final week report (lfs1827)
TRANSCRIPT
Indian Academy of Sciences, Bangalore Indian
National Academy of Sciences, New Delhi
The National Academy of Sciences India, Allahabad
SUMMER RESEARCH FELLOWSHIPS — 2015
Format for the final Report*
Name of the candidate : SAI PREETHI NAKKINA
Application Registration no. : LFS1827
Date of joining : 27-05-2015
Date of completion : 22-07-2015
Total no. of days worked : 57
Name of the guide : Prof. ANUPAM DIKSHIT
Guide’s institution : UNIVERSITY OF ALLAHABAD
Project title : In Vitro Study of Antimicrobial and Antioxidant Activity of Some
Secondary Metabolites of Selected Plants
Address with pin code to which the certificate could be sent:
SAI KRUPA, #204, 9TH A MAIN, 1ST BLOCK, KALYAN NAGAR, BANASWADI, HRBR LAYOUT, BANGALORE- 560043
E-mail ID: [email protected]
Phone No: 080-25425327
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Signature of the candidate Signature of the guide
Date: 23-07-2015 Date: 23-07-2015 *The final report could be anywhere between 20 and 25 pages including tables, figures etc.
This format should be the first page of the report and should be stapled with the main report.
(For office use only; do not fill/tear)
Candidate’s name: Fellowship amount:
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Others A/c holder’s name: IMPORTANT NOTES: A soft copy of this report should be uploaded in the online page of our website by making use of the userid/password
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UNIVERSITY OF ALLAHABAD …: +91-532-2546200,(R), Tele fax: 2461887(O)(A Central University Under the Act of Parliament) 09453254221; 09335108519(Mobile)D E P A R T M E N T O F B O T A N Y Email: [email protected]
BIOLOGICAL PRODUCT LABORATORY Allahabad-211002, IndiaResidence: A-307, Sai Nilayam, Mehdauri Avas Vikas Colony, Teliarganj, Allahabad-211 002
Professor Anupam Dikshit Date: 22.07.2015M.Sc. D.Ph., Ph.D., FPSI, FBS, F.N.A.Sc.
Head, Department of BotanyCoordinator, Environmental SciencesMember- IPR Centre
TO WHOM IT MAY CONCERN
This is to certify that Ms. SAI PREETHI NAKKINA, B.Tech., BIOTECHNOLOGY,
5th Semester, student of PES Institute of Technology, Bengaluru, Karnataka, completed her
project work entitled, “IN VITRO STUDY OF ANTIMICROBIAL AND ANTIOXIDANT
ACTIVITY OF SOME SECONDARY METABOLITES OF SELECTED PLANTS” in the
Biological Product Laboratory, Department of Botany, University of Allahabad, Allahabad
(U.P.) during the period from 27th May 2015 to 21st July 2015 as Summer Research Fellow
(LFS1827) of Science Academies’. It is further certified that, the matter embodied in the report
has not been submitted by any one previously to the best of my knowledge. She is enthusiastic,
diligent and dedicated to her work. The candidate has potential and research aptitude. I wish her
all the success in her career.
(Anupam Dikshit)Supervisor
Dedication Page
I would like to dedicate the project to my beloved parents and sister.
Acknowledgments
Firstly, I would like to thank The Indian Sciences Academies’ for offering summer fellowships
and providing a wonderful opportunity for students to move ahead in their research career.
My sincere thanks goes to my guide, Prof. Anupam Dikshit (FNASc.), Biological Product
Laboratory, Head of Department, Department of Botany, University of Allahabad for selecting
me and providing his laboratory facilities to carry out my work. I am extremely grateful for his
guidance and support throughout my stay in Allahabad. This work would not have been possible
without his valuable guidance, support and endless encouragement.
I am thankful to Dr. Rohit Kumar Mishra, Research Scientist, for providing help in initiating my
work and his support in various ways.
My special thanks to Mr. Ashutosh Pathak, Research Scholar, Biological Product Laboratory,
Department of Botany, University of Allahabad, because of whom my project work has been one
that I will cherish forever. He supported and guided me in all stages of my work.
My heartiest thanks to all the research scholars of the Biological Product Laboratory, Dr. Anand
Pandey, Mr. Rajesh Kumar, Mr. Shashi Kant Shukla, Mrs. Madhu Pandey, Ms. Afifa Qidwai and
Mrs. Manisha Pandey for treating me well. Their support during my stay and inputs regarding
my work have been invaluable. I would also like to thank Mr. Raghuraj Pratap “Raghu Bhaiya”
for his kind co-operation and help during my work.
Words seem to be limiting to thank my parents Mr. Venkata R K Nakkina, Mrs. Lakshmi
Padmaja Nakkina, my dearest sister Sai Shruthi Nakkina, and all my family members for their
love and support without which this work would not have been possible.
I would also like to thank my relatives, friends and well wishers who have helped me directly
and indirectly in every step of the way.
Finally, I thank the Almighty God for helping surpass all the trials and helping me pursue this
study.
(Sai Preethi Nakkina)
LIST OF ABBREVIATIONS
ml -------------------- milliliter
cm -------------------- centimeter
mg -------------------- milligram
hrs -------------------- Hour
mg/ml -------------------- milligram per milliliter
MIC -------------------- Minimum Inhibitory Concentration
i.e. -------------------- id est; that is
⁰C -------------------- degree Celsius
appox. -------------------- Approximately
CFU -------------------- Colony Forming Unit
IC₅₀ -------------------- Half Maximal Inhibitory Concentration
µl -------------------- microliter
CLSI --------------------- Clinical and Laboratory Standards Institute
DPPH -------------------- Di-phenyl Picryl hydrazine
EC50 ---------------------- Concentration at which 50% of DPPH was used
Contents 1. INTRODUCTION ..................................................................................................................................... 1
1.1 Antimicrobial Assays .......................................................................................................................... 1
1.1.1 Microbes: Omnipotent in environment ........................................................................................ 1
1.1.2 Pathogens: Bacteria and Fungus ................................................................................................. 2
1.1.3 Antibiotics: Related concerns ...................................................................................................... 2
1.1.4 Complementary and Alternative sources: Plant Extracts ............................................................ 4
1.2 Antioxidant Assay ............................................................................................................................... 5
1.2.1 Free radicals ................................................................................................................................ 5
1.2.2 Antioxidants ................................................................................................................................. 6
2. REVIEW OF LITERATURE ........................................................................................................................ 7
2.1 Antimicrobial activity of Plant Extracts .............................................................................................. 7
2.2 Antioxidant activity of Plant Extracts ................................................................................................. 8
3. MATERIALS AND METHODS ................................................................................................................ 11
3.1 Place and Duration of Study ............................................................................................................. 11
3.2 Materials ........................................................................................................................................... 11
3.2.1 Culture Media Used: Name and Composition ........................................................................... 11
3.2.2 Chemical Agents ........................................................................................................................ 12
3.2.3 Microbial Strains ....................................................................................................................... 13
3.2.4 Laboratory Equipment ............................................................................................................... 13
3.2.5 Extracts/Compounds/oil ............................................................................................................. 14
3.3 Methods............................................................................................................................................. 14
3.3.1 Cleaning of Glass Equipment .................................................................................................... 14
3.3.2 Washing ...................................................................................................................................... 15
3.3.3 Sterilization ................................................................................................................................ 15
3.3.4 Antifungal assay ......................................................................................................................... 16
3.3.5 Antibacterial assay ..................................................................................................................... 17
3.4 Extraction of Trachyspermum ammi Oil ........................................................................................... 18
3.4.1 Determination of Scavenging Activity of Oils and Plant Extracts by DPPH Assay .................. 18
4. RESULTS AND DISCUSSION .................................................................................................................. 19
4.1 Antibacterial tests .............................................................................................................................. 19
4.1.1 Gentamicin ................................................................................................................................. 19
4.1.2 BPL-11 ...................................................................................................................................... 19
4.1.3 BPL-14 ...................................................................................................................................... 20
4.1.4 BPL-16 ...................................................................................................................................... 21
4.2 Antifungal tests ................................................................................................................................. 22
4.2.1 BPL-Ua ...................................................................................................................................... 22
4.2.2 Sertaconazole NitrateBP ............................................................................................................ 23
4.3 Percent Yield of Trachyspermum ammi essential oil ........................................................................ 24
4.4 Antioxidant tests ............................................................................................................................... 25
4.4.1 Vitamin C ................................................................................................................................... 25
4.4.2 Trachyspermum ammi ................................................................................................................ 25
4.4.3BPL-Ua ....................................................................................................................................... 26
4.4.4 BPL-Ma ..................................................................................................................................... 26
4.4.5 BPL-Pn ...................................................................................................................................... 27
5. CONCLUSION AND FUTURE PROSPECTS ............................................................................................. 28
6. BIBLIOGRAPHY .................................................................................................................................... 29
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1. INTRODUCTION
During the past decade, Plant products have been reported as to possess antibacterial,
antifungal, antiviral, insecticidal and antioxidant properties (Kordali et al., 2005; Sylvestre et al.,
2006). Utilization expanding from cancer treatment, food preservation, aroma therapy to
fragrance industries, they are a rich source of biologically active compounds (Faid et al., 1995;
Buttner et al., 1996; Van de Braak et al., 1999; Milhau et al., 1997). To evaluate newer plant
extracts for antimicrobial and antioxidant activity is reasonable having witnessed a large number
of reports regarding the same (Darokar et al., 1998; Martini et al., 1998).
