant Extracts against Human Vascular

Endothelial Cells Effect on cell viability was checked by 3-(4,5-dimethylythiazole-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-tetrazolium (MTS) assay. After

24 h incubation, MTS assay revealed the strong proliferative qualities of plant

extracts for human vascular endothelial cells (ECV304). Instead of exerting

toxic effects, aqueous and methanolic extracts of camellia sinensis at 10-

0.62mg/ml helps the cells to proliferate. In case of control wells where cells

were in growth medium average of A595 was 0.5, whereas A595 in test wells was

not more 0.3 suggestive of proliferative properties (Fig #58a).

In case of Juglans regia both aqueous and methanolic extract did not surpass

the cut off value of toxicity at any concentration tested. A595 of cells 0.7 in case

of methanolic extract at 2.5mg/ml was higher than 5mg/ml but in the range of

normal cut off value (Fig # 58b). Fig # 58c also illustrates the non-toxic nature

of aqueous and methanolic extracts Hippophae rhamnoides at concentrations

ranged from 10-0.625mg/ml on ECV304 cells. Rather an increase in cell

proliferation was observed.

3.4.3 Free Radical Scavenging Activity of Plant Extracts The ability of aqueous and methanolic plant extracts to scavenge reactive

oxygen species of Human Endothelial cells (ECV304) was assessed using

fluorescence probe (2', 7'-dichlorofluorescin {DCF} assay. ECV304 cells

loaded with 10 µM DCFH-DA, were treated with different concentrations of

plant extracts ranging from 0.625-2.5mg/ml.fluorescence was measured

continuously over the period of 80 min. cells added with PBS Plus instead of

plant extracts, served as control.

The ability of Hippophae rhamnoides to scavenge ROS produced by ECV304

cells was proved. An immediate fall in DCF-fluoursence after the addition of

plant extract was observed. However, fluorescence was increased over the

period of 80min in treated cells but rate of progression was slower than control

cells. A concentrations dependent scavenging activity of aqueous and

methanolic extracts is shown in Fig #59. Therefore, we can say that

fluorescence assay confirmed very strong antioxidant activity in aqueous and

methanolic extracts of Hippophae rhamnoides in a dose dependent manner. Due

to the interference in fluorescence by the color or nature of green tea and

dandasa, we were unable to get reliable results.

Fig # 56: Anti-Trichomonas Activity of Plant Extracts

.5mg/ml

Aq6.25

Aq3.12

Aq1.25

Aq0.75

Aq0.31

2mg/ml

Juglans regia

30min 180min

0102030405060708090

Juglans regia

30min 180min

Anti-Trichomonas activity of (a) aqueous and (b) methanolic extracts of

Juglans regia against seven clinical strains of Trichomonas vaginalis carried out

by trypan blue assay over the period of 3 hours. Different concentrations of

extracts ranged from 2.5 to 0.625mg/ml were tested.

Cytotoxicity of Plant Extracts against Human Vascular Endothelial Cells

Concentration of Camellia sinensis (mg/ml)

CTRL H2O:1.25mg/ml

H2O:2.5

H2O: 5mg/ml

H2O:10

MetOH:0.6

MetOH:1.25mg/ml

MetOH:2.5

Concentration of Juglans regia (mg/ml)Ab

Concentration of Hippophae rhamnoides (mg/ml)

Fig # 59: Free Radical Scavenging Activity of

y = 1.9203x + 331.16

y = 1.5584x + 291.44

y = 1.9689x + 224.54y = 1.0115x + 267.87

y = 1.8788x + 147.15y = 0.7762x + 201.96y = 0.8086x + 181.84

0 20 40 60 80 100Hippophae rhamnoides

Aq 2.5mg/mlAq 1.25mg/mlAq 0.625mg/mlMeOH 2.5mg/mlMeOH 1.25mg/mlMeOH 0.625mg/mlcontrol

Reactive Oxygen Species (ROS) scavenging activity of Hippophae rhamnoides. (a): Effect of different concentrations of aqueous and methanolic extracts on DCF fluorescence (in a.u.) in ECV304 cells was observed up to 80 min. (b): Comparative data was expressed in the form of Slope obtained by linear regression analysis of DCF- fluorescence by using formula; y = m x + b.

CTRL H2O 2.5 H2O 1.25 H2O 0,625 MetOH 2,5 MetOH 1,25 MetOH 0.625

Slope or m

b (Slope)

3.4.4 Effect of Plant Extracts on Cell Proliferation by 3H

Thymidine Incorporation

In order to see the effect of plant extracts on endothelial cell proliferation, total

DNA synthesis was estimated by measuring [3H]thymidine incorporation into

cellular DNA. To observe any DNA synthesis arrest, readings were taken at two

different time points of cell cycle i.e. 3 hours and 24 hours. The findings of

proliferation assay were in accordance to DCF fluorescence assay. Fig # 60

shows that aqueous and methanolic extracts of Hippophae rhamnoides had no

adverse effect on the incorporation of radioactive thymidine into endothelial

cells. In addition, an increase in DNA synthesis was observed in case of

concentration 1.25mg/ml of both extracts. Significant increase was observed

after 24 hours compared to control cells. Our data not only prove anti-oxidant

activity of Hippophae rhamnoides but also indicates its help to continue the

cells stay alive.

3.5 Immunopharmacological Studies of Plants

3.5.1 Animal Toxicity Studies of Plant Extracts

Non-toxic nature of the extracts was finally confirmed by acute and subacute

toxicity experiments, conducted on BALB/C mice. In case of Camellia sinensis,

single intraperitoneal administration of graded doses ranging from 1000 to 100

mg/kg did not induce any remarkable alterations in the behavior pattern and

physical appearance of mice. No evidence of mortality was observed.

Hematological and biochemical parameters (shown in Table# 25) were not

significantly affected. Lack of any sign of sickness and death over one week

time period suggested no apparent acute toxicity exerted by Camellia sinensis.

For sub-acute toxicity studies, multiple doses of extract were administered by

intraperitoneal (i.p) route on every alternate day over the period of 2 weeks.

There were no deaths and no sign of toxicity observed till 28 days. There was

no significant difference in biochemical and hematological parameters of

animals taken green tea as compared to those in control group (Table # 26).

Fig # 60: Effect of Plant Extracts on Cell Proliferation

Aq 2.5mg/ml

Aq 1.25m

Aq0.62

5mg/ml

H 2.5mg/ml

5mg/ml

Aq 2.5mg/ml

Aq 1.25m

Aq0.62

5mg/ml

H 2.5mg/ml

5mg/ml

Hippophe rhamnoides

Effect of aqueous and methanolic extract of Hippophae rhamnoides on cell proliferation by [3H] thymidine incorporation into endothelial cells. Cells were incubated with different concentrations of extracts for (a) 3 hours and (b) 24 hours. Cells were pulsed with 1µCi/ ml of (3H) Thymidine and radioactivity was measured after 24 hours. Control cells did not receive any extract treatment. Results are given as an average of three experiments.

Table # 25: Hematological and Biochemical Parameters

during Acute Animal Toxicity Studies of Camellia sinensis

Summarized results of hematological and biochemical parameters during acute

toxicity studies of aqueous extract of Camellia sinensis carried out in BALB/C

mice. Single dose of each concentration was given and blood samples were

collected after 7 days. Data is expressed as average ± SD (n = 6). No statistical

difference was observed

Parameters Dose Concentration of Camellia sinensis (Aqueous Extract)

(mg/kg of body weight)

Control (N/S)

1000 500 300 100 WBC Count (106/L)

5.2± 0.01 4.8 ± 0.13 5.1 ± 0.12 4.9 ± 0.28 5.1 ± 0.96

RBC count (109/L)

8.5 ± 0.23 8.6 ± 0.32 8.5 ± 0.55 8.5 ± 0.48 8.7 ± 0.44

SGPT (U/L)

16.0 ± 0.2 12 ± 1 15 ± 1.5 12 ± 2 15 ± 1

Alk Phos (IU/L)

69 ± 3 48 ± 1.5 53 ± 1 67 ± 2 64 ± 2

BUN (mg/dl)

18 ± 1.3 20 ± 1 20 ± 0.5 16 ± 2 16 ± 1.5

Creatinine (mg/dl)

0.30 ± 0.02 0.2 ± 0.02 0.2 ± 0.01 0.2 ± 0.12 0.3 ± 0.02

Albumin (g/dl)

2.46 ± 0.33 2.40 ± 0.34 2.40 ± 0.08 2.75 ± 0.02 2.6 ± 0.01

Total Protein (g/dl)

1.9 ± 0.42 2.8 ± 0.22 3.3 ± 0.1 3.4 ± 0.12 3.4 ± 0.2

Amylase (U/dl)

20 ± 1 19 ± 2 12 ± 4 23 ± 1 21 ± 2

Single dose of Juglans regia at concentrations 1000mg/kg, 500 mg/kg, 300

mg/kg and 100 mg/kg did not exert any change in the psychological and

physical appearance of mice. Hematological (Table # 27) and biochemical

(Table # 28) parameters including liver function, kidney function profiles and

amylase were appeared to be comparable to normal group. In sub-acute toxicity

experiments, multiple doses of extract did not show any behavior change. No

death was observed in any of the case. Lab findings of hematology (Table # 29)

and biochemistry were within normal range but with a little decrease in serum

amylase activity in dose dependent manner as shown in Table # 30. No apparent

sign of toxicity was observed in different organs removed after 28 days. Our

data suggest that the plant extract is relatively safe or non-toxic for mice.

In case of Hippophae rhamnoides, there was no death observed in acute and

sub-acute toxicity experiments. Graded doses of plant extract from 2000 to

100mg/kg of the body weight were found to be safe for BALB/C mice. Animals

were kept under observation till 2 months to see any sign of chronic toxicity. No

change in psychology, weight, physical appearance was observed.

Table # 26: Hematological and Biochemical Parameters

during Sub-acute Animal Toxicity Studies of Camellia

sinensis

Summarized results of hematological and biochemical parameters during

Subacute toxicity studies of aqueous extract of Camellia sinensis carried out in

BALB/C mice. Multiple doses of each concentration were given up to 14 days.

Blood samples were collected at 28th day. Data is expressed as average ± SD (n

= 6). No statistical difference was observed

Parameters Dose Concentration of Camellia sinensis (Aqueous Extract)

(mg/kg of body weight)

Control (N/S)

1000 500 300 100 WBC Count (106/L)

5.4± 0.2 4.7 ± 0.22 5.1 ± 0.3 4.9 ± 0.45 5.1 ± 0.3

RBC count (109/L)

8.9 ± 0.12 8.9 ± 0.3 8.5 ± 0.21 8.7 ± 0.5 8.6 ± 0.2

SGPT (U/L)

12 ± 1 10 ± 1 12 ± 1 11 ± 2 12 ± 1

Alk Phos (IU/L)

69 ± 2 48 ± 3 53 ± 4 67 ± 1 61 ± 3

BUN (mg/dl)

15 ± 2 14 ± 0.5 18 ± 1.5 15 ± 3 16 ± 2.5

Creatinine (mg/dl)

0.20 ± 0.01 0.3 ± 00 0.3 ± 00 0.2 ± 0.02 0.3 ± 0.04

Albumin (g/dl)

2.2 ± 0.12 2.2 ± 0.1 1.8 ± 0.1 1.9 ± 0.02 2.0 ± 0.01

3.1 ± 0.2 3.2 ± 0.15 3.1 ± 0.2 3.1 ± 0.12 3.3 ± 0.3

Amylase (U/dl)

18 ± 3 21 ± 2 22 ± 2 22 ± 3 21 ± 2

Table # 27: Hematological Parameters during Acute Animal

Toxicity Studies of Juglans regia

Summarized results of hematological parameters during acute toxicity studies of

aqueous extract of Juglans regia carried out in BALB/C mice. Single dose of

each concentration was given and blood samples were collected after 7 days.

Data is expressed as average ± SD (n = 6). No statistical difference was

observed

Parameters Dose Concentration of Juglans regia Extract (mg/kg of body weight)

Control Group 1000 500 300 100

WBC Count (106/L)

3.55 ± 0.25 2.58 ± 0.03 3.35 ± 0.52 3.58 ± 0.38 3.14 ± 0.96

RBC count (109/L)

8.10 ± 0.40 8.25 ± 0.72 8.47 ± 0.27 8.05 ± 0.48 7.56 ± 0.12

Hemoglobin (g/dl)

15.31 ± 0.30 15.5 ± 0.40 15.3 ± 0.30 14.8 ± 0.90 14.98 ± 0.40

Haematocrit (%)

43.20 ± 1.10 45.3 ± 0.90 44.5 ± 1.14 42.4 ± 1.30 43.7 ± 0.50

Platelets (106/L)

5.48 ± 0.67 3.32 ± 0.25 5.78 ± 0.07 4.55 ± 0.04 4.28 ± 0.23

Table # 28: Biochemical Parameters during Acute Animal

Toxicity Studies of Juglans regia

Summarized results of biochemical parameters during acute toxicity studies of

aqueous extract of Juglans regia carried out in BALB/C mice. Single dose of

each concentration was given and blood samples were collected after 7 days.

Data is expressed as average ± SD (n = 6). No statistical difference was

observed.

Normal Saline (Control Group)

1000 500 300 100

SGPT (U/L)

21.0 ± 1.26 20.33 ± 1.03 21.50 ± 2.94 20.0 ± 2.28 26.33 ± 1.03

SGOT (U/L)

15.66 ± 0.51 14.16 ± 1.83 17.0 ± 2.0 15.83 ± 0.98 17.5 ± 1.63

BUN (mg/dl)

16.83 ± 1.32 15.66± 1.03 14.83 ± 0.98 16.83 ± 1.83 17.5 ± 1.64

Creatinine (mg/dl)

0.50 ± 0.06 0.51 ± 0.07 0.61 ± 0.09 0.56 ± 0.12 0.51 ± 0.04

Albumin (g/dl)

2.46 ± 0.33 2.40 ± 0.34 2.40 ± 0.08 2.75 ± 0.02 2.6 ± 0.01

4.86 ± 0.50 4.45 ± 0.34 4.37 ± 0.36 4.5 ± 0.03 3.96 ± 0.01

Amylase (g/dl)

1447 ± 85 1478 ± 61 1590 ± 116 1568 ± 132 1694 ± 108

Table # 29: Hematological Parameters during Sub-acute

Animal Toxicity Studies of Juglans regia

Summarized results of hematological parameters during Subacute toxicity

studies of aqueous extract of Juglans regia carried out in BALB/C mice.

Multiple doses of each concentration were given up to 14 days. Blood samples

were collected at 28th day. Data is expressed as average ± SD (n = 6). No

statistical difference was observed.

Control Group 1000 500 300 100

WBC Count (106/L)

3.86 ± 0.28 2.83 ± 0.30 3.65 ± 0.35 3.71 ± 0.36

3.66 ± 0.28

RBC count (109/L)

7.89 ± 0.39 8.20 ± 0.57 8.42 ± 0.26 7.59 ± 0.63

7.06 ± 0.10

Hemoglobin (g/dl)

15.00 ± 0.40 15.7 ± 0.30 15.3 ± 0.24 14.75 ± 0.45

14.93 ± 0.19

Haematocrit (%)

43.50 ± 1.39 43.5 ± 1.70 44.2 ± 2.15 41.7 ± 1.11

43.3 ± 1.42

Platelets (106/L)

5.95 ± 0.70 3.52 ± 0.27 5.57 ± 0.07 4.66 ± 0.03

4.22 ± 0.23

Table # 30: Biochemical Parameters during Sub-acute Animal Toxicity Studies of Juglans regia

Summarized results of biochemical parameters during Sub-acute toxicity studies

of aqueous extract of Juglans regia carried out in BALB/C mice. Multiple

doses of each concentration were given up to 14 days. Blood samples were

collected at 28th day. Data is expressed as average ± SD (n = 6). No statistical

difference was observed

Normal Saline (Control Group)

1000 500 300 100

SGPT (U/L)

27.0 ± 2.19 26.1± 1.16 24.66 ± 0.81 28.16 ± 2.9 26.33 ± 1.03

SGOT (U/L)

18.16 ± 1.16 20.6 ± 1.86 17.16 ± 1.6 17.5 ± 0.8 17.5 ± 1.64

BUN (mg/dl)

15.66 ± 0.51 15.6± 0.81 16.66 ± 0.81 16.66 ± 1.3 17.5 ± 1.64

Creatinine (mg/dl)

0.57 ± 0.08 0.5 ± 0.01 0.50 ± 0.06 0.54 ± 0.1 0.50 ± 0.04

Albumin (g/dl)

2.53 ± 0.08 2.6 ± 0.08 2.56 ± 0.08 2.56 ± 0.1 2.6 ± 0.01

3.74 ± 0.24 3.9 ± 0.03 4.02 ± 0.02 4.5 ± 0.2 3.96 ± 0.01

Amylase (g/dl) 1403 ± 34 1493 ± 58 1583 ± 118 1638 ± 71 1694 ± 108

3.5.2 In-Vivo Antimicrobial Activity

Antimicrobial activity of Camellia sinensis and Hippophae rhamnoides was

confirmed in murine models.

A In Vivo Antimicrobial Activity of Camellia sinensis

against MRSA in Experimental Murine Septicemia A single dose of 156mg/kg of aqueous extract of Camellia sinensis was given to

the test group of neutropenic mice only after 2 hrs of systemic MRSA infection.

The effect of Camellia sinensis treatment on organ dislocation of MRSA was

observed till 6 hours after the administration. Noticeable reduction in the CFU/

organ was observed in test group than control or infected group. > 2 log10

reduction in bacterial load of heart and lungs was observed in 6 hours of green

tea treatment. In liver CFU started reducing from 2nd hour but at 6th hour, we

were unable to find any living organism (Fig # 61). Due to the limitation of the

procedure, CFU < 2 x log10 was considered as 0. Over all effect of Camellia

sinensis on organ dislocation of MRSA was significant and we can say that in

vivo findings were in accordance with the in vitro activity.

B In Vivo Antimicrobial Activity of Hippophae rhamnoides

against Pasteurella multocida

i) LD50 of Pasteurella multocida In order to determine lethal dose (LD50) of Pasteurella multocida serotype B2,

groups of mice were challenges i.p. with graded doses of bacteria ranges from 2

x 108 to 2 x 102. Results indicated that 20 CFU per mouse for this strain was

sufficient to kill 50% population of mouse present in one group within 48 hours

of infection. Therefore, LD50 was considered as 2 x 102. The virulence of

higher doses was very evident from experiment in which all mouse injected

with 2 x 103 or more were died within 48 hours. Especially doses ≥ 2 x 107 were

capable of killing mice within 18 hours. mice given these doses showed severe

shivering, lack of activity and drowsiness after 6 hours of infection. Symptoms

In Vivo Antimicrobial Activity of Camellia sinensis against MRSA in

Experimental Murine Septicemia (a-b)

0 2 4 6

Time (hours)

Test Group Control Group

0 2 4 6Time (hours)

Fig # 61 (a-b): In-Vivo antimicrobial activity of Camellia sinensis on multiorgan dislocation of MRSA (strain #3443) in systemic mouse model.

Significant reduction in CFU was observed in (a) Heart (b) Liver of test group (treated with single dose of 156mg/kg of Camellia sinensis extract

after 2hours of MRSA infection). MRSA infected group of mice that did not receive any treatment was served as Control. Results are given as

average standard deviation for three experiments.

In Vivo Antimicrobial Activity of Camellia sinensis against MRSA in

Experimental Murine Septicemia (c-d)

0 2 4 6Time (hours)

0 2 4 6

Time (hours)

Fig # 61 (c-d): In-Vivo antimicrobial activity of Camellia sinensis on multiorgan dislocation of MRSA (strain #3443) in systemic mouse model.

Significant reduction in CFU was observed in (c) Spleen and (d) Lungs of test group (treated with single dose of 156mg/kg of Camellia sinensis

extract after 2hours of MRSA infection). MRSA infected group of mice that did not receive any treatment was served as Control. Results are

given as average standard deviation for three experiments.

became severe after 2 hours. The maximum death time observed was 48 hours

in case of 2 animals given 2 x 103 CFU per mouse. Results confirm the

virulence of clinical strain of Pasteurella multocida serotype B2 isolated from

water buffalo suffering from hemorrhagic septicemia, for laboratory mice.

ii) Effect of Different Concentrations of Hippophae

rhamnoides on Organ Dislocation of Organisms Graded doses of aqueous extract of Hippophae rhamnoides ranged from 100-

5mg/kg were given to different groups of mice, challenged by 2 x 104 CFU (100

x LD50) by intraperitoneal route. Single dose of Hippophae rhamnoides at

100mg/kg was dramatically found to be protective for the mouse having 106

CFU (104 x LD50) of virulent strain. Treatment with 80mg/ kg of extract

resulted in complete eradication of organisms from liver, heart and spleen after

24 hours of onset of symptoms. There was no bacterial growth observed in

pleural fluid at any time point which indicates the immediate eradication of

organisms. Fig # 62 showed the dose dependent effect of Hippophae

rhamnoides on organ dislocation of virulent strain of Pasteurella multocida

serotype B2 in murine model of Hemorrhagic septicemia. > 2 log10 reduction in

bacterial load of spleen, liver, heart and kidneys, in comparison with control

group (untreated), was seen among animals treated with 50mg/kg of extract.

This concentration completely inhibited the bacterial load of heart after 24

hours of infections. Treatment with 10mg/kg also caused reduction of bacterial

count in liver, pleural fluid and lungs but with lesser extent.

Fig # 62: Effect of Hippophae rhamnoides on Organ Dislocation of Pasteurella multocida

6 12 24

Concentration of Hippophae rhamnoides

log CFU

/ liver

HR 80mg/kg HR 50mg/kg HR10mg/kgHr 5mg/kg Control Group

6 12 24

Concentration of Hippophae rhamnoides

Fig #62 (A-B): In-Vivo antimicrobial activity of Hippophae rhamnoides on dislocation of Pasteurella multocida serotype B2 in A: liver, B: heart of mouse model of hemorrhagic septicemia. Pasteurella multocida serotype B2 infected group of mice that did not receive any treatment was served as Control. Results are given as average standard deviation for three experiments.

6 12 24

Concentrations of Hippophae rhamnoides

log CFU

/ pair o

f kidne

6 12 24

log CFU

/ splee

(C-D): In-Vivo antimicrobial activity of Hippophae rhamnoides on dislocation of Pasteurella multocida serotype B2 in C:kidneys, B:spleen of mouse model of hemorrhagic septicemia. Pasteurella multocida serotype B2 infected group of mice that did not receive any treatment was served as Control. Results are given as average standard deviation for three experiments.

6 12 24

/ ml o

f Pleur

6 12 24

log CFU

/ pair o

(E-F): In-Vivo antimicrobial activity of Hippophae rhamnoides on dislocation of Pasteurella multocida serotype B2 in E: Peritoneal Fluid, F: Lungs of mouse model of hemorrhagic septicemia. Pasteurella multocida serotype B2 infected group of mice that did not receive any treatment was served as Control. Results are given as average standard deviation for three experiments.

3.5.3 Intracellular Killing in Phagocytic Cells in

the Presence of Plants Aqueous extracts of Camellia sinensis, Juglans regia and FACS II (new

purified compound from Camellia sinensis) were tested for their effect on

intracellular killing of MRSA by human PMNLs. Our results revealed that

intracellular growth of MRSA in human PMNLs was affected as a result of

cell activation in response of priming with plants tested. In cells primed with

graded concentrations (2 x MIC, 1 x MIC and 0.5 x MIC) of Camellia sinensis

extract, intracellular bacterial growth was decreased compared to the control. It

was worthwhile to note that a sudden decrease in bacterial growth was observed

in case of concentrations 2 x MIC and 1 x MIC, however bacterial remained

alive in static conditions till 180 min. in contrast, normal PMNLs were unable

to stop multiplication of MRSA strain (Fig # 63).

When we tested FA-CS II, new pure compound of Camellia sinensis, it showed

intracellular antimicrobial activity against MRSA. The concentrations even less

than MIC (0.5 x MIC) exhibited a significant difference in bacterial count of

surviving bacteria intracellularly as shown in Fig # 63. Rate of bacterial growth

between 90-180 minutes was according to the graded doses of FA-CS II, used in

priming of PMNLs, which proved its contribution of control of intracellular

bacterial growth.Similar results were obtained, when cells were primed with

Juglans regia extract. Fig # 64 shows a dose dependent effect of Juglans regia

on intracellular growth of MRSA after 180 minutes of phagocytosis.

Fig # 63: Intracellular Killing in Phagocytic Cells in the Presence of Camellia sinensis and FA-CS II

0 30 45 90 180

Time (min)

2x MIC 1 x MIC 0.5 x MIC Control

0 30 45 90 180

Time (min)

Effect of (a) Camellia sinensis extract and (b) FA-CS II on the intracellular growth of methicillin-resistant Staphylococcus aureus (strain # 3443) in human PMNLs. Graded concentrations of extract/ compound were added after 20min of phagocytosis and further incubated for 180min. Cells received same amount HBSS served as Control. Samples were taken out at different time intervals to observe surviving CFU inside PMNLs. A difference in bacterial count was observed in primed cells than control. Results are given as average ± SD of three separate experiments.

3.5.4 Effect of Plants on Humoral Immune

Response

The effect of plants and plant derived compounds on humoral immune response

was investigated by Hemolytic Plaque Assay. Any change in antibody

production and antibody producing spleen cells was observed. SRBCs were

used as antigen. Group of animals received normal saline instead of plant

extract was served as control. Our results indicate a two-fold increase in PFCs

in animals primed with Camellia sinensis as compare to control animals.

Animals received Camellia sinensis extract (10mg/kg) produced 256 ± 25

PFCs/ 105 cells whereas control animals showed 75 ± 20 PFCs/ 105 cells.

However, there was no significant difference observed in spleen weights of each

group. The numbers of PFCs per 105 spleen cells were 116 ± 21 and 125 ± 25 in

animals primed with Juglans regia aqueous extract (10mg/kg) and FA-CS II

(5mg/kg) respectively showing a considerable difference as compare to control.

Results are illustrated in Fig# 65.

Furthermore, α-SRBCs antibody titer was observed in the peripheral blood after

5 days of immunization by hemeagglutination method. Antibody titer in control

group was 1: 32. an 8-fold increase (1: 256) in antibody titer was observed in

animals primed with Camellia sinensis or Juglans regia, suggesting that both

plant extracts boost up in-vivo antibody production, thus verified the results of

hemolytic plaque assay. However in case of FA-CS II a weak effect on antibody

titer (1:64) was observed.

Fig # 64: Intracellular Killing in Phagocytic Cells in the Presence of Juglans regia

0 30 45 90 180

Time (min)

Effect of Juglans regia extract on the intracellular growth of methicillin-resistant Staphylococcus aureus (strain # 3443) in human PMNLs. Graded concentration of extract was added after 20min of phagocytosis and further incubated for 180min. Cells received same amount HBSS served as Control. Samples were taken out at different time intervals to observe surviving CFU inside PMNLs. A difference in bacterial count was observed in primed cells than control. Results are given as average ± SD of three separate experiments.

Fig # 65: Effect of Plants on Humoral Immune Response

Camelliasinensis

Juglans regia FA-CS II Control

Fig # 65: Effect of Plants and Plant derived substances on humoral immune response was investigated by Hemolytic Plaque Assay. SRBCs were used as antigen. Group of animals received normal saline instead of plant extract was served as control. Results are given as average ± SD of three separate experiments.

DISCUSSION

Despite remarkable advances in medical research during last century, infectious

diseases remain among the leading causes of death worldwide. Serious illnesses

due to a number of extracellular and intracellular pathogens are major health

problems in developing countries like Pakistan. Septicemia, toxic shock

syndrome and various skin infections due to Staphylococcus aureus, diarrheal

diseases due to Escherichia coli, gastroenteritis and invasive Salmonelloses due

to Salmonella Typhi and Tuberculosis due to Mycobacterium tuberculosis are

top health hazards of our country. According to WHO estimate diarrheal

diseases constitute 27% of total infectious diseases in Eastern Mediterranean

Regional (EMRO) countries (296), whereas, 80% of total typhoid cases were

occurred in Indian-sub continent during 1996-2005 (30). On the other hand

south-east Asian region has been declared as most vulnerable for new TB cases

in 2005 with 34% of the global incidents in that year (45). In addition with these

on-going challenges, our country also faces the emergence of new pathogens

and re-emergence of old enemies. Situation become worsen after the emergence

of antibiotics resistance among these pathogens.

Use of antibiotics was considered as golden strategy to combat infections,

however, in this era microbes are challenging us in ways we would not have

imagined 20 years ago. Natural genetic variation, recombination, adaptations,

transfer of resistant genes (R-determinant) from one strain to others via

plasmids, transposons and integrons and a number of unlying bacterial

mechanims like absence or alteration in drug target, enzymatic inactivation of

drug and over expression of efflux pump proteins are major elements of drug

resistance. Especially, Efflux pump genes present in all organisms, regardless of

their susceptibility pattern, become induced by a variety of substrates that

increase their expression. Over-expression of efflux proteins results in the

decrease of intracellular concentration of the substrate antibiotic and makes the

organism resistant against multiple drugs (125). Several other factors like

poverty, unsafe health practices, over-crowding, lack of education, misuse and

over the counter availability of antibiotics also contribute to make the wonder

weapons useless. A number of multidrug resistant (MDR) extracellular and

intracellular pathogens are increasingly observed in normal community and/ or

hospital settings including Methicillin Resistant Staphylococcus aureus, MDR

Salmonella Typhi, MDR and XDR-Mycobacterium tuberculosis and drug

resistant Escherichia coli that make formerly easy-treatable infections severe

life threats. Therefore, it is important to look for new treatment options.

In order to combat MDR organisms, many approaches are in consideration by

scientific community. Some believe on the preventive approach thus working

on vaccines, some want to discover novel drug targets whereas others to restore

the activity of old antibiotics by using a synergistic combination approach. For

intracellular organisms, search of immunostimulating compounds is also on

rise. In our opinion, although vaccines have their own importance in disease

prevention but still we need to have alternatives for treatment of MDR

infections. Either we talk about novel class of antimicrobial drugs or synergistic

antimicrobial combinations; we need to look for new antimicrobial compounds

from a source known to be rich in variety e.g. plants.

Plants have long been used as an alternative regime in traditional medicine all

over the world. Use of herbal medicines is becoming famous day by day due to

better activity, less side effects and common availability. Broad profile of

bioactive compounds associated with common herbs makes them a possible

alternatives of old and useless antibiotics. In recent years, after realizing the

popularity of herbal medicine in different regions of the world, for instance, in

AFRO and WPRO member countries as first line therapy and in others as

complementary or alternative medicine, WHO recommended member countries

to set up strategy for the use traditional medicines. They urge policy-makers and

health professionals to set guidelines and policies addressing the issues of

safety, efficacy, quality, access and rational use of local herbs (297).

In the view of present scenario of high infection rate, emergence of drug

resistance among intracellular and extracellular pathogens in our country, we

decided to explore alternative antimicrobials from three indigenous plants i.e.

Camellia sinensis (Green Tea), Juglans regia (Dandasa) and Hippophae

rhamnoides (Sea buckthorn). The rationale behind the choice was their wide

consumption as food and cosmetic product, common use in traditional medicine

without any significant report of toxicity and easily availability. Moreover,

promising preliminary data provided us basis to carry out detailed study. A

total of 377 clinical and 11 reference isolates of different intracellular and

extracellular bacterial pathogens were included in this study. After

identification, characterization and determination of antibiotic resistance

pattern, they were screened for antimicrobial susceptibility against indigenous

plants. In-vivo antimicrobial activity, mechanism of antimicrobial action and

immunopharmacological studies were also carried out.

1. Plant Antimicrobials and Extracellular Pathogens

A variety of gram positive and gram negative extracellular bacterial pathogens

were tested for antimicrobial susceptibility patterns against aqueous and organic

plant extracts and pure compounds alone and in combination with antibiotics

formerly used to treat such infections. Susceptibility data from our study

demonstrated a clear shift of antimicrobial activity towards gram positive

pathogens than gram negatives, likewise other studies on phytocompounds. A

four-fold difference in MICs was observed in case of aqueous extract of

Camellia sinensis i.e. MIC against Staphylococcus aureus ranged from 0.19-

0.78 mg/ml and against different genotypes of Escherichia coli ranged from

1.56-3.12mg/ml. Similar was the case of Juglans regia (MIC = 0.31-

1.25mg/ml) for Staphylococcus aureus and MIC = 2.5-5mg/ml for

Enteriobacteriaceae. Same trend was observed for FA-CS II, who showed a

two-fold lower MIC level against gram positive extracellular bacteria than gram

negatives.

It was interesting to note that both plants exhibited better activity against MDR

pathogens like MRSA as compared to those sensitive to common antibiotics. A

number of previous studies reported the antimicrobial potential of Camellia

sinensis (190, 193) and Juglans regia (225, 226, and 231). Some workers

observed antimicrobial effect of Camellia sinensis on MRSA only after the

combination with β-lactum antibiotics (192, 194). Unlike these studies, our data

suggest cidal nature of Camellia sinensis alone, as shown by MIC/MBC 0.5.

We observed a clear difference in MIC level of Camellia sinensis i.e.

0.19mg/ml for MRSA (n = 99) and 0.78mg/ml for clinical strains of MSSA (n=

59) and a reference strain. This was confirmed by time kill kinetics which

demonstrated inhibition of organisms at concentrations MIC and above. Indeed,

concentration dependent manner of antimicrobial action was also worthwhile to

make this conclusion. Our results are similar to previous studies carried out on

antibiotics and plant origin compounds (298). Drastic effect on cell morphology

of MRSA i.e. presence of thick intercellular masses, as observed by

transmission electron microscopy further confirmed our findings and

demonstrated the interference in bacterial cell walls by Camellia sinensis. We

further confirmed antimicrobial activity in-vivo. The organ load reduction was

observed in murine model of disseminated septicemia. Study outcome

confirmed the capability of Camellia sinensis to treat systemic MRSA

infections. A single dose of 156mg/kg was able to reduce > 2 log10 in bacterial

load in different body organs. This is first time report of in-vivo efficacy of

green tea against MRSA, though, there had been a report about in-vivo

antibacterial effect against intestinal pathogens (190). The activity of new pure

compound, FA-CS II with MIC 125µg/ml and MIC/MBC 0.5 against MRSA

was comparable to epigallocatechin gallate(EGCG)-the main tea catechin

activity (MIC 100µg/ml), however, EGCG was found to be antagonistic with

commonly used anti MRSA drugs i.e. vancomycin and teicoplanin (299).

In previous studies (300, 190), there was conflict about the antimicrobial

activity of green tea against extracellular gram negative rods. Some authors

reported MIC of crude extract against EPEC as high as 88.30 mg/ml (300)

whereas others showed activity as low as 30µg/ml (190). It is a matter of fact

that at high concentration any compound or the extract exhibited antimicrobial

activity therefore, it is important to set a criterion of susceptibility. In our point

of view, as shown in other studies also, crude extracts usually exhibited higher

MIC level due to presence of trace amount of bioactive component, although

the level should be much lower than the dose showed toxicity to mammalian

cells. In our study Camellia sinensis extract showed antibacterial activity at

MIC 3.12mg/ml against a wide range of gram negative extracellular pathogens

including different genotypes of Escherichia coli. Furthermore, unlike gram

positive strains, extract behaved bacteriostatic in a concentration dependent

manner against most of gram negatives except ETEC.

It is not always important to kill an organism completely. Suppression of

bacterial virulence is another way to interrupt establishment of infection.

Therefore, a number of antibiotics found effective clinically irrespective of their

cidal or static behavior. There had been many reports about the effect of

macrolides and fluoroquinolones on virulence factors of extracellular pathogens

(301, 302). Static but promising effect of Camellia sinensis on gram negative

rods especially ETEC and on MRSA at sub-inhibitory concentrations drew our

attention to check the status of bacterial virulence factors under stressed

conditions caused by green tea. Collectively, the approach provides an easy way

to carry out a proteome based preliminary mechanistic studies. In case of

MRSA, green tea at sub-inhibitory concentrations showed a dramatic inhibition

of high molecular weight exoproteins. According to the molecular sizes,

likewise other studies (303), down regulated bands may be speculated as

autolysin (97kda), α-hemolysin (33kda), lipase/ glycerol ester hydrolase (90

kda) and Protein A (60kda)-a cell wall associated surface protein. Inhibition of

145 and 97kda proteins bands indicates the absence of bifunctional autolysin

(145kda), autolysin (97kda) from the bacterial cells treated with green tea.

Although, our data is not sufficient to give final conclusion but it clearly

indicates the inhibition of surface expressed proteins rather than exoproteins

that supports our observations of electron microscopy as shown in Fig # 53A.

In contrast with MRSA, Camellia sinensis exhibited inhibition of most of the

exoproteins whereas a dose dependent effect was observed on cell associated

protein profile of ETEC. Complete inhibition of secretary products including

LT and ST at 2 x MIC of green tea, theoretically exerts severe effects in the

disease establishment process of ETEC. Persistence of 37kda protein,

presumably ompA- a major ETEC outer membrane protein, in the supernatant

of ETEC after the treatment with graded concentrations of green tea was

observed (Fig # 54A ). Outer membrane protein A (OmpA) is a porin that plays

a vital role in the structural integrity of the organism and is known to be

survived under stressed condition like sodium dodecyl sulfate (SDS), cholate,

acidic environment, high osmolarity, and pooled human serum (304). On the

basis of pervious investigations and our current results, we can put forward a

hypothesis that green tea may inhibit the virulence factors of gram negative

bacteria completely, leaving the organism alive.

Unlike previous studies where Juglans regia bark was reported to have broad

spectrum antimicrobial activity (225), we found it antimicrobial in nature only

against gram positive organisms. Although, it showed inhibition of ETEC at

MIC 2.5mg/ml but the bacteria appeared to be in long static phase and after

sometime started multiplying again. For gram positive organisms, there are few

reports stated about antimicrobial activity of this plant against Staphylococcus

aureus, Streptococcus mutans, Streptococcus salivarius, Lactobacillus casei and

Actinomyces viscosus (225, 226) but none of them claimed about antimicrobial

activity against MDR organisms like MRSA. In this study, likewise Camellia

sinensis, lower MIC level aqueous extract (0.31mg/ml), n-hexane fraction

(32µg/ml) and sub-fraction PP 1 (25µg/ml) of Juglans regia against MRSA

were observed. Juglans regia exhibited bactericidal activity against MSSA at

higher concentrations (1.25- 2.5mg/ml).

It is already known that the cell wall in gram positive bacteria consists of multi

sheets of peptidoglycan that plays an essential role cell integrity and division

whereas comparatively thin cell wall of gram negative bacteria is overlaid by an

outermembrane mainly composed of LPS. Therefore, due to the less affinity

towards LPS, antibiotics specifically used against gram positive organisms like

β-lactams and glycopeptides usually target bacterial peptidoglycan. Keeping

this in mind, we checked the effect of Juglans regia on bacterial cell wall and

structural proteins. Significant changes in bacterial cell morphology of MRSA

including the presence of unknown material on cell surface, swollen, de-shaped

and completely hollow bacterial cells, interpreted as structural defect by other

authors (305-307),as well as inhibition of cell associated/ structural proteins of

MRSA strains, observed by SDS-PAGE permit us to hypothesize the presence

of anti-staphylococcal component in this plant targeting bacterial cell wall.

Previously, a number of studies reported the synergistic activity of methicillin

with either inhibitors of cell wall synthesis or (8, 9) or cell membrane

modulators (10), therefore, we attempted to see interaction of Juglans regia

with oxacillin. Our hypothesis is further supported by strong ability of Juglans

regia to react synergistically with oxacillin, a cell wall inhibitor (FICI 0.193),

we observed by a variety of in-vitro studies. The combination not only restores

the activity of oxacillin against MRSA but also made MSSA more susceptible.

Synergism against both strains indicates no direct effect of Juglans regia on

PBP2´, which is specific for MRSA. It is important to identify nature of

bioactive component(s) present in Juglans regia, their interaction with other

inhibitors of peptidoglycan synthesis to better understand mechanism of

synergy.

Extracellular bacteria are not only limited to human diseases. Their firm

association with animal health hazards is the area of growing concern. In our

country, frequent outbreaks of Hemorrhagic septicemia among large ruminants

is one of the major economic losses caused by Pasteurella maltocida, an

extracellular pathogen. despite of the prophylactic use of antibiotics in animal

feed, the disease resulting in 9% mortality and 78% case fatality rates, thus

contributes significantly in lowering down livestock and diary industry in

Pakistan (40). Although, antibiotic susceptibility rate of Pasteurella multocida

is very impressive,unfortunately available antibiotics are sub-standard and

costly. Therefore, it is important to search more effective and economical

alternatives from natural resources. Our studies on Hippophae rhamnoides (Sea

buckthorn berries) revealed presence of an effective alternative from our natural

resources. The plant, abundantly found in Northern Areas of Pakistan and used

as major ingredient of jam, jelly and juices showed strong antimicrobial activity

(MIC 50µg/ml and MBC 100µg/ml) against virulent clinical strains of

Pasteurella multocida serotype B2. In-vitro findings were further confirmed by

in-vivo experiments where single dose of 100mg/kg Hippophae rhamnoides

dramatically protected the mice infected with 106 CFU (104 x LD50). Treatment

with 80mg/ kg of extract results an effect on multiple organ dislocation of

virulent strain 24 hours of onset of symptoms whereas an early eradication of

organisms was observed at the site of infection. Previous studies on several

other varieties of Sea buckthorn berries e.g. Finnish and Indian, showed

antimicrobial activity only against commensale bacteria (311, 312). Our results

strongly suggest the possible use of Hippophae rhamnoides as prophylactic

supplement in animal feed. Since it is easily growing crop in northern areas of

Pakistan, we can suggest its cultivation more in grazing lands of that region so

that animals can consume it naturally as food, if antimicrobial activity is not

restricted to berries.

2. Plant Antimicrobials and Intracellular Pathogens

Intracellular bacteria are always considered as more tricky bugs than

extracellular pathogens. Most of the antibiotics that are best known for their

efficacies appear to be useless against serious intracellular bacterial pathogens,

sometimes due to their inability to pass through host cell membrane like

aminoglycosides or sometimes due to their inability to survive in the harsh

environment of host cell where pathogen persists (313). Among intracellular

bacterial pathogens, Mycobacterium species are on the top of serious threats. In

the scenario where no TB drug has been introduced since 30 years, emergence

of MDR and XDR TB cases are unavoidable concerns. The situation provides

the rationale to search for new antimycobacterial drugs. A number of attempts

have been made to look for novel anti-TB agent from natural products (133, 135

and 137). Our efforts were in the same direction. Our results of

antimycobacterial testing showed these plants as very promising

antimycobacterial candidates. Methanolic extracts of Green Tea showed better

activity against XDR TB strain (1.25 mg/ml) than MDR TB (MIC 2.5 mg/ml)

whereas methanolic extract of Hippophae rhamnoides (Sea buckthorn) was

found to be inhibitory against reference and clinical MDR and XDR strains at

0.75mg/ml. to the best of our knowledge this is first report stated about the

anitmycobacterial potential of these plants.

In case of Juglans regia, methanolic extract inhibited all species of

Mycobacterium tested whereas aqueous extract have no activity against M.

bovis and M. avium. MIC of methanolic extract against reference and MDR,

XDR clinical strains of M. tuberculosis was found to be 0.75mg/ml. Although,

we did not check other fractions of Juglans regia but recently, a Mexican group

reported presence of anti-tuberculosis activity of hexane extract of Juglans

regia bark. But their report did not indicate any bioactivity against MDR strains

(234). In developing countries like Pakistan where TB burden is exceptionally

high and people can not afford costly treatment regime, our preliminary data

indicates the potential of these indigenous plants to serve as source of affordable

antimycobacterial drugs.

Salmonella enterica serovar Typhi (S. typhi), a causative agent of typhoid fever,

is another facultative intracellular pathogen. Their entry and survival in non-

phagocytic cells is considered as an essential factor for their pathogenicity. High

frequencies of drug resistance and disease severity enlist us among the list of

countries with highest mortality rate (38, 314) and left us with very limited

choices of treatment. Realizing the situation, we carried out genotypic

characterization of S. Typhi and S. Paratyphi A to elucidate the genetic basis of

multi-drug resistance. Our observations are in agreement of previous studies

(32) stated the presence of 98.6 mega-dalton, self-transmissible R-plasmid

belongs to the H1 incompatibility group in MDR isolates. Class 1 integron with

3´ conserved segments (3´-CS) containing drug-resistance cassettes was also

found to be associated with MDR S. Typhi. Previous studies demonstrated

significant homogeneity among MDR Salmonella isolates from various regions

of Pakistan i.e. two PFGE patterns in case of MDR S. Typhi (32) and single

pattern in case of S. Paratyphi A (315) were observed. In contrast with previous

reports, we found significant genetic diversity (11 PFGE patterns) among all

isolates in general and especially among those sensitive to first line drugs that

suggest the circulation of multiple clones of Salmonellae in Pakistan.

In order to find out alternate treatment regime, we tested natural plant extracts

against genetically diverse variety of Salmonellae. Among all Green Tea was

found to be most promising candidate alone (MIC 1.56mg/ml) and in

combination with nalidixic acid (FICI 0.37) against MDR Salmonellae (R-type:

AmpCSxtTNA). Different MICs of green tea extract against Salmonella have

been reported in previous studies (190, 300). In contrast with previous findings,

we observed stronger synergistic activity with Nalidixic acid and in different

effect of green tea extract with chloramphenicol and tetracycline. Difference in

anti-Salmonella activity of green tea can be explained due to difference in test

bacterial strain, variety of tea and method of extraction. Significance of our data

lie in anti-Salmonella activity of our local tea with lower MIC levels against a

genotypically diverse group of MDR + fluoroquinolones resistant strains.

Presence of different target site in bacteria can be the simplest explanation of

the mechanism of synergy between green tea and Nalidixic acid that opens the

doors of detailed studies to understand real mechanism.

3. Immunomodulation and Plants

In the eradication of extracellular and intracellular pathogens, host immune

response plays a pivotal role. It is a fact that most of the antibiotics have ability

to stimulate immune cell functions, thus act as dual-edged sword to eradicate

infections (155, 316). Therefore, it is always desired by a microbiologist to

evaluate the effect of new antimicrobial sources on host immune system. A

portion of our study was focused on the evaluation of plant and plant derived

substances, found to be antimicrobial in this study, on various aspects of host

immune response like phagocytosis. This is the key mechanism of non-specific

immune response presented by the host to combat variety of foreign agents. Due

to impairment in phagocytosis, not only intracellular bacteria but many

extracellular bacteria have a chance to survive inside the cell. Organisms like

MRSA are known to survive in the acidic environment of phagolysosome (317),

thus capable of hiding from a number of antibiotics that cannot enter in

macrophages and PMNLs. Our present data revealed the significant dose

dependent effect of Camellia sinensis and FA-CS II on intracellular growth of

MRSA in human PMNLs. Especially FA-CS II also found to be successful to

stop the growth of intracellular MRSA at concentrations less than MIC. Our

results are in agreement of recent report by Kohda et al that stated the effect of

tea catechin on intracellular growth of Listeria monocytogenes (207). A decline

in intracellular growth of pathogen indicates the ability of green tea and FA-CS

II to accumulate and survive in the vulnerable environment of phagocytic cell.

Also, intracellular antibiotic activity of FA-CS II below MIC indicates their

ability to accumulate in the cell irrespective of extracellular concentration.

These results, taken together with observations of extracellular antimicrobial

response, demonstrate the comprehensive view of therapeutic potential of green

tea and FA-CS II.

Activation of antibody producing B-lymphocytes by plants and plant derived

substances is another useful approach to eradicate infections caused by

extracellular bacteria. A number of studies about this quality of natural plants

have already been carried out (166, 168 and 169). To evaluate the effect of

plants and plant derived compounds antibody producing spleen cells, we used

hemolytic plaque assay, a widely used method for these studies (318). Among

the plants tested, Camellia sinensis was found to be most promising. A two-fold

increase in PFCs and eight-fold increase in antibody titer of the animals primed

with multiple doses of Camellia sinensis as compared to control animals

indicated the presence of immunostimulatory component(s) in green tea. It was

surprising to note that FA-CS II, that was able to increase antibody producing

cells, was unable to exert significant effect on antibody production. Change in

antibody production and number of antibody producing cells indicate the

possible role of green tea to enhance B cell differentiation. We may suggest

further evaluation of green tea response on cytokines responsible for B cell

differentiation like IL-4.

A number of infectious diseases like gastric ulcers, tuberculosis, ulcerative

colitis and pertussis are associated with inflammatory process that exert adverse

effect on host cells that is no longer repairable. Increased formation of Reactive

Oxygen Species (ROS) during inflammatory processes leads the host cell

towards oxidative cell death (171). Therefore, to prevent host cell death, it is

important to scavenge excess amount of ROS. Anti-oxidants have the capability

to terminate the series of oxidation reaction occurred inside the cell during

inflammation, thus prevent cell death. In order to assess anti-oxidant potential

of our plants, we performed 2', 7'-dichlorofluorescin {DCF} assay on Human

Endothelial cells (ECV304) to see free radical scavenging activity followed by

their effect on host cell DNA by [3H] thymidine incorporation method. A

concentration dependent free radical scavenging activity of Hippophae

rhamnoides with no adverse effect on thymidine incorporation was observed. In

addition, an increase in DNA synthesis was observed in case of cells exposed to

1.25mg/ml of Sea buckthorn extract which indicates that the plant led to an

improvement in normal cell survival thus prevent them from any future damage.

However, data is already available about the anti-oxidant property of this plant

which suggest protective role only in case of chromium-induced damage on

lymphocytes (251). Unfortunately, due to the interference in fluorescence by the

color or nature of green tea and dandasa, we were unable to get reliable results.

4. Toxicity Studies of Plants

Before suggesting therapeutic potential, it is always important to know the

effect of candidate plant or compound on host body. Despite of the fact that

these plants are already very famous food or cosmetic items in Pakistan, toxicity

studies were carried out at three different levels to fulfill the basic criterion.

First of all, direct membrane toxic effect of plants was determined. RBCs are

considered as most fragile cells of the mammalian body therefore, we examined

the ability of Camellia sinensis, Juglans regia and FA-CS II to cause hemolysis

of human RBCs. Absence of any significant hemolytic activity in doses > 100

times higher than their therapeutic concentrations exclude the presence of any

component in the plants that is toxic for cell membrane e.g. melittin which

exerts adverse osmotic effects on RBCs and induces the release of membrane

permeability markers thus responsible of direct membrane toxicity (319). Since

these observations do not preclude the presence toxic effect on other

mammalian cell types, we examined the ability of plant extracts at cell culture

level. Effect on human vascular endothelial cell line (ECV304) was observed by

MTS assay. Our data revealed proliferative nature of Camellia sinensis, Juglans

regia and Hippophae rhamnoides for mammalian cells rather than any toxic

effect. Proliferative potential of Hippophae rhamnoides was also confirmed by

their strong anti-oxidant nature. Non-toxic effect on ECV304 cells confirmed

the absence of toxic elements that exert adverse effect on basic cell functions

that are expressed in similar way in all cell types but it does not exclude the

presence of any toxicity on certain cellular mechanisms related to specific cell

types. Due to absence of complex intercellular environment in cell culture

system, one can not expect the behavior of test compound inside host body.

Sometimes toxic effects are produced in extracellular environment rather than

intracellular (320). The issue stresses the need of conducting animal toxicity

studies. We performed acute and subacute toxicity studies of aqueous extracts in

adult BALB/C mice. Intraperitoneal administration of graded doses as high as

1g/kg, single in case of acute toxicity and multiple in sub-acute toxicity, did not

claim any mortality in experimental animals. Although, the concept of LD50 is

only limited to the number of animals died due to toxic effect of drug, it is more

important to see immediate or delayed toxic effects of test extract/ compound on

other physical sign and symptoms, effect on target organs and their functions.

We therefore, closely observed experimental animals for considerably longer

period of time to notice change in their psychological behavior and physical

appearance. In addition, hematological parameters, liver and kidney function

profiles were checked at different time intervals. No significant change in either

case confirmed the non-toxic nature of Camellia sinensis, Juglans regia and

Hippophae rhamnoides.

5. Characterization of Plant Antimicrobials

As a part of our dissertation, we attempted to locate bioactive component(s)

present in plants by bio-assay guided chemical analysis. Although, conventional

bio-assay guided fractionation and purification is a widely used method in

phytomedicine (81), we undertook a novel combinatorial approach of

bioautography and MALDI-TOF-MS to characterize antimicrobial components

present in plants. In previous studies, extracts subjected to column

chromatography, Sephadex LH-20, thin-layer chromatography (TLC) etc were

analyzed by HPLC and HPLC/LC-MS (321) but in our study, due to ability of

method to analyze complex samples, direct crude extracts and bioactive spots

on TLC plate, located by bioautography, were subjected to MALDI-TOF-MS.

Spectrum achieved in either case can be directly linked with antimicrobial

activity of plant to its components. Analysis of crude extracts of plants by

MALDI-TOF is increasingly famous (90, 91 and 322), however, to the best of

our knowledge, this is first time when this approach is used in combination with

bioautography to locate antimicrobial components in plants.

Bioactive spots of all three plants, showed antimicrobial activity against MDR

Salmonella Typhi, separated by solvent system containing CHCl3: Ethyl acetate:

MeOH (50:40:10), was composed of several low molecular weight peaks under

positive ionization mode for example, molecular masses 416, 438, 854 and 861

with high intensity were observed in Camellia sinensis. Peaks with m/z 416,

444, 655, 860, 861, 862 were found with high intensity signals in all bioactive

spots of Juglans regia. Masses 416, 440 were on height in Hippophae

rhamnoides. Without structure analysis and detailed study on comparison of

relative ionization abilities, it is not possible to give any quantitative data;

however signal intensity can be generally co-related to the amount of

components (322).

Extensive work has been done on the phytochemistry of Chinese Green Tea,

however very less is known about Pakistani variety therefore, in addition with

the above mentioned approach, conventional approach of bio-assay guided

fractionation and NMR spectra was also undertaken for the analysis of Camellia

sinensis. We were successful to isolate a new purine class alkaloid, FA CS-II

from aqueous crude extract of Pakistani Green Tea, found to be antimicrobial

against wide range of pathogens. Presence of antimicrobial compound other

than catechins opens new insights about contributing factor of antimicrobial

activity in Pakistani variety. Even in case of Chinese variety, there is confusion

about actual contributing compounds of antimicrobial activity. Difference lies in

the degree of antimicrobial activity of crude extract, organic fraction and

purified catechins. Some groups preferred the use of crude extracts (198, 323

and 324) whereas some are restricted to catechins (int326- Stapleton-IJAA-

2004) that indicates the possibility of the presence of minor components with

more antimicrobial potential or their synergistic contribution with major

catechins to exhibit an antimicrobial effect (321).

Conclusions

A number of life threatening infections due to various multidrug resistant

bacterial pathogens are major cause of death in Pakistan. In Pakistan, frequently

isolated serious pathogens include MRSA, MDR Salmonella, MDR and XDR

Mycobacterium tuberculosis. The situation necessitates the need of more

effective, safer and less toxic alternate treatment options from natural resources

like plants.

• Three different indigenous plants including Camellia sinensis

(Green Tea), Juglans regia (Dandasa) and Hippophae

rhamnoides (Sea buckthorn berries) were selected from Pakistani

herbal flora due to their common availability and wide human

consumption.

• Plants were subjected to bio-assay guided chemical analysis and

compound purification using modern techniques like novel

combinatorial approach of bioautography and MALDI-TOF-MS.

• A total of 12 low molecular weight compounds with molecular

size ranged from m/z 400-800 were observed from plants. In

addition, we were successful to isolate a new purine class

alkaloid, FA CS-II from Camellia sinensis of Pakistani origin.

• Plants and plant derived substances were screened for

antimicrobial properties against 377 clinical and 11 reference

isolates of different intracellular and extracellular bacterial

pathogens.

• Camellia sinensis was found to be bactericidal in nature against

gram positive organisms including MRSA by in vitro and in vivo

studies. Camellia sinensis and FA-CS II also inhibited

intracellular growth of MRSA at below MIC levels using human

polymorphonuclear leucocytes test system.

• Bacteriostatic effect was observed against gram negatives like

ETEC, however Green Tea was able to change protein profile of

• Camellia sinensis was also found to be most promising candidate

against genetically diverse variety of Salmonella enterica serovar

Typhi with MIC 1.56mg/ml.

• A novel synergistic antimicrobial combination of green tea with

nalidixic acid was formulated against Nalidixic acid resistant and

MDR Salmonellae (FICI 0.37).

• FA-CS II was found to be highly antimicrobial in nature against

MDR pathogens for example its activity against MRSA (MIC

125µg/ml) was comparable to other tea catechins.

• Organic and aqueous extracts of Juglans regia were found to be

more against MRSA with MIC ranged from 25-312 µg/ml.

antimicrobial activity was further confirmed by damaged cell

morphology which indicates the presence of cell wall inhibiting

anti-staphylococcal component(s) in the plant.

• Another novel synergistic combination was formulated using

Juglans regia extract and oxacillin against various clinical strains

of MRSA (FICI 0.193).

• In general Camellia sinensis and Jugalns regia extracts were

more active against multidrug resistant pathogens as compared to

their respective sensitive strains.

• Hippophae rhamnoides revealed most promising activity (MIC

50µg/ml and MBC 100µg/ml) against Pasteurella maltocida

serotype B2, isolated from water buffloes with Hemorrhagic

septicemia (HS) infection. Antimicrobial activity was further

confirmed by in vivo mouse model of hemorrhagic septicem.

This observation strongly suggested the possible use of

Hippophae rhamnoides as prophylactic supplement in animal

feed to prevent HS among large ruminants, a major economic

loss of livestock and dairy industry.

• All three plants exhibited antimicrobial acitivity against

reference and clinical strains of Mycobacterium tuberculosis and

MOTT. However, antimycobacterial activity was better against

Mycobacterium tuberculosis (MIC 0.75mg/ml) than MOTT.

Methanolic plant extracts exerts better antimycobacterial effects

than their respective aqueous extract.

• All three plants exhibited antimycobacterial activity against

clinical strains of MDR and XDR Mycobacterium tuberculosis

(MIC 1.25-2.5mg/ml).

• Camellia sinensis and FA-CS II were found to be stimulatory for

humoral immune response.

• Strong anti-oxidant activity was observed in Hippophae

rhamnoides.

• Aqueous and organic extracts of the plants tested were found to

be non-toxic in nature by hemolytic, cytotoxic and animal

toxicity studies.

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Appendix

Acrylamide bis acrylamide (30:0.8): Acrylamide 30mg Bis-acrylamide 0.8mg Filter the solution (store at 4’C in dark bottle)

Agarose solution (for Hemolytic Plaque Assay) Agarose 1g Hank’s Balance Salt Solution 100ml

Alsever’s Solution Glucose 2.05g Sodium Citrate 0.8g Citric acid 0.055g NaCl 0.42g D/W 100ml Filter with membrane filter 0.2µm

1% Ammonium per sulfate (SDS): APS 0.05gm Distilled water 5ml (prepare fresh before use) (N,N,N’,N’- tetramethylethylenediamine TEMED is used as supplied) Blotting buffer 0.025 M Tris-HCl/20% (v/v) methanol, pH 8.3), 50mM Tris-HCl / 0.196M glycine pH 8.3) with 20% methanol

CLysis B Tris 50mM EDTA 50mM Sarkosyl 1% Adjust pH 8.0 with HCl and autoclave

CSB Tris 100mM EDTA 100mM Adjust pH 8.0 with HCl and autoclave Destaining Solution Acetic acid 100ml Distilled water 900ml (store at room temperature) Formalized Saline Formaline Solution 3ml NaCl 8.5 g D/W 1000ml

Hank’s Balance Salt Solution CaCl2 0.14g NaCl 8g KCl 0.4g 0.8mM MgSO4.7H2O 0.2g 0.4mM K2H2PO4.6H2O 0.2g 1.4mM Na2HPO4 0.24g Glucose 1g D/W 1 Lit Filter with membrane 0.2µm

Lysis Buffer 1M Tris 1.25 ml 10% SDS 7.5ml 2M NaOH 1.025ml make up the volume upto 25 ml with distilled water PBS Plus 1 X PBS 200 ml CaCl2 0.5mM MgCl2 1mM Glucose 1.08g

PMNLs Separating Solution 0.85% NH4Cl 0.08% NaHCO3 (Mix them with the ratio of 1:1)

Reservoir BufferESERVOIR BUFFER: (pH 8.3) Tris 3.025gm Glycine 14.4gm SDS 1mg Distilled water 500ml (make up to 1 litre.store at 4’C) Restriction Mix (100µl) MiliQ wter 87µl 10 x restriction buffer 10µl BSA 1µl Sample Diluting Buffer (pH:6.8) HCL(0.5M) 12.5ml SDS 2gm 2-mercaptoethanol 5ml Glecerol 10ml (make up to 100ml.store at 4’C) 1% Sodium Dodecl Sulfate (SDS): SDS 1mg (make up to 100ml with distilled water) (store at room temperature)

Staining Solution Comasse Blue 0.5gm Acetic acid 18.75ml Methanol 12.5ml (make up to 250ml with distilled water.filter it and store at room temperature)

Suspending Buffer 1M Tris 1.25 ml 0.5M EDTA 50µl make up the volume upto 25 ml with distilled water TBE 5 X Tris Base 54g Boric Acid 27.5g 0.5mM EDTA 20ml pH should be 8.0

TBS Tween20 10 mM Tris-Cl 150 mM NaCl containing 0.05% Tween-20

TE Tris 10Mm EDTA 1mM Adjust pH 8.0 with HCl and autoclave

3.0 M Tris-HCL (pH 8.8): Tris 36.3gm HCL 48ml (adjust pH with 0.1M HCL) (makeups volume up to 100ml with distill water) (store at 4’C) 0.5M Tris- HCL(pH 6.8): Tris 6.05gm Distilled water 40ml (pH to 6.8 with 1M HCL.) (Volume upto 100ml store at 4’C) Tracking Dye Diluting buffer 5ml (dissolve few crystals of bromophenol blue)

Trypan Blue Trypan Blue Powder 4g D/W 10ml Filter by Watman’s filter paper # 1 before use

17. Veronal Buffer (5X) NaCl 42.5g Sodium Barbitone 1.87g Barbituric acid 2.87g

Make up the volume upto 1 lit.

Abbreviations

AIDs Acquired Immunodeficiency Syndrome

Amp Amplicillin

BHS Beta hemolytic Streptococci

C Chloramphenicol

CA-MRSA Community acquired Methicillin Resistant Staph. Aureus

CLSI Clinical and Laboratory Standards Institute

CNS Coagulase Negative Staphylococci

Co Co-trimoxazole

COPD Chronic obstructive pulmonary disease

DCFH-DA 2', 7'-dichlorofluorescin diacetate

dNTPs Deoxynucleoside triphosphates

EC Epicatechin

ECG Epicatechin gallate

EGC Epigallocatechin

EGCG Epigallocatechin gallate

EPIs Efflux Pump Inhibitors

ESBL Extended Spectrum Beta Lactamases

FBS Fetal Bovine Serum

FDA Food and Drug Administration

FIC Fractional Inhibitory Concentration

FICI Fractional Inhibitory Concentration Index

HIV Human Immunodeficiency Virus

HTS High throughput Screening

hVISA Heterogeneous VISA

LB Luria Bertani

MALDI-TOF-MS Matrix assisted Laser Desorption/ Ionization-Time-of-Flight

mass spectrometry

MBC Minimum Bactericidal Concentration

MDR-TB Multidrug resistant Tuberculosis

MHA Muller Hinton Agar

MHA Muller Hinton Broth

MIC Minimum Inhibitory Concentration

MJ Methyl jasmonate

MRSA Methicillin Resistant Staphyloccus aureus

NA Nalidixic acid

NH4OH Ammonium Hydroxide

PBS Phosphate Buffered Saline

PMNLs Polymorphonuclear leucocytes

RBCs Red Blood Cells

ROS Reactive Oxygen Species

R-type Resistant type

SRBCs Sheep Red Blood Cells

SXM Sulfonamide

T Tetracycline

TB Tuberculosis

TBE Tris borate EDTA

TCA Trifluroacetic acid

TE Tris EDTA

TFA Trifluoro acetic acid

TLC Thin layer Chromatography

TTO Tea Tree Oil

UV Ultra Violet

VISA Vancomycin-intermediate S. aureus

VRSA Vancomycin-resistant S. aureus

WHO World Health Organization

XDR-TB Extremely drug resistant Tuberculosis

studies on the antimicrobial and immunomodulating

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