the study of pharmacognostical, phytochemical and...
TRANSCRIPT
THE STUDY OF PHARMACOGNOSTICAL,
PHYTOCHEMICAL AND BIOLOGICAL ACTIVITIES
OF RUBUS RACEMOSUS FAMILY ROSACEAE (ROXB)
Thesis submitted to
THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY
CHENNAI-600032
For the award of degree of
DOCTOR OF PHILOSOPHY
By
P. R. KUMAR, M.Pharm.,
Under the guidance of
Prof. V.VAIDHYALINGAM, M.Pharm,Ph.D.
C.L.BAID METHA
COLLEGE OF PHARMACY
CHENNAI – 600 097 APRIL 2009
CERTIFICATE
I certify that the thesis entitled “THE STUDY
OF PHARMACOGNOSTICAL, PHYTOCHEMICAL AND
BIOLOGICAL ACTIVITIES OF RUBUS RACEMOSUS FAMILY
ROSACEAE (ROXB)” submitted for the award of degree of Doctor of
Philosophy by Mr.P.R.Kumar is the record of research work carried out
by him during the period of 2005 – 2009 under my guidance and
supervision and this work has not formed the basis for the award to the
candidate of any degree, diploma, associateship, fellowship or other titles
in this or any other University or Institution of Higher Learning.
Date: Place:
(V.VAIDHYALINGAM)
DECLARATION I declare that the thesis entitled “THE STUDY
OF PHARMACOGNOSTICAL, PHYTOCHEMICAL AND
BIOLOGICAL ACTIVITIES OF RUBUS RACEMOSUS FAMILY
ROSACEAE (ROXB)” submitted by me for the award of degree of
Doctor of Philosophy is the record work carried out by me during the
period from 2005-2009 under the guidance of
Prof.V.VAIDHYALINGAM and has not formed the basis for the award
of any degree, diploma, associateship, fellowship or other titles in this or
any other University or Institution of Higher Learning.
Date: Place:
(P.R.KUMAR)
ACKNOWLEDGEMENT
I express my deep sense of gratitude and most respectful regards to my
guide Prof.V.Vaidhyalingam M.Pharm., Ph.D., Director, K.K.College of
Pharmacy, Chennai, for his excellent guidance, encouragement and continuous
inspiration throughout my dissertation work.
I am extremely grateful and express my heartful thanks to
Prof. Mrs. Grace Ratnam, M.Pharm, (PhD), Head of the Department,
Department of Pharmaceutics and Director of C.L.Baid Metha College of
Pharmacy, for her valuable contribution to my dissertation work.
I express my sincere thanks and gratitude to Dr.A.Shantha Arcot,
Principal, C.L.Baid Metha College of Pharmacy, Chennai for all the
encouragement and useful suggestions given by her throughout my research
work.
I am greatly indebted to my co-guide Prof.P.Muthusamy Ph.D. for his
valuable guidance.
I express my heartfelt thanks to Prof.P.Muralidharan, Head of the
Department, Pharmacology, C.L.Baid Metha College of Pharmacy, Chennai for
his valuable support in pharmacological studies.
I am very much thankful to Dr.S.Rajan, Field Botanist, Survey of
Medicinal Plants & Collection Unit, (Central Council for Research in
Homoeopathy), Department of AYUSH, Ministry of Health & Family Welfare,
Govt. of India, 112, Government Arts College Campus, Udhagamandalam,
Pin – 643 002 for his valuable suggestions about the plant.
I am highly thankful to Dr.S.Ravi, Senior Scientist, Centre for Medical
Plants Research, Kottakkal for his valuable suggestions for my thesis work.
It is my privilege to express my heartful sense of gratitude and immense
respect to Prof.V.Jayasingh, Professor, Jamia Salafia College of Pharmacy,
Kerala for his valuable suggestions and constant encouragement in my thesis
work.
I am extremely thankful to Prof.P.Jayaraman, Director, Plant Anatomy
Research Centre, Chennai- 45 for the helpful suggestions given during the
progress of my work.