1.1 Antimicrobial Assays
1.1.1 Microbes: Omnipotent in environment
Micro-organisms are living forms too small to be seen clearly with the naked eye and
lack highly differentiated cells and distinct tissues. They comprise of organisms living not only
in the ocean and soil but also human bodies and play important roles in the recycle of nutrients,
degradation of toxins and the maintenance of human health (Figure 1) (Okeke et al., 2005; Bryce
1992).
Figure 1: Cosmopolitan distribution of microbes.
They are plenteous natural sources of around 23000 unmatchable secondary metabolites
with biological activities, many commercialized. Firstly antibacterial, antifungals, antiprotozoal
as well as antivirals; secondly, pharmacological agents showing important activities as
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antitumor, immune modulators, neurological agents and enzyme inhibitors; thirdly, as agro
biological comprising insecticides, pesticides and herbicides and fourthly, compounds with
regulatory activities (Berdy, 2005; Duraipandiyan et al., 2010; Zotchev 2011; Finch 1998).
Hence, one can conclude that Microorganisms are closely associated with the health and welfare
of human beings, of which many are beneficial. However, many are detrimental (Dismukes,
2006; Gibbons, 2005).
1.1.2 Pathogens: Bacteria and Fungus
Human microbe interactions are beneficial as well as sometimes harmful to the human
health. The host–micro biota interactions being the beneficial ones pathogen-host interaction
results in host damage (Brodsky and Medzhitov, 2009). Specific virulence factors manipulating
host molecular activity is a result of host–pathogen interactions further causing responses from
the host, including the production of antibacterial factors by the mammalian innate immune
system (Diacovich and Gorvel, 2010; Hammer et al., 1999). Even after the extensive progress in
medical sciences, infectious diseases caused by bacteria, fungi, viruses and parasites are still a
leading cause of worldwide morbidity and mortality, causing approximately one-half of all
deaths and that of up to 3 million pre-school children each year (Farthing and Kelly, 2007;
Graser et al., 2000). They have their largest impact in the developing world due to relative
unavailability of medicines and the emergence of widespread drug resistance (Anonymous,
2011; Curtis, 1998).
Due to the genetic ability of microbes, they transmit resistance against present antibiotics.
Because of treatment using the same routinely used drugs, reports state that micro-organism have
become multi resistant to other medications available in the market. To tackle the same, in recent
year’s researcher and pharmaceutical companies have been motivated to develop new
antimicrobial (Sakagami and Kajimura, 2002; Thomashow et al., 1997).
1.1.3 Antibiotics: Related concerns
An antimicrobial agent is a compound that kills or inhibits the growth of microbes such
as bacteria and fungi (Li et al., 2008). Chemically, antibiotics are heterogeneous group of
organic, low-molecular weight compounds produced by microorganisms that are deleterious to
the growth or metabolic activities of other microorganisms (Kamali and Amir, 2010).
In the 20th Century, Antibiotics are undeniably one of the most important therapeutic
discoveries and with advancement in field of medicine, remarkable progress has been made with
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the discoveries of many natural and synthetic drugs. However, only one third of the infectious
diseases known have been treated from these synthetic products. Regrettably, widespread
indiscriminate use, incessant overuse, underuse and general misuse of antibiotics are major
factors in the emergence and dissemination of resistance (Westh et al., 2004; Moellering et al.,
2007; Maisnier-Patin and Andersson, 2004).
The concept of drug resistance is more complex than it seems. Microbial susceptibility is
a continuum that reflects phenotypic and genotypic variations in natural microbial populations
(Andersson and Hughes, 2010). Microbial resistance to antimicrobials may occur through the
emergence of pre-existing but previously unexpressed resistance phenotypes or through inherent
insusceptibility to antibiotics as a consequence of general adaptive processes. However, the most
commonly described form of bacterial resistance occurs either by genomic mutation or through
the acquisition of new genetic information encoding for resistance elements (Wright, 2005;
Gilbert et al., 2002; Simoes et al., 2008; Brehm-Stecher and Johnson, 2003; Kumar and
Schweizer, 2005). The major mechanisms of bacterial resistance to antimicrobials are
demonstrated in Figure 2 and include drug inactivation, target modification, alteration in the
accessibility to the target through drug efflux and decreased uptake (Wright, 2005; Simoes et al.,
2008; Dantas et al., 2008; Abreu et al., 2012; Jagessar et al. 2008).
Figure 2: Mechanisms of resistance to antimicrobials: active drug efflux systems from the cell via a
collection of membrane-associated pumping proteins that effectively remove toxic compounds from cells;
mutations resulting in altered cell permeability; enzymatic degradation of antimicrobials by the synthesis
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of modifying- or inactivating-enzymes that selectively target and destroy these compounds;
alteration/modification of the target site (Abreu et al., 2012).
As a result of the alarming rate of appearance and emergence of multidrug resistant
strains, especially by bacteria and fungi, treatment failures are still the major cause of morbidity
and mortality worldwide (Kuete, 2013; Goossens, 2005; Prashant et al., 2006). Treatment of
infectious diseases with antimicrobial agents continues to present problems not only as resistance
towards them but also toxicity and many common side effects (Iwu et al., 1999; Kunin, 1993;
Burt, 2004; Martini and Eloff, 1998), thus limiting the uses of conventional antimicrobial agents
due to their common side effects such as hepatotoxicity, nausea, diarrhea and impotency (Milhau
et al., 1997; Nascimento et al., 2000). The situation is alarming in developing as well as
developed countries. Therefore, alternative antimicrobial strategies are urgently needed, leading
to a re-evaluation of the therapeutic use of ancient remedies, such as plants (Mandal et al., 2010;
Basualdo et al., 2007).
1.1.4 Complementary and Alternative sources: Plant Extracts
Many reports reveal use of these plants by the local tribal people, from ancient time
(Subashkumar et al., 2013; Kunin, 1993). Traced as far back as the beginning of human
civilization, earliest being those found in “Rigveda”, written between 4500 - 1600 B.C, tells
Ayurveda, the foundation of medicinal science being purely based on plants as medicines
(Cowan, 1999). Plant extract, the richest resource of drugs of traditional systems of medicine,
modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical
intermediates and chemical entities for synthetic drugs has a potential application as natural
medicine and to treat diseases as well as the microbiological safety of the human health
(Gibbons, 2005; Burt, 2004; Rastogi and Mehrotra, 2002).
In Ayurveda and Homeopathic sciences plant extracts are used extensively, similarly they
are also used for extraction of pharmacological medicines (Murugesan et al., 2011;
Ahameethunisa and Hooper, 2010; Robbers et al., 1996). From about 250 to 500 thousand plant
species are estimated to exist on the planet, several are used for the treatment of various ailments
ranging from minor infections to dysentery, skin diseases, asthma, malaria and a horde of other
infections. However, this constitutes only 1 and 10% of the overall species opening ample scope
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to search for newer and better plants which are yet to be explored (Watve et al., 2001;
Nascimento et al., 2000).
Plants species have pharmacological properties as they possess various secondary
metabolites like glycosides, saponins, flavonoids, steroids, tannins, alkaloids, tirpenes which
have antimicrobial properties (Lalitha et al., 2010; Hussain et al., 2011; Enne et al., 2001).
According to World Health Organization’s report plants have long been used as the primary
source for human treatment by approximately 80% of the world population. These traditional
medicine practices of using extracts of different plant species demonstrates the presence of
antibacterial and antifungal agents (Anonymous, 1993; McGaw et al., 2000; McGaw et al., 2001;
Masoko et al., 2005; Masoko and Eloff, 2005; Casadevall and Pirofski, 2000; Schelz et al.,
2010). Also, the antimicrobial proprieties of various plant extracts against certain pathogen have
primary benefit of being relatively safer and reliable, good therapeutic benefit, and helpful in
overcoming the resistance problems besides affordable treatment (Papadoupoulo et al., 2005;
Elakkia and Venkatesalu, 2013; Sibaram et al., 2012; Finch, 1998).
1.2 Antioxidant Assay
1.2.1 Free radicals
In the recent decades there has been a surge of interest in disease prevention, particularly
the area pertaining to the role of oxygen-free radicals, commonly known as “reactive oxygen
species (ROS)” and “reactive nitrogen species (RNS)” (Halliwell and Gutteridge, 1999;
Devasagayam et al., 2004). A free radical is any molecular species that is capable of independent
existence and contains an unpaired electron in an atomic orbital (Halliwell and Gutteridge,
1989). Free radicals have very brief periods, with half-lives in milli-, micro- or nano seconds
(Devasagayam et al., 2004). Production of free radicals occurs as a part of normal cellular
function, in all cells (Young and Woodside, 2001). Production of free radicals also occurs as a
deleterious effect of irradiation by UV light, X-rays and by gamma-rays (Gilbert, 1981; Cadenas,
1989; Fang et al., 2002). ROS and RNS are well recognized for playing a dual role in biological
systems (Kohen and Nyska, 2002; Valko et al., 2006; Valko et al., 2004). At lower
concentrations ROS show beneficial effects and act as signaling molecules, mediating cell
growth and differentiation (Valko et al., 2006; Valko et al., 2007). However, at higher
concentrations, they induce senescence and apoptosis (Chandra et al., 2000). They can cause cell
damage, also referred to as oxidative stress (Poli et al., 2004). Examples of free radicals include
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superoxide, peroxyl, alkoxyl, trichloromethyl and hydrogen peroxide (Pier-Giorgio, 2000;
Halliwell et al., 1992; Blokhina et al., 2003).
1.2.2 Antioxidants
Antioxidants are any substances which neutralize free radicals. An antioxidant can be
defined as: “any substance that, when present in low concentrations compared to that of an
oxidisable substrate, significantly delays or inhibits the oxidation of that substrate” (Halliwell
and Gutteridge, 1995). The deleterious effects of ROS are neutralized by the antioxidant action
of non-enzymatic antioxidants in addition to antioxidant enzymes (Halliwell, 1994). Cells
possess antioxidant defense systems to counteract oxidative damage from ROS (Halliwell and
Gutteridge, 1999; Halliwell, 1994). It has been long established that oxidative stress, which is
caused by free radicals, is a key component in numerous human diseases, tumor development, as
well as ageing (Ziech et al., 2010; Valko et al., 2006; Franco et al., 2008; Halliwell and
Gutteridge, 1997; Harman, 1956). A large body of literature is in favor of the idea that diet-
derived antioxidants may be useful radioprotectors and play an important role in helping us to
stay healthier for longer (Fang et al., 2002; Halliwell, 1996). Since the percentage of people who
eat the recommended five servings of fruits and vegetables per day, is low, the opportunity for
preventing oxidative damage by improving diet is great (Ames et al., 1993). Foods with
characteristic red and blue colors, such as berries and certain vegetables, have been known for
their excellent antioxidant properties (Hammerstone et al., 2000; Carando et al., 1999; Hara et
al., 1995; Kreft et al., 1999; Rousseff et al., 1987; Reinli and Block, 1996). Data shows that diets
rich in fruits and vegetables show decreased risks for cancer (Seifried et al., 2007).
1.2.3 Antioxidants from Natural extracts
Certain spices and spice extracts have been known for their antioxidant effect (Gerhardt and
Schroter, 1983). A number of components with antioxidant properties have been identified, from
several spices (Halliwell, 1996). Over the last decade, increasing restriction in the use of
synthetic antioxidants has increased interest in the study of natural antioxidants, particularly
those present in spices (Helle et al., 1996). The antioxidant activities of spices suggest that they
possess potential health benefits apart from imparting flavor to the food (Shobana et al., 2000).
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2. REVIEW OF LITERATURE
2.1 Antimicrobial activity of Plant Extracts
The roots, leaves and stalk extracts of Rheum ribes showed significant antibacterial activities
suggesting their effective use against clinical isolates (Bibi et al., 2005).
Hydroalcoholic extracts of eight species of medicinal plants, namely, Acokanthera schimperi
(Apocynaceae), Calpurnia aurea (Leguminosae), Kalanchoe petitiana (Crassulaceae), Lippia
adoensis (Verbenaceae), Malva parviflora (Malvaceae), Olinia rochetiana (Oliniaceae),
Phytolacca dodecandra (Phytolaccaceae) and Verbascum sinaiticum (Scrophulariaceae),
were screened for antimicrobial activity. The results indicated the potential of these herbal
drugs in treating microbial infections (Hailu et al., 2005).
Twelve medicinal plants were screened, namely Abrus precatorius L., Caesalpinia
pulcherrima Swartz., Cardiospermum halicacabum L., Casuarina equisetifolia L., Cynodon
dactylon (L.) Pers., Delonix regia L., Euphorbia hirta L., E. tirucalli L., Ficus benghalensis
L., Gmelina asiatica L., Santalum album L., and Tecomella undulata (Sm.) Seem, for
potential antibacterial activity. The plant extracts were more active against Gram-positive
bacteria than against Gram-negative bacteria (Parekh et al., 2005).
A study in which 21 essential oils were tested, showed that 19 essential oils had antibacterial
activity against one or more strains of four gram-negative bacteria. Cinnamon, clove,
geranium, lemon, lime, orange and rosemary oils exhibited significant inhibitory effect.
Majority of the oils showed antibacterial activity against the tested strains hence can be a
potential source for antibacterial agents (Seenivasan et al., 2006).
Leaf extracts of seven South African plant species, Cussonia zuluensis, Vepris reflexa,
Curtisia dentata, Trichilia emetica, Terminalia phanerophlebia, Terminalia sambesiaca and
Kigelia africana, were evaluated for antibacterial and antifungal activities using microplate
dilution method. The acetone and dichloromethane extracts of all plant leaves were active
against some or all of the tested microorganisms. Some extracts had the highest activities
against both bacterial and fungal test organisms with minimal inhibitory concentration (MIC)
values as low as 0.02 mg/ml (Shai et al., 2008).
Crude extracts of various Agapanthus africanus plant parts were screened in vitro against
eight economically important plant pathogenic fungi radial mycelial growth was inhibited
significantly in five test organisms (Tegegnea et al., 2008).
8
In another study, 10 wild plants namely Mesembryanthemum crystallinum, Blackiella aellen,
Arthrocnemon glaucum, Atriplex halimus, Thymelaea hirsute, Carduus getulus, Nicotiana
glauca, Alhagi maurorum, Atractylis carduus and Echinops spinosissimus hexane and
methanol extracts showed strong antibacterial activity (Salwa et al., 2011).
Inhibitory activities of both aqueous and methanolic extracts of the root, stem bark, and leaf
of Morinda lucida on Escherichia coli, Salmonella typhi, Salmonella paratyphi, and
Salmonella typhorium was investigated in vitro. The results of this study show that the
extracts of M. lucida has the potentials of inhibiting the growth of E. coli and Salmonella
spp., thereby suggesting its potency in the treatment of infections in which E. coli and
Salmonella spp. are implicated (Fakoya et al., 2014).
The leaf extracts of Adhatoda vasica and Crotolaria verrucosa were subjected for screening
of in vitro antibacterial activity against selected major human pathogenic bacterial strains like
Bacillus substilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and
Pseudomonas aeruginosa by agar well diffusion method. The results of antibacterial activity
revealed that the A. vasica and C. verrucosa leaf extracts showed good activity on the
selected bacterial strains (Prasad et al., 2015).
2.2 Antioxidant activity of Plant Extracts
A recent review reported about spices and herbs being used for thousands of years for
flavour, aroma, as colouring in foods and as preservatives. They contain powerful
antioxidants that have been proven to be effective in inhibiting lipid oxidation or slowing
down the onset of rancidity in foods. Antioxidants from spices and herbs possess desirable
properties such as being natural, non-GMO and having clean label ingredients (i.e., can be
listed as spice or herb or flavouring). Antioxidant activities and antioxidant capacities of
compounds from spices and herbs have been determined and well published in the scientific
literature. Interests in food antioxidants from spices and herbs will continue to increase as
well as research and technology that will develop better ways of growing spices and herbs
that contain higher amounts of antioxidants (Milda, 2015).
Another research analyzed the methanolic crude extracts of 12 traditionally used Indian
medicinal plants for their antioxidant and free radical scavenging properties using α-
tocopherol and butylated hydroxy toluene (BHT) as standard antioxidants. Free radical
scavenging activity was evaluated using diphenyl picryl hydrazyl (DPPH) radicals. Seven
9
plants, namely Terminalia chebula, Mangifera indica, Terminalia bellerica, Punica
granatum, Ocimum sanctum, Cichorium intybus and Camellia sinensis showed strong free
radical scavenging activity with the DPPH method. The tested plant extracts showed
promising antioxidant and free radical scavenging activity (Aqil et al., 2006).
Another study evaluated the antioxidant activity of spice extracts such as ginger, turmeric
and garlic by 2, 2’-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Method. The
antioxidant activities when compared among ginger, turmeric and garlic the potency of these
spices was found to be in the order of Vit C > Ginger > Turmeric ≥ Dry garlic > Fresh garlic.
The study indicates that the spices like ginger, garlic and turmeric have antioxidant activity.
Further studies are needed to study the biological effects of antioxidant-rich herbs and spices
on oxidative stress related diseases (Virendra et al., 2013).
The aim of another study was to evaluate the antioxidant power of curcumin by two methods
i.e., 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) assay and reducing power activity (RPA),
compared with ascorbic acid, a well known antioxidant. Percentage of free radical
scavenging of curcumin and ascorbic acid was more than 69 and 62 % at concentration 0.1
mM, respectively. No difference was observed between curcumin and ascorbic acid in
antioxidant potencies. These results showed excellent antioxidant activity for curcumin and it
is main reason for biological activities of curcumin (Asouri et al., 2013).
Dealing with Zingiberaceae family to explore their antioxidant activities determined by
measuring 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity of spice
extracts including turmeric (Curcuma longa L. ), white saffron (C. mangga Val.), temulawak
(C. xanthorrhiza Roxb), ginger (Zingiber officinale Roscoe) were used. Ten concentrations
of extracts 100; 50; 25; 12.5; 6.25; 3.125; 1.563; 0.781; 0.391 and 0.195 µg/mL were
performed to determine the DPPH scavenging activities. Results showed that Inhibitory
Concentrations (IC)-50 of DPPH were as followed C. longa 8.33 µg/mL; C. mangga 277.79;
C. xanthorrhiza 39.58 µg/mL µg/m; Z. officinale 10.51 µg/mL; and curcumin 7.85 µg/mL..
In the study, C. longa extract showed the highest antioxidant activity among all tested
extracts and the lowest antioxidant activity was C. mangga (Wahyu et al., 2011).
In another research, the antioxidant activity of curcumin employing antioxidant assays 1,1-
diphenyl-2-picryl hydrazyl free radical (DPPH) scavenging was measured. Curcumin had an
effective DPPH scavenging. Also, BHA, BHT, α-tocopherol and trolox, were used as the
10
reference antioxidant and radical scavenger compounds. According to that study, curcumin
was proposed to be used in the pharmacological and food industry because of these
properties (Tuba and Ilhami, 2008).
A study was performed to examine the antioxidant activity of Trachyseprmum ammi (L)
Sprague (Ajwain) essential oil. It was determined that the tested essential oil possessed a
high degree of FRAP (ferric reducing antioxidant power), a good DPPH (2, 2-diphenyl-1-
picryl-hydrazyl) radical scavenging activity and a moderate H2O2 radical scavenging activity.
The study found that the essential oil of Ajwain could serve as a significant bio resource of
antioxidants, for use in the food and pharmaceutical industry (Ak and Gulc, 2008).
The acidic and neutral polyphenolic fractions of the oils from ajwain, mustard, fenugreek and
poppy seeds were tested for antioxidant activity. The highest amount of polyphenols was
found in ajwain, followed by mustard seeds, fenugreek and poppy seeds. The study
concludes that oil seeds are potential sources of natural antioxidants which may replace
synthetic ones (Shaguftalshiaque et al., 2013).
The study was done to determine the antioxidant and free radical scavenging potential of
ethanolic seed extract of Trachyspermum ammi (L) Sprague (ESETA). Antioxidant potential
of ESETA was evaluated by different scavenging models including DPPH, nitric oxide,
superoxide, and hydroxyl radical as well as its lipid peroxidation ability in bovine brain
extract. The presence of various phyto-constituents including alkaloids, glycosides,
terpenoids, saponins, phenols and steroids were revealed upon phytochemical analysis of
ESETA. ESETA displayed concentration-dependent reducing power ability and remarkable
ferric ion-induced lipid peroxidation inhibitory effect in bovine brain extract. These results
confirm the efficacy of ESETA as a potential source of antioxidant (Bajpai and Agrawal,
2015).
The above reports about the studies done on various plant species have clearly stated the
potential of plants as an effective and promising source of antimicrobial and antioxidant
agents. Inspired by these, the study was focused on exploring the same for some plant
extracts.
11
3. MATERIALS AND METHODS
3.1 Place and Duration of Study
The experiments were carried out in the Biological Product Laboratory, Department of
Botany, University of Allahabad, Allahabad, during the period from 27th May 2015 to 22nd July
2015.
3.2 Materials
3.2.1 Culture Media Used: Name and Composition
(i) Autoclavable media
a) Solid Media
Nutrient Agar (28.0g/L)
Ingredients Grams/Liter
Beef Extract 1.0
Yeast Extract 2.0
Peptone 5.0
Sodium Chloride 5.0
Agar 15.0
Distilled Water 1.0 L
Sabouraud Dextrose Agar (SDA) (65.0g/L)
Ingredients Grams/Liter
Mycological peptone 10.0
Dextrose 40.0
Agar 15.0
Distilled Water 1.0 L
12
b) Broth or Liquid Media
Mueller Hinton Broth (MHB) (38.0g/L)
Ingredients Grams/Liter
Casein Acid hydrolysate 17.5
Beef Extract 3.0
Starch 1.5
Distilled Water 1.0 L
Preparation
o Mentioned composition of each constituent was carefully measured using a Physical balance.
o All the constituents were put in an Erlenmeyer flask covered using sterile cotton plug and
mixed with required amount of distilled water.
o The mixture was made homogenous by gentle heating and constant mixing until turbidity of
the solution was not visible.
o The medium was sterilized using wet sterilization technique at once after homogenizing. For
this media was kept at 121⁰C and 15 psi for a period of 20 minutes in an Autoclave chamber.
o The media was only opened under complete sterile environment such as Laminar Air Flow
chamber for further use.
(ii) Non- Autoclavable:
RPMI– 1640 ( Rosewell Park Memorial Institute Media) (16.4g/L)
o Desired amount of RPMI was measured and put in an Erlenmeyer flask to mix with required
amount of distilled water.
o Mixing was done to achieve homogenized solution. The media was not heated at all to avoid
denaturation of biomolecules present.
o Sterilization method employed was Filter Sterilization done using Vacuum filter and
Millipore filter paper of pore size 0.22 µm.
3.2.2 Chemical Agents
Chromic Acid:
13
o 20 g of Potassium Dichromate was added with small amount of water.
o Sufficient mixing to obtain a paste of chromate salt was done.
o To the paste 300ml of concentrated H2SO4 was added.
Double Distilled Water
Ethanol
Formaldehyde
Acetone
Methanol
DPPH(Di-phenyl Picryl hydrazine)
DMSO (Dimethyl sulphoxide)
3.2.3 Microbial Strains
a) Bacteria
Vibrio cholera
Salmonella typhimurium
Klebsiella pneumoniae
b) Fungi
Epidermophyton floccosum
Microsporum fulvum
Microsporum gypseum
3.2.4 Laboratory Equipment
Autoclave
Laminar Air Flow Chamber
Micropipette
Physical Balance
96 well Plates
UV – Visible Spectrophotometer (Figure 3A)
B.O.D. Incubators
Clevenger (Figure 3B)
Erlenmeyer Flasks
Petri Dishes
Measuring Jars
14
Nonabsorbent Cotton Plugs
Inoculation Loop and Spatula
Figure 3: SpectraMax Plus384, Molecular Devices Corporation, U.S.A. (A); Clevenger
apparatus (B).
3.2.5 Extracts/Compounds/oil
BPL-11; BPL-14; BPL-16; BPL-Ua were tested for Antimicrobial Activity and obtained
from Biological Product Laboratory, Department of Botany, University of Allahabad. The
concentration of stock was 50 mg/mL.
BPL-Ua; BPL-Ma; BPL-Pn extracts were used for Antioxidant Assays and obtained from
Biological Product Laboratory, Department of Botany, University of Allahabad. The
concentration of stock was 50 mg/mL.
Oil of Trachyspermum ammi (L) Sprague was extracted using Clevenger Apparatus from
fruit pod of T. ammi for Antioxidant Assay. The concentration of stock was 50 µL/mL.
3.3 Methods
3.3.1 Cleaning of Glass Equipment
Microbes, being the masters of adaptation are omnipotent resulting in random growth of
multiple species together. Contamination in a growth media is one of the biggest menaces a
microbiologist faces. To tackle the same, one must sterilize the glass wares and the medium
which is being used for the growth of microbes. By this, not only one avoids random growth but
A B
15
also can achieves a pure colony easily. For the study, following methods were employed for
sterilization and cleaning the glass wares.
3.3.2 Washing
o The glassware such as culture tubes, Petri plates, Erlenmeyer Flasks, beakers etc. were
washed with soap solution using running water.
o After washing with detergent, they were dipped in 5% Chromic Acid solution for at least 30
minutes.
o The glassware were further washed with tap water and dried in air.
3.3.3 Sterilization
Sterilization is the process of rendering a medium or material free of all forms of life which can
be achieved by three basic methods. They are summarized as follows:
i. Wet Heat Sterilization
The most useful approach is autoclaving, in which items are sterilized by exposure to steam at
121°C and 15 lbs of pressure for 15 minutes or longer, depending on the nature of the item.
Under these conditions, microorganisms, even endospores, will not survive.
o The glassware such as conical flasks, test tubes, etc., was plugged with nonabsorbent cotton.
o Petri dishes were wrapped in a clean wrapping paper or aluminum foil.
o Forceps, scalpels, glass rods were kept in test tubes and plugged with nonabsorbent cotton,
and covered with wrapping paper.
o The pipette tip boxes were wrapped with paper.
o All the equipment’s were put in an autoclave for 20 minutes 121°C under 15 lbs pressure.
ii. Dry Sterilization
Often, dry glassware’s are affected by steam as it tends to etch glassware and also leaves it
damp. Therefore, such items are generally dry-heat sterilized.
o The glassware were wrapped carefully and plugged with cotton if necessary.
o The glassware were then kept in an oven at 140°C–160°C for 2 h. it was made sure the
temperature not rises above 180⁰C, which leads to burning of cotton.
iii. Filter Sterilization
Thermo labile compounds such as amino acids, vitamins, number of plant growth hormones,
etc., are usually destroyed during autoclaving. And thus are sterilized by filtration through
Millipore filtration membranes of 0.22 or 0.45μm porosity which physically removes bacteria
and larger microorganisms from the solution and thereby sterilizes them without heat.
o A vacuum filter chamber was cleaned and autoclaved.
16
o A clean Erlenmeyer flask was taken and autoclaved after being plugged with non-absorbent
cotton.
o Vacuum filter or the Millipore filtration unit was fixed with a new bacteriological
membranes (0.22 or 0.45μm) in place.
o The media was passed through the filter by the use of a vacuum pump to obtain a sterile
media which was further used (Bryce, 1992; Graser et al., 2000).
3.3.4 Antifungal assay (Rex et al., 2008)
1) Preparation of media
RPMI-1640 medium (Roswell Park Memorial Institute medium) supplemented with
MOPS buffer [3-(N-morpholino) propanesulfonic acid] was the media used. The media was
mixed thoroughly without using heat to prevent denaturation of the biomolecules present in the
media.
2) Sterilization of media
RPMI-1640 medium was sterilized using a vacuum filter.
3) Preparation of inoculum
i. One loop full of colony was dispensed in saline media and homogenized.
ii. Once the optical density equivalent to 0.5 McFarland was obtained, it was taken as stock.
4) Testing format
I. Negative control
40% formaldehyde in inoculum was added to kill the cells. 100µL of this culture suspension was
added to each well in column 1. This served as a negative control.
II. Broth control
200µL of media was added to each well in media/broth control lane per well. No organism and
no test samples were added. This was done to check contamination in the media.
III. Positive control
For positive control, contains 100µL of broth and 100 µL of inoculum prepared.
IV. Drug control
Drug control well contains 190 µL of broth and 10 µL of drug/compound/extract/oil.
5) Calculation of MIC
Optical density (O.D.) was recorded with a spectrophotometer (SpectraMax Plus384, Molecular
Devices Corporation, USA) at 530 nm after 96 hrs incubated at 35 ± 2 ⁰C. Per cent inhibition
was calculated using the following equations:
17
Per cent Inhibition (%) = [(O.D. positive control- O.D. drug treated)/(O.D. positive control)] x
100.
Minimum Inhibitory Concentration was obtained statistically and graphically using a computer
programmed software.
3.3.5 Antibacterial assay (Wilker et al., 2006)
1) Preparation of media
Mueller Hinton broth (MHB) was the media used. The media was mixed thoroughly using heat
to obtain homogenized solution.
2) Sterilization of media
The Erlenmeyer Flask containing the media was sealed with a cotton plug and autoclaved at
121⁰C and 15 lbs for 15 minutes.
3) Preparation of inoculum
i. One loop full of colony was dispensed in the saline media and homogenized.
ii. Once the desired optical density equivalent to 0.5 McFarland is obtained, it was taken as
stock.
4) Testing format
i. Negative control
40% formaldehyde in inoculum was added to kill the cells. 100µL of this culture suspension was
added to each well in column 1. This served as a negative control.
ii. Broth control
200µL of media was added to each well in media/broth control lane per well. No organism and
no test samples were added. This was done to check contamination in the media.
iii. Positive control
For positive control, contains 100µL of broth and 100 µL of inoculum prepared.
iv. Drug control
Drug control well contains 190 µL of broth and 10 µL of drug/compound/extract/oil.
v. Obtaining MIC
Optical density (O.D.) was recorded with a spectrophotometer (SpectraMax Plus384, Molecular
Devices Corporation, USA) at 492 nm after 24 hrs incubated at 35 ± 2 ⁰C. Per cent inhibition
was calculated using the following equations:
Per cent Inhibition (%) = [(O.D. positive control- O.D. drug treated)/(O.D. positive control)] x
100.
18
Minimum Inhibitory Concentration was obtained statistically and graphically using a computer
programmed software.
3.4 Extraction of Trachyspermum ammi (L) Sprague Oil
T. ammi oil for DPPH Antioxidant Assay was extracted from fruits (cremocarp) using the
clevenger apparatus.
About 250 grams of T. ammi fruit pods were purchased, cleaned and filled in clevenger
apparatus.
Tap water was put in the round bottom flask such the water level was equal to heating
mantle.
The heating unit was switched on at a temperature of 60⁰C till boiling was observed.
As boiling started, the temperature was reduced to 40⁰C and maintained for a period of
72 hrs.
After 72 hrs, oil was taken out and the per cent yield was measured using the formula
Per cent Yield = (Weight of Oil obtained/ Weight of turmeric taken.) x 100
The T. ammi oil was stored for further analysis.
3.4.1 Determination of Scavenging Activity of Oils and Plant Extracts by DPPH Assay
Protocol:
i. In a 96 well plate, the antioxidant tests were conducted in replicates.
ii. The first two columns contained 180µL of DPPH and 20µL of DMSO. These two
columns were taken as blank.
iii. The sample to be tested was taken in varying concentrations, 20µL, 17.5µL, 15µL,
12.5µL and so on till 5µL. In each of these wells the volume was made up to 20µL using
DMSO.
iv. 180µL of DPPH (100 µg/mL in methanol) is added in each well.
v. Vitamin C (1 mg/ml) was taken as a standard.
vi. The entire setup was kept incubated in the dark for 30 minutes.
vii. Optical density (O.D.) was recorded with a spectrophotometer (SpectraMax Plus384,
Molecular Devices Corporation, USA) at 517 nm.
Calculations:
Per cent Scavenging ability = [1-(O.DSample/O.DBlank)]x100
19
4. RESULTS AND DISCUSSION
4.1 Antibacterial tests
Three pathogens were chosen for the antibacterial tests, V. cholera, K. pneumonia and S.
typhimurium. Gentamicin, a synthetic drug was taken as the standard. The other drugs tested
were BPL-11, BPL-14 and BPL-16.
4.1.1 Gentamicin
The susceptibility of bacteria against gentamicin is represented in Figure 4 and Table 1. The
minimum concentration for 90% inhibition against any pathogen was 0.692 mg/mL. Gentamicin
was found to be most effective against Vibrio cholerae and least against Klebsiella pneumoniae.
Figure 4: The concentration of Gentamicin vs Per cent inhibition of three pathogens V. cholerae,
K. pneumoniae and S. typhimurium.
4.1.2 BPL-11
The growth of all three test pathogens was effectively inhibited by BPL-11 and most effective
against S. typhimurium and least against K. pneumonia and represented in Figure 5 and Table 1.
20
Figure 5: The concentration of BPL-11 vs Per cent inhibition of three pathogens V. cholerae, K.
pneumoniae and S. typhimurium.
4.1.3 BPL-14
BPL-14 was the least effective against the three test pathogens and individual susceptibility of
bacteria towards BPL-14 were represented in Figure 6 and Table 1.
Figure 6: The concentration of BPL-14 vs Per cent inhibition of three pathogens V. cholerae, K.
pneumoniae and S. typhimurium.
21
4.1.4 BPL-16
BPL-16 was found to have a lower minimum concentration for 90% inhibition than the standard
drug, Gentamicin. All the test pathogens were effectively inhibited, however, BPL-16 was not as
effective as BPL-14. The efficacies of BPL-16 against bacteria tested were represented in Figure
7 and Table 1.
Figure 7: The concentration of BPL-16 vs Per cent inhibition of three pathogens V. cholera, K.
pneumonia and S. typhimurium.
Table 1: IC50 and IC90 gentamicin and drugs tested against three bacteria.
Gentamicin BPL-16 BPL-14 BPL-11
Pathogens IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90
V. cholera 0.197 0.629 0.230 0.502 0.672 1.133 0.256 0.478
K. pneumonia 0.412 1.139 0.215 0.585 0.765 1.162 0.275 0.511
S. typhimurium 0.328 0.897 0.219 0.533 0.701 1.130 0.225 0.393
* All the values were represented in mg/ml.
22
Figure 8: Comparative IC50 and IC90 of Test drugs.
The antibacterial activity of the compounds BPL-11 and BPL-16 was more effective when
compared with the standard used (Figure 8), i.e. Gentamicin. This shows promise for use of the
compounds as antibiotics.
4.2 Antifungal tests
Three pathogens were chosen for the antifungal tests, E. floccosum, M. fulvum and M. gypseum.
Sertaconazole NitrateBP was taken as the standard. BPL-Ua was tested against the same
pathogens. The antifungal activity has been recorded, as seen below:
4.2.1 BPL-Ua
BPL-Ua was found to have a lower minimum concentration for 90% inhibition than the standard
drug tested, i.e. Sertaconazole, against the three test pathogens, E. floccosum, M. fulvum and M.
gypseum. The relationship between the BPL-Ua concentration and percent inhibition of
aforementioned fungi were represented in Figure 9 and Table 2.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90
Gentamicin BPL-16 BPL-14 BPL-11
Co
nce
ntr
atio
n (
mg/
mL)
V. cholerae
K. pneumoniae
S. typhimurium
23
Figure 9: The concentration vs Per cent inhibition of BPL-Ua against three pathogens E.
floccosum, M. fulvum and M. gypseum.
4.2.2 Sertaconazole NitrateBP
Sertaconazole was the standard drug taken. It has higher minimum concentration for 90%
inhibition of all pathogens when compared to BPL-Ua. The relationship between the
Sertaconazole concentration and aforementioned fungi tested were represented in Figure 10 and
Table 2.
Figure 10: The concentration vs Per cent inhibition of Sertaconazole NitrateBP against three
pathogens E. floccosum, M. fulvum and M. gypseum.
24
Table 2: Inhibitory concentrations (mg/mL) of Sertaconazole and BPL-Ua.
Sertaconazole BPL-Ua
Pathogens IC50 IC90 IC50 IC90
E. floccosum 0.66 0.70 0.02 0.03
M. fulvum 0.63 0.66 0.35 0.57
M. gypseum 0.99 1.32 0.29 0.32
Figure 11: Comparative IC50 and IC90 of BPL-Ua acetone extract and Sertaconazole NitrateBP.
The antifungal activity of BPL-Ua was more effective when compared with the standard
used (Figure 11), i.e. Sertaconazole NitrateBP. The results obtained show that the future
prospects of BPL-Ua as an antifungal are promising.
4.3 Per cent Yield of Trachyspermum ammi (L) Sprague essential oil
Weight of fruit pods taken = 250 g
Yield of oil = 10 mL
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
IC50 IC90 IC50 IC90
Sertaconazole BPL-Ua
Co
nce
ntr
atio
n (
mg/
mL)
E. floccosum
M. fulvum
M. gypseum
25
Per cent Yield = 4%
4.4 Antioxidant tests
4.4.1 Vitamin C
EC50 (Amount of antioxidant necessary to decrease the initial DPPH concentration by 50%)
of Vitamin C was calculated graphically and found to be 0.021 mg/mL and calculated on the
basis of equation present in the Figure 12.
Figure 12: Per cent Scavenging ability of Vitamin C
4.4.2 Trachyspermum ammi (L) Sprague
Figure 13: Per cent Scavenging ability of Trachyspermum ammi extract
y = 2.8979x - 12.747R² = 0.9685
0
20
40
60
80
100
120
0 10 20 30 40 50
Scav
en
gin
g ab
ility
Concentration of Vitamin C (µg/mL)
% Scavenging ability of Vitamin C
% Scavenging ability ofVitamin C
y = 2.6624x + 19.678R² = 0.9586
0
20
40
60
80
100
0 5 10 15 20 25
Scav
en
gin
g ab
ility
Concentration of Trachyspermum ammi extract (µL/mL)
% Scavenging ability of Trachyspermum ammi extract
% Scavenging ability ofTrachyspermum ammiextract
26
EC50 of the extract of T. ammi was calculated graphically and found to be 11.3937 µL/mL
and calculated on the basis of equation present in the Figure 13.
4.4.3BPL-Ua
Figure 14: Per cent Scavenging ability of BPL-Ua.
EC50 of BPL-Ua was calculated graphically and found to be 3.3067 mg/mL and calculated on
the basis of equation present in the Figure 14.
4.4.4 BPL-Ma
Figure 15: Per cent Scavenging ability of BPL-Ma
y = 11.642x + 11.511R² = 0.9791
0
10
20
30
40
50
60
70
80
0 2 4 6
Scav
en
gin
g ab
ility
Concentration of BPL-Ua extract (mg/mL)
% Scavenging ability of BPL-Ua
% Scavenging ability ofBPL-Ua
y = 12.753x + 18.809R² = 0.8371
0
10
20
30
40
50
60
70
80
90
0 2 4 6
Scav
en
gin
g ab
ility
Concentration of BPL-Ma extract (mg/mL)
% Scavenging ability of BPL-Ma
% Scavenging ability ofBPL-Ma
27
EC50 of BPL-Ma was calculated graphically and found to be 2.447 mg/mL and calculated on
the basis of equation present in the Figure 15.
4.4.5 BPL-Pn
Figure 16: Per cent Scavenging ability of BPL-Pn
EC50 of BPL-Pn was calculated graphically and found to be 3.878 mg/mL and calculated on
the basis of equation present in the Figure 16.
In the order of antioxidant activity, we have, Vitamin C > BPL-Pn > BPL-Ua > BPL-Ma. The
extract from the fruits of Trachyspermum ammi (L) Sprague, commonly used in Indian cooking,
showed good antioxidant activity. The results show great promise for future use of
aforementioned extracts/oil as antioxidants.
y = 13.413x - 2.0084R² = 0.976
0
10
20
30
40
50
60
70
0 2 4 6
Scav
en
gin
g ab
ility
Concentration of BPL-Pn extract (mg/mL)
% Scavenging ability of BPL-Pn
% Scavenging ability ofBPL-Pn
28
5. CONCLUSION AND FUTURE PROSPECTS
Based on the tests conducted it was found that, BPL-11 has the best antibacterial activity and BPL-
Ua has the best antifungal activity among the tested drugs. BPL-Pn was found to have good
antioxidant activity along with the essential oil of Trachyspermum ammi (L) Sprague. Natural
products have been used since time immemorial for their health benefits. Ethnomedical uses of
some natural products have been reported in many countries, though it seems that many of these
have been forgotten over time. Till date, many of these are used as home remedies in both
developed and developing parts of the world. This study has shown that they have scope for use as
antifungal, antibiotics and antioxidants. Further research in this field will help identify the abilities
of different plant extracts and progress towards using them for the benefit of mankind.
29
6. BIBLIOGRAPHY
Abreu, A.C, McBainb, A.J., and Sim~oes, M. (2012). Plants as sources of new antimicrobials and
resistance-modifying agents. Nat. Prod. Rep., 29, 1007.
Ahameethunisa, A.R., and Hooper, W. (2010). Antibacterial activity of Artemisia nilagirica leaf extracts
against clinical and phytopathogenic bacteria, BMC Complementary and Alternative Medicine, 10, 1-6.
Ak, T., and Gulc, I. (2008). Antioxidant and radical scavenging properties of Curcumin. Chemico-
Biological Interactions 174 27–37. Chatterjee, S., Goswami, N., & Kothari, N. (2013). Evaluation of
antioxidant activity of essential oil from Ajwain (Trachyspermum ammi) seeds. Int J Green Pharm, 7,
140-144.
Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993). Oxidants, antioxidants, and the degenerative
diseases of aging. 90(17), 7915–7922. http://www.pnas.org/content/90/17/7915.short
Andersson, D.I., and Hughes, D. (2010). Antibiotic resistance and its cost: is it possible to reverse
resistance? Nat. Rev. Microbiol., 8, 260–271.
Anonymous. (1993). Summary of WHO guidelines for assessment of herbal medicines. Herbal Gram, 28,
13-14.
Anonymous. (2011). Regional Committee for Europe, European strategic action plan on antibiotic
resistance, Copenhagen,
Aqil, F., Ahmad, I., and Mehmood, Z. (2006). Antioxidant and Free Radical Scavenging Properties of
Twelve Traditionally Used Indian Medicinal Plants. Turk J Biol., 30, 177-183.
Asouri, M., Ataee, R., Ahmadi, A.A., Amini, A., and Moshaei, M.R. (2013). Antioxidant and Free
Radical Scavenging Activities of Curcumin. Asian Journal of Chemistry, 25(13), 7593-7595.
Bajpai, V.K., and Agrawal, P. (2015). Studies on Phytochemicals, Antioxidant, Free Radical Scavenging
and Lipid Peroxidation Inhibitory effects of Trachyspermum ammi seeds. Indian Journal of
Pharmaceutical Education and Research, 9(1).
Basualdo, C., Sgroy, V., Finola, M.S., and Juam, M. (2007). Comparison of the antibacterial activity of
honey from different provenance against bacteria usually isolated from skinwounds. Veterinary
Microbiology, 124, 375-381.
Berdy, J. (2005). Bioactive microbial metabolites. J Antibiot, 58, 1–26.
Bibi, S.F.B., Mehrangiz, K., and Hamid, R.S. (2005). In Vitro Antibacterial Activity of Rheum ribes
Extract Obtained from Various Plant Parts against Clinical Isolates of Gram-Negative Pathogens. Iranian
Journal of Pharmaceutical Research, 2, 87-91.
Blokhina, O., Virolainen, E., and Fagerstedt, K.V. (2003). Antioxidants, Oxidative Damage and Oxygen
Deprivation Stress: a Review. Annals of Botany, 91, 179-194.
Brehm-Stecher, B.F., and Johnson, E.A. (2003). Sensitization of Staphylococcus aureus and Escherichia
coli to Antibiotics by the Sesquiterpenoids Nerolidol, Farnesol, Bisabolol, and Apritone. Antimicrob.
Agents Chemother.,47, 3357–3360.
30
Brodsky, I.E., and Medzhitov, R. (2009). Targeting of immune signaling networks by bacterial pathogens.
Nat. Cell Biol., 11, 521–526.
Bryce, K. (1992). The Fifth kingdom. J Mycologue Publications Ontario, 451.
Burt, S.A. (2004). Essential oils: their antibacterial properties andpotential applications in foods: a review.
Inter J Food Microbiol, 94, 223-253.
Buttner, M.P., Willeke, K., and Grinshpun, S.A. (1996). Sampling and analysis of airborne
microorganisms. In Manual of Environmental Microbiology Edited by: Hurst CJ, Knudsen GR,
McInerney MJ, Stetzenbach LD,Walter MV. ASM Press: Washington, DC; 629-640.
Cadenas, E. (1989). Biochemistry of oxygen toxicity. Ann. Rev. Biochem., 58, 79–110.
Carando, S., Teissedre, P.L., Pascual-Martinez, L., and Cabanis, J.C. (1999). Levels of flavan-3-ols in
French wines. J Agr Food Chem, 47, 4161–4166.
Casadevall, A., and Pirofski, L.A. (2000). Host–pathogen interactions:basic concepts of microbial
commensalism, colonization, infection, and disease. Infect. Immun., 68, 6511–6518.
Chandra, J., Samali, A., and Orrenius, S. (2000). Triggering and modulation of apoptosis by oxidative
stress. Free Radic. Biol., 29, 323–333.
Cowan, M.M. (1999). Plant products as antimicrobial agents. ClinMicrobiol Rev., 12(4), 564-82.
Curtis, C. (1998). Use and abuse of topical dermatological therapy in dogs and cats. Part 1. Shampoo. J
TherPract., 20, 244–51.
Dantas, G., Sommer, M.O., Oluwasegun, R.D., and Church, G.M. (2008). Bacterial Subsisting on
Antibiotics. Science, 320, 100–103.
Darokar, M.P., Mathur, A., Dwivedi, S., Bhalla, R., Khanuja, S.P.S., and Kumar, S. (1998). Detection of
antibacterial activity in the floral petals of somehigher plants. CurrSci, 75, 187.
Devasagayam, Tilak, J.C., Boloor, K.K., Sane, K.S., Ghaskadbi, S.S., and Lele, R.D. (2004). Free
Radicals and Antioxidants in Human Health: Current Status and Future Prospects TPA. JAPI, 52.
Diacovich, L., and Gorvel, J.P. (2010). Bacterial manipulation of innateimmunity to promote infection.
Nat. Rev. Microbiol., 8, 117–128.
Dismukes, W.E. (2006). Antifungal therapy: lessons learned over the past 27 years. ClinInfect Dis, 42,
1289–1296.
Duraipandiyan, V., Sasi, A.H., Islam, V.I.H., Valanarasu, M., and Ignacimuthu, S. (2010). Antimicrobial
properties of actinomycetes from the soil of Himalaya. J Med Mycol, 20, 15.
Elakkia, S.A., and Venkatesalu, V. (2013). Antimicrobial activity of different solvent extracts of some
Cassia species. Int J Pharma Bio Sci., 4, 728‑736.
Enne, V.I., Livermore, D.M., Stephens, P., and Hal, L.M.C. (2001). Persistence of sulphonamide
resistance in Escherichia coli in the UK despite national prescribing restriction. The Lancet, 28, 1325-
1328.
31
Faid, M., Bakhy, K., Anchad, M., and Tantaoui-Elaraki, A. (1995). Physicochemical and microbiological
characterizations and preservation with sorbic acid and cinnamon. J Food Prod, 58, 547-550.
Fakoya, A., Owojuyigbe, O.S, Fakoya, S., and Adeoye, S.O. (2014). Possible antimicrobial activity of
Morinda lucida stem bark, leaf and root extracts. 13(3), 471-475.
Fang, Y., Yang, S., and Wu, G. (2002). Free radicals, antioxidants, and nutrition. Nutrition, 18(10), 872–
879.
Farthing, M.J.G., and Kelly, P. (2007). Infectious diarrhoea. Medicine Gastroenterology Part 3 of 4, 35,
251–256.
Finch, R.G. (1998). Antibiotic resistance. J. Antimicrobial Chemotherapy, 42, 125-128.
Franco, R., Schoneveld, O., Georgakilas, A., and Panayiotidis, M. (2008). Oxidative stress, DNA
methylation and carcinogenesis. Cancer Lett, 266, 6–11.
Gerhardt, U., and Schroter, A. (1983). Antioxidative Wirkung von Gewtirzen. Gordian, 9, 171-176.
Gibbons, S. (2005). Plants as a source of bacterial resistance modulators and anti‑infective agents.
Phytochem Rev, 4, 63‑78.
Gilbert, D.L. (1981). Oxygen and living processes: an interdisciplinary approach, Springer, NY.
Gilbert, P., Maira-Litran, T., McBain, A.J., Rickard, A.H., and Whyte, F.W. (2002). The physiology and
collective recalcitrance of microbial biofilm communities. Adv. Microb. Physiol., 46, 203–256.
Goossens, H. (2005). European status of resistance in nosocomialinfections. Chemotherapy, 51, 177–181.
Graser, Y., Kuijpers, A.F.A., Presber, W., and Hoo, G.S. (2000). Molecular taxonomy of the
Trichophytonrubrum complex. J ClinMicrobiol., 38, 3329-36.
Hailu, T., Endris, M. Kaleab, A., and Tsige, G. (2005). Antimicrobial activities of some selected
traditional Ethiopian medicinal plants used in the treatment of skin disorders. Journal of
Ethnopharmacology, 100, 168–175.
Halliwell, B. (1994). Free Radicals and Antioxidants: a Personal View. Nutrition Reviews. 52(8), 253-
265.
Halliwell, B. (1996). Antioxidants in human health and disease. Ann. Rev. Nutr., 16, 33–50.
DOI: 10.1146/annurev.nu.16.070196.000341
Halliwell, B., and Gutteridge, J.M.C. (1989). Free radicals in biology and medicine, 2nd ed. Oxford:
Clarendon Press, 1989.
Halliwell, B., and Gutteridge, J.M.C. (1995). The definition and measurement of antioxidants in
biological systems. Free Radic Biol Med, 18, 125–6.
Halliwell, B., and Gutteridge, J.M.C. (1997). (eds), Free Radicals in Biology and Medicine, Oxford
University Press, Oxford.
Halliwell, B. and Gutteridge, J.M.C. (1999). Free Radicals in Biology and Medicine. (3rd ed.) Oxford
University Press, Oxford.
32
Halliwell, B., Gutteridge, J.M.C., and Cross, C.E. (1992). Free radicals, antioxidants, and human disease:
Where are we now? Translational Research, 119(6), 598-620.
Hammer, K.A., Carson, C.F., and Riley, T.V. (1999). Antimicrobial activity of essential oils and other
plant extracts. J. Appl. Microbiol., 86(6), 985.
Hammerstone J.F., Lazarus, S.A., and Schmitz, H.H. (2000). Procyanidin content and variation in some
commonly consumed foods. J Nutr, 130, 2086S–2092S.
Hara, Y., Luo, S.J., Wickremasinghe, R.L., and Yamanishi, T. (1995). Special issue on tea. Food Rev Int,
11, 371–542.
Harman, D. (1956). Ageing: a theory based on free radical and radiation chemistry. J Gerontol, 11, 298-
300.
Helle, L.M., Bo, R.N., Grete, B., and Leif, H.S. (1996). Screening of antioxidative activity of spices. A
comparison between assays based on ESR spin trapping and electrochemical measurement of oxygen
consumption. Food Chemistry, 57(2), 331-337.
Hussain, H., Badawy, A., Elshazly, A., Elsayed, A., Krohn, K., Riaz, M., and Schulz, B. (2011).
Chemical Constituents and Antimicrobial Activity of Salix subserrata. Rec. Nat. Prod., 5(2), 133-137.
Iwu, M.W., Duncan, A.R., and Okunji, C.O. (1999). New antimicrobials of plant origin. In: Janick J. (Ed)
Perspectives on New Crops and New Uses. ASHSpress, Alexandria 457-462.
Jagessar, R.C., Mars, A., and Gomes, G. (2008). Selective Antimicrobial properties of Phyllanthusacidus
leaf extract against Candida albicans, Escherichia coli and Staphylococcus aureus using Stokes Disc
diffusion, Well diffusion, Streakplate and a dilution method. Nature and Science, 6(2), 24-38. ISSN:
1545-0740.
Kamali, H.H.E.L., and Amir, M.Y.E.L. (2010). Antibacterial Activity and Phytochemical Screening of
Ethanolic Extracts Obtained from Selected Sudanese Medicinal Plants. Curr. Res. J. of Bio. Sci., 2(2),
143-146.
Kohen, R., and Nyska, A. (2002). Invited Review: Oxidation of Biological Systems: Oxidative Stress
Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification. Toxicol Pathol.,
30(6), 620-650. DOI: 10.1080/01926230290166724
Kordali. S., Kotan, R., Mavi, A., Cakir, A., Ala, A., and Yildirim, A. (2005). Determination of the
chemical composition and antioxidant activity ofthe essential oil of Artemisia dracunculus and of the
antifungal and antibacterial activities of Turkish Artemisia absinthium, A. dracunculus, Artemisia
santonicum, and Artemisia spicigera essential oils. J Agric Food Chem, 53, 9452-9458.
Kreft, S., Knapp, M., and Kreft, I. (1999). Extraction of rutin from buckwheat (Fagopyrum
esculentum Moench) seeds and determination by capillary electrophoresis. J Agric Food Chem, 47, 4649–
4652.
Kuete, V. (2013). Medicinal Plant Research in Africa: Chapter 5, Page 212, Pharmacology and
Chemistry, Elsevier, Oxford.
Kumar, A., and Schweizer, H.P. (2005). Bacterial Resistance to antibiotics: active efflux and reduced
uptake. Adv. Drug Delivery Rev., 57, 1486–1513.
33
Kunin, C.M. (1993). Resistance to antimicrobial drugs a worldwide calamity. Annals of Internal
Medicine, 118, 557-561.
Lalitha, P., Arathi, K.A., Shubashini, K., Sripathi, Hemalatha S., and Jayanthi, P. (2010). Antimicrobial
Activity and Phytochemical Screening of an Ornamental Foliage Plant, Pothosaurea (Linden ex Andre).
An Int. J. of Chem. 1(2), 63-71.
Li, M., Wang, B., and Zhang, M. (2008). Symbiotic gut microbes modulate human metabolic phenotypes.
Proceedings of the National Academy of Sciences, 105(6), 2117-2122.
Maisnier-Patin, S., and Andersson, D.I. (2004). Adaptation to the deleterious of antimicrobial drug
resistance mutations by compensatory evolution. Res. Microbiol., 155, 360–369.
Mandal, S., Mandal, D.M., and Pal, N.K. (2010). Synergistic anti-Staphylococcus aureus activity of
amoxicillin in combination with Emblica officinalis and Nymphae odorata extracts. Asian Pacific Journal
of Tropical Medicine, 3, 711-714.
Martini, N., and Eloff, J.N. (1998). The preliminary isolation of several antibacterial compounds from
Combretum erythrophyllum — (Combretaceae). Journal of Ethnopharmacology, 62, 255–263.
Masoko, P., and Eloff, J.N. (2005). The diversity of antifungal compounds of six South African
Terminalia species (Combretaceae) determined by bioautography. African Journal of Biotechnology, 4,
1425–1431.
Masoko, P., Picard, J., and Eloff, J.N. (2005). Antifungal activities of six South African Terminalia
species (Combretaceae). Journal of Ethnopharmacology, 99, 301–308.
McGaw, L.J., Jäger, A.K., and Van Staden, J. (2000). Antibacterial, anthelmintic andanti-amoebic activity
in South African medicinal plants. Journal of Ethnopharmacology, 72, 247–263.
McGaw, L.J., Rabe, T., Sparg, S.G., Jäger, A.K., Eloff, J.N., and Van Staden, J. (2001). An investigation
on the biological activity of Combretum species. Journal of Ethnopharmacology, 75, 43–50.
Milda, E.E. (2015). Spices and herbs: Natural sources of antioxidants – a mini review, Journal of
Functional Foods, DOI: 10.1016/j.jff.2015.03.005.
Milhau, G., Valentin, A., Benoit, F., Mallie, M., Bastide, J., Pelissier, Y., and Bessiere, J. (1997). In vitro
antimicrobial activity of eight essential oils. J EssentOil Res, 9, 329-333.
Moellering, J.R.C., Graybill, J.R., McGowan, J.J.E., and Corey, L. (2007). Antimicrobial resistance
prevention initiative—an update: proceedings of an expert panel on resistance. The American Journal of
Medicine, 120, S4–S25.
Murugesan, S., Pannerselvam, A., and Tangavelou, A.C. (2011). Phytochemical screening and
antimicrobial activity of the leaves of Memecylon umbellatumburm. F. Journal of Applied
Pharmaceutical Science, 1, 42-45.
Nascimento, G.G.F., Locatelli, J., Freitas, P.C., and Silva, G.L. (2000). Antibacterial activity of plant
extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol., 31(1), 247-56.
34
Okeke, I.N., Laxmaninarayan, R., Bhutta, Z.A., Duse, A.G., Jenkins, P., and O’Brien, T.F. (2005).
Antimicrobial resistance in developing countries. Part 1: recent trends and current status. J Lancet Infect
Dis, 5, 481-93.
Papadoupoulo, C., Soulti, K., and Roussis, I.G. (2005). Potential antimicrobial activity of red and white
wine phenolic extracts against strains of Staphyloccocus aureus, Escherichia coli and Candida albicans.
F. Tech-Biotech., 43, 41-46.
Pier-Giorgio, P. (2000). Flavonoids as Antioxidants. J. Nat. Prod., 63, 1035-1042.
Poli, G., Leonarduzzi, G., Biasi, F., and Chiarpotto, E. (2004). Oxidative stress and cell signaling. Curr.
Med. Chem., 11, 1163–1182.
Prashant, V.K., Chauhan, N.S., Padh, H., and Rajani, M. (2006). “Search for antibacterial and antifungal
agents from selected Indian medicinal plants,” J Ethnopharmacol, 107(2), 182-188.
Rastogi, R.P., and Mehrotra, B.N. (2002). Glossary of Indian Medicinal Plants. National Institute of
science communication, New Delhi, India.
Reinli, K., and Block, G. (1996). Phytoestrogen content of foods: a compendium of literature values. Nutr
Cancer Int J, 26, 123–148.
Rex, J.H., Alexander, B.D., Andes, D., Arthington-Skaggs, B., Brown, S.D., Chaturveli, V., Espinel-
Ingroff, A., Ghannoum, M.A., Knapp, C.C., Motyl, M.R., Ostrosky-Ziechner, L., Pfaller, M., Sheehan,
D.J., and Walsh, T.J. (2008). Reference method for broth dilution antifungal susceptibility testing of
filamentous fungi; Approved Standard-Second Edition. Clinical and Laboratory Standard Institute
(CLSI), M38A2 28(16).
Robbers, J., Speedie, M., and Tyler, V. (1996). Pharmacognosy and Pharmacobiotechnology. Baltimore:
Williams and Wilkins; 1-14.
Rousseff, R.L., Martin, S.F., and Youtsey, C.O. (1987). Quantitative survey of narirutin, naringin,
heperidin, and neohesperidin in citrus. J Agric Food Chem, 35, 1027–1030.
Sakagami, Y., and Kajimura, K. (2002). Bactericidal activities of disinfectants against vancomycin
resistant enterococci. J HospInfec., 50(2), 140-4.
Salwa, M.A.R., Sawsan, A.A., Sahar, F.D., and Ashraf, A.K. (2011). Antibacterial activity of some wild
medicinal plants collected from western Mediterranean coast, Egypt: Natural alternatives for infectious
disease treatment. African Journal of Biotechnology, 10(52), 10733-10743.
Schelz, Z., Hohmann, J., and Molnar, J. (2010). In Ethnomedicine: A Source of Complementary
Therapeutics, ed. D. Chattopadhyay, Research Signpost, 1st edn, 6, 179–201.
Seenivasan, P., Manickkam, J., and Savarimuthu, I. (2006). In vitro antibacterial activity of some plant
essential oils. BMC Complementary and Alternative Medicine, 6, 39, DOI:10.1186/1472-6882-6-39.
Seifried, H.E., Anderson, D.E., Fisher, E.I., and Milner, J.A. (2007). A review of the interaction among
dietary antioxidants and reactive oxygen species. The Journal of Nutritional Biochemistry, 18(9), 567–
579.
35
Shaguftalshiaque, K.N., Siddiqui, M.A., Siddiqi, R., and Shahina, N. (2013). Antioxidant Potential of the
Extracts, Fraction and Oils Derived from Oilseeds. Antioxidants, 2(4), 246-256.
Shai, L.J., McGaw, L.J., Masoko, P., and Eloff, J.N. (2008). Antifungal and antibacterial activity of seven
traditionally used South African plant species active against Candida albicans. South African Journal of
Botany, 74, 677–684.
Shobana, S., Akhilender, K., and Naidu. (2000). Antioxidant activity of selected Indian spices.
Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA), 62(2), 107–110.
Sibaram, P., Subhasish, M., and Mookerjee, M. (2012). Phytochemial investigation and evaluation of
anthelmintic activities of V. Negundo leaf extract. Int J Res Pharm Biomed Sci., 3, 1143‑1146.
Simoes, M., Rocha, R., Coimbra, M.A., and Vieira, M. (2008). Enhancement of Escherichia coli and
Staphylococcus aureus antibiotic susceptibility using sesquiterpenoids. Med. Chem., 4, 616–623.
Subashkumar, R., Sureshkumar, M., Babu, S., and Thayumanavan, T. (2013). Antibacterial effect of
crude aqueous extract of Piper betle L. Against pathogenic bacteria. Int J Res Pharm Biomed Sci., 4, 42-
46.
Sylvestre, M., Pichette, A., Longtin, A., Nagau, F., and Legault, J. (2006). Essential oil analysis and
anticancer activity of leaf essential oil of Crotonflavens L. from Guadeloupe. J Ethnopharmacol, 103, 99-
102.
Tegegnea, G., Pretoriusa, J.C., and Swartb, W.J. (2008). Antifungal properties of Agapanthus africanus
L. extracts against plant pathogens. Crop Protection, 27, 1052–1060.
Thomashow, L.S., Bonsall, R.E., and Weller, D.M. (1997). Antibiotic production by soil and rhizosphere
microbesin situ. In Manual of Environmental Microbiology, ed. CJ Hurst, GR Knudsen, MJ McInerney,
LD Stetzenbach, MV Walter, 493–99.
Tuba, A.K., and Ilhami, G. (2008). In Antioxidant and radical scavenging properties of Curcumin.
Chemico-Biological Interactions 174 (2008) 27–37.
Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J., and Telser, J. (2004). Role of oxygen radicals in DNA
damage and cancer incidence. Mol. Cell. Biochem, 266, 37–56.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.D., Mazur, M., and Telser, J. (2007). Free radicals and
antioxidants in normal physiological functions and human disease. The International Journal of
Biochemistry and Cell Biology, 39(1), 44–84.
Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., and Mazur, M. (2006). Free radicals, metals and
antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact., 160, 1–40.
Van de Braak, S.A.A.J., and Leijten, G.C.J.J. (1999). Essential Oils and Oleoresins: A Survey in the
Netherlands and other Major Markets in the European Union. CBI, Centre for the Promotion of
Importsfrom Developing Countries, Rotterdam. 116.
Virendra, V., Panpatil, S., Tattari, N., Kota, C., and Nimgulkar, K.P. (2013). In vitro evaluation on
antioxidant and antimicrobial activity of spice extracts of ginger, turmeric and garlic. Journal of
Pharmacognosy and Phytochemistry, 2(3), 143-148.
36
Wahyu, W., Caroline, T.S., Laura, W., Dian, R.L. and Lusiana, D. (2011). Free Radicals Scavenging
Activities of Spices and Curcumin. ISBN 978-979-25-1209-0.
Watve, M.G., Tichoo, R., Jog, M.M. and Bhole, B.D. (2001). How many antibiotics are produced by the
genus Streptomyces. Arch Microbiol, 176, 386-90.
Westh, H., Zinn, C.S. and Rosdahl, V.T. (2004). An international multicenter study of antimicrobial
consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microb.
Drug Resist., 10, 169-176.
Wilker, M.A., Low, D.E., Cockerill, F.R., Sheehan, D.J., Craig, W.A., and Tenover, F.C. (2006).
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically;
approvedstandard-seventh edition, Vol 26. Clinical and Laboratory Standards Institute (CLSI) M7-A7,
Wayne.
Wright, G.D. (2005). Bacterial Resistance to Antibiotic: Enzymatic Degradation and Modifications. Adv.
Drug Delivery Rev., 57, 1451–1470.
Young, I.S., and Woodside, J.V. (2001). Antioxidants in health and disease. J Clin Pathol., 54, 176–186.
Ziech, D., Franco, R., Georgakilas, A.G., Georgakila, S., Schoneveld, V.M.O., Pappa, A., and
Panayiotidis, M.I. (2010). The role of reactive oxygen species and oxidative stress in environmental
carcinogenesis and biomarker development. Chemico-Biological Interactions, 188(2), 334–339.
Zotchev, S.B. (2011). Marine actinomycetes as an emerging resource for the drug development pipelines.
J Biotechnol, http://dx.doi.org/10.1016/j.jbiotec.2011.1006.1002.