I express my heartfelt thanks to Dr.Padma Venkat and
Mr.Chandrasekhar, Foundation for Revitalization of Local Health Traditions
(FRLHT), Bangalore for their help to collect the literature survey.
I owe my gratitude to Mrs.Banumathi, Librarian, C.L.Baid Metha
College of Pharmacy, Chennai., Mrs.Pratima Mathur, Drug Information
Centre for their help to collect the literature.
I take the privilege to thank Mr.Srinivasaraghavan, Mrs.Usha,
Mrs.Kalpakam, Mrs.Valli and Mrs.Radha the administration staff, C.L.Baid
Metha College of Pharmacy for their help.
Words are inadequate to express my gratitude and indebtedness to my
parents Mr.P.S.Ramachandran and Mrs.K.Padma.
I acknowledge deep sense of gratitude to my wife Mrs.V.Andal and my
beloved Daughters Harini and Deeptha for their abundant support.
Above all my humble thanks and prayers to Almighty who gave me
strength, confidence and capacity to complete my work.
Dedicated
to my
beloved parents
CONTENTS
CHAPTER TITLE PAGE NO. NO.
I INTRODUCTION 1 1.1 AIM AND OBJECTIVES OF THE STUDY 17
II REVIEW OF LITERATURE 18
III MATERIALS AND METHODS 26
3.1 GLASSWARE AND CHEMICALS 26
3.2 PLANT PROFILE 27
3.3 PLAN OF WORK 28
3.4 ANATOMICAL STUDIES 32
3.5 POWDER ANALYSIS 34
3.6 QUANTITATIVE MICROSCOPY 35
3.7 DETERMINATION OF LEAF
CONSTANTS 37
3.8 DETERMINATION OF
PHYSIOCHEMICAL CONSTANTS 40
3.9 PLANT MATERIAL AND EXTRACTION 44
CHAPTER TITLE PAGE NO. NO.
3.10 PRELIMINARY PHYTOCHEMICAL
SCREENING 46
3.11 THIN LAYER CHROMATOGRAPHY 50
3.12 ISOLATION OF CONSTITUENTS BY
COLUMN CHROMATOGRAPHY 51
3.13 HPTLC STUDIES 53
3.14 EXPERIMENTAL ANIMALS 54
3.15 TOXICOLOGICAL EVALUATION 55
3.16 ANTIDIABETIC ACTIVITY 60
3.17 BIOCHEMICAL ESTIMATIONS 65
3.18 ESTIMATION OF ANTIOXIDANT
ENZYME LEVELS IN VARIOUS TISSUES 68
3.19 ANTIOXIDANT STUDIES 73
3.20 ANTIEPILEPTIC ACTIVITY 76
3.21 ANTIMICROBIAL METHODS 80
CHAPTER TITLE PAGE NO. NO.
IV RESULTS 84
4.1 PHARMACOGNOSTICAL STUDIES 84
4.1.1 Macroscopy 84
4.1.2 Microscopy 85
4.1.3 Powder analysis 88
4.1.4 Fluorescence analysis 89
4.1.5 Quantitative microscopy 90
4.1.6 Determination of Leaf constants 91
4.1.7 Determination of physiochemical
constant 92
4.2 PHYTOCHEMICAL STUDIES 92
4.2.1 Extractive Values of different extracts 93 4.2.2 Preliminary Phytochemical Screening 93
4.2.3 Fluorescence analysis of different
extracts 96
4.2.4 Thin layer chromatography 96
CHAPTER TITLE PAGE NO. NO.
4.2.5 Isolation of constituents by column
Chromatography 98
4.2.6 Spectral studies 100
4.2.6.1 Compound – I 100
4.2.6.2 Compound – II 103
4.2.7 HPTLC Studies 106
4.3 PHARMACOLOGICAL STUDIES 107
4.3.1 Acute oral toxicity studies 107
4.3.2 Sub acute toxicity studies 107
4.3.3 Haematological Parameters 108
4.3.4 Antidiabetic activity 108
4.3.4.1 Effect of MERR on blood glucose level in normal rats 108
4.3.4.2 Effect of MERR on blood glucose level on glucose fed hyperglycemic rats 108
4.3.4.3 Effect of acute treatment of MERR on blood glucose level in streptozotocin induced diabetic rats 109
CHAPTER TITLE PAGE NO. NO.
4.3.4.4 Effect of sub-acute treatment of
MERR on blood glucose level in
streptozotocin induced diabetic rats 109
4.3.4.5 Biochemical Estimation 110
4.3.4.5.1 Total bilirubin 110
4.3.4.5.2 Serum glutamate
oxalocetate transminase 110
4.3.4.5.3 Serum glutamate
pyruvate transminase 110
4.3.4.5.4 Serum total protein 111
4.3.4.5.5 Alkaline phosphatase 111
4.3.4.5.6 Serum total cholesterol 111
4.3.4.5.7 Serum HDL cholesterol 112
4.3.4.5.8 Serum triglyceride 112
4.3.4.5.9 Serum LDL-Cholesterol 112
4.3.4.6 Antioxidant enzymes in liver
Homogenate 113
4.3.4.6.1 Superoxide dismutase 113
4.3.4.6.2 Catalase 113
4.3.4.6.3 Glutathione peroxidase 113
CHAPTER TITLE PAGE NO. NO.
4.3.4.6.4 Glutathione reductase 114
4.3.4.6.5 Lipid peroxidation 114
4.3.5 In vitro anti-oxidant studies 115
4.3.5.1 Free radical scavenging activity
by DPPH reduction 115
4.3.5.2 Nitric oxide scavenging activity 115
4.3.5.3 Hydroxyl radicals scavenging activity 115
4.3.5.4 Determination of reducing power 116
4.3.5.5 Determination of total phenolic
compounds 116
4.3.6 Antiepileptic activity 117
4.3.6.1 Maximal electroshock induced
convulsion 117
4.3.6.2 Effect of MERR on neurotransmitter
levels in MES induced rats 117
4.3.6.2.1 Serotonin 117
4.3.6.2.2 Nor-adrenaline 117
4.3.6.2.3 Dopomine 118
CHAPTER TITLE PAGE NO. NO.
4.4 ANTIMICROBIAL STUDIES 119
4.4.1 Antibacterial Activity 119
4.4.2 Antifungal Activity 120
V DISCUSSION 161
VI SUMMARY AND CONCLUSION 173
VII REFERENCES 176
LIST OF TABLES
TABLE TITLE NO.
1. Fluorescence analysis
2. Preliminary phytochemical screening
3. Fluorscence analysis of different extracts
4. TLC for flavanoids
5. TLC for glycoside
6. TLC for tannins
7. Examination of eluates
8. TLC for isolated compound
9. Spectral studies – compound II
10. HPTLC studies
11. Acute toxicity class method OECD guidelines 423
12. Haematological parameters of MERR
treatment on sub-acute toxicity study
TABLE TITLE NO.
13. Histopathology report of various organs on
MERR treatment on sub-acute toxicity study
14. Effect of Rubus Racemosus treatment on blood
glucose level in normoglycaemic rats
15. Effect of Rubus Racemosus on blood
glucose fed hyperglycemic rats
16. Effect of acute treatment of Rubus Racemosus
on blood glucose in STZ induced diabetic rats
17. Effect of sub-acute treatment of Rubus Racemosus
on blood glucose in STZ induced diabetic rats
18. Effect of Methanolic Extract of Rubus Racemosus on
Serum Total Bilirubin in STZ induced diabetic rats
19. Effect of Methanolic Extract of Rubus Racemosus
on SGOT in STZ induced diabetic rats
TABLE TITLE NO.
20. Effect of Methanolic Extract of Rubus Racemosus
on SGPT in STZ induced diabetic rats
21. Effect of Methanolic Extract of Rubus Racemosus
on Serum Total Protein in STZ induced diabetic rats
22. Effect of Methanolic Extract of Rubus Racemosus
on Serum Alkaline phosphatase in STZ induced
diabetic rats
23. Effect of Methanolic Extract of Rubus Racemosus
on Serum Total Cholesterol in STZ induced
diabetic rats
24. Effect of Methanolic Extract of Rubus Racemosus
on Serum HDL- Cholesterol in STZ induced
diabetic rats
25. Effect of Methanolic Extract of Rubus Racemosus
on Serum Triglyceride in STZ induced
diabetic rats
TABLE TITLE NO.
26. Effect of Methanolic Extract of Rubus Racemosus
on Serum LDL- Cholesterol in STZ induced
diabetic rats
27. Effect of Methanolic Extract on Superoxide
dismutase in STZ induced diabetic rats
28. Effect of Methanolic Extract on Catalase in STZ
induced diabetic rats
29. Effect of Methanolic Extract on Glutathione
Peroxidase in STZ induced diabetic rats
30. Effect of Methanolic Extract on Glutathione
reductase in STZ induced diabetic rats
31. Effect of Methanolic Extract on Lipid
Peroxidation in STZ induced diabetic rats
TABLE TITLE NO.
32. Histopathological studies of the Liver
33. Histopathological Studies of the Pancreas
34. Free radical scavenging activity of MERR
by DPPH reduction
35. Nitric oxide scavenging activity of MERR
36. Hydroxyl radical scavenging activity of MERR
37. Effect of MERR and BHT on reducing power
38. Effect of MERR on MES induced epilepsy
39. Effect of MERR on Serotonin levels in MES
induced epilepsy
40. Effect of MERR on Non adrenaline levels in
MES induced epilepsy
41. Effect of MERR on Dopamine levels in MES induced epilepsy
TABLE TITLE NO.
42. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Staphylococcus aureus
43. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Staphylococcus epidermidis
44. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Bacillus cereus
45. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Micrococcus luteus
46. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Klebsiella pneumoniae
TABLE TITLE NO.
47. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Pseudomonos aeruginosa
48. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Escherichia coli
49. In vitro evaluation of Anti-fungal activity
of different extracts of Rubus racemosus on
Aspergillus Niger
50. In vitro evaluation of Anti-fungal activity
of different extracts of Rubus racemosus on
Aspergillus fumigatus
LIST OF FIGURES
FIGURE TITLE NO.
1. Rubus racemosus - Habitat
1.1 Flower of Rubus Racemosus
1.2 Leaves of the plant
2. OECD guideline
3. Cross Sectional View of young folded Leaf
4. Anatomy of the Leaf
4.1 Transverse Section of Leaf through midrib with lamina
4.2 Transverse Section of Midrib
4.3 Transverse Section of Lamina
5. Anatomy of the midrib and Trichomes
5.1 Transverse section of young leaf showing midrib
and lateral vein
5.2 Glandular and Non-glandular trichomes in sectional view
5.3 Glandular and Non-glandular trichomes enlarged
FIGURE TITLE NO.
6. Anatomy of the Petiole
6.1 Transverse Section of petiole entire view [Distal region]
6.2 Transverse Section of petiole ground Plane [Proximal region]
7. Structure of the Petiole – Proximal region
8. Anatomy of the Young Stem
8.1 Transverse Section of Stem under low magnification
8.2 Transverse Section of Stem under high magnification
9. Glandular trichome of the Stem
9.1 Transverse Section of Stem showing a glandular trichome
9.2 Glandular trichome enlarged
10. Trichome Morphology
10.1 Non-glandular trichome stained with Toluidine Blue
10.2 Non-glandular trichome stained with Safranin
11. Histopathology of various organs in toxicity studies
FIGURE TITLE NO.
12. Effect of Rubus Racemosus treatment on blood
glucose level in normoglycaemic rats
13. Effect of Rubus Racemosus treatment on blood
glucose fed hyperglycemic rats
14. Effect of acute treatment of Rubus Racemosus
on blood glucose level in STZ induced diabetic rats
15. Effect of sub-acute treatment of Rubus Racemosus
on blood glucose in STZ induced diabetic rats
16. Effect of Methanolic Extract of Rubus Racemosus on
Serum Total Bilirubin in STZ induced diabetic rats
17. Effect of Methanolic Extract of Rubus Racemosus
on SGOT in STZ induced diabetic rats
18. Effect of Methanolic Extract of Rubus Racemosus
on SGPT in STZ induced diabetic rats
FIGURE TITLE NO.
19. Effect of Methanolic Extract of Rubus Racemosus
on Serum Total Protein in STZ induced diabetic rats
20. Effect of Methanolic Extract of Rubus Racemosus
on Serum Alkaline phosphatase in STZ induced
diabetic rats
21. Effect of Methanolic Extract of Rubus Racemosus
on Serum Total Cholesterol in STZ induced
diabetic rats
22. Effect of Methanolic Extract of Rubus Racemosus
on Serum HDL- Cholesterol in STZ induced
diabetic rats
23. Effect of Methanolic Extract of Rubus Racemosus
on Serum Triglyceride in STZ induced diabetic rats
FIGURE TITLE NO.
24. Effect of Methanolic Extract of Rubus Racemosus
on Serum LDL- Cholesterol in STZ induced
diabetic rats
25. Effect of Methanolic Extract on Superoxide
dismutase in STZ induced diabetic rats
26. Effect of Methanolic Extract on Catalase
in STZ induced diabetic rats
27. Effect of Methanolic Extract on Glutathione Peroxidase in STZ induced diabetic rats
28. Effect of Methanolic Extract on Glutathione
reductase in STZ induced diabetic rats
29. Effect of Methanolic Extract on Lipid Peroxidation
in STZ induced diabetic rats
FIGURE TITLE NO.
30. Histopathology of the liver section
31. Histopathology of the pancreatic section
32. Free radical scavenging activity of MERR
by DPPH reduction
33. Nitric oxide scavenging activity of MERR
34. Hydroxyl radical scavenging activity of MERR
35. Effect of MERR and BHT on reducing power
36. Effect of MERR on MES induced epilepsy
51. Effect of MERR on Serotonin levels in MES
induced epilepsy
52. Effect of MERR on Non adrenaline levels in
MES induced epilepsy
53. Effect of MERR on Dopamine levels in MES
induced epilepsy
FIGURE TITLE NO.
54. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Staphylococcus aureus
55. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Staphylococcus epidermidis
56. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Bacillus cereus
57. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Micrococcus luteus
58. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Klebsiella pneumoniae
FIGURE TITLE NO.
59. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Pseudomonos aeruginosa
60. In vitro evaluation of Anti-microbial activity
of different extracts of Rubus racemosus on
Escherichia coli
61. In vitro evaluation of Anti-fungal activity
of different extracts of Rubus racemosus on
Aspergillus Niger
62. In vitro evaluation of Anti-fungal activity
of different extracts of Rubus racemosus on
Aspergillus fumigatus
LIST OF SPECTRA
SPECTRA TITLE NO.
1. Compound I
1.1 I.R. Spectrum
1.2 1H NMR Spectrum
1.3 13C NMR Spectrum
1.4 MASS Spectrum
2. Compound II
2.1 I.R. Spectrum
2.2 I.R. Spectrum
2.3 1H NMR Spectrum
2.4 13C NMR Spectrum
2.5 MASS Spectrum
3. HPTLC Studies
3.1 Spectrum scan parameters
3.2 Spectrum for extract
3.3 Spectrum for isolated compound
LIST OF ABBREVIATIONS USED
Ab - Accessary bundle
AbE - Abaxial Epidermis
Abs - Abaxial side
AdG - Adaxial groove
Ads - Adaxial side
ALP - Alkaline Phosphatase
ANOVA - Analysis of Variance
AR - Analytical Reagent
B - Body of the trichome
beta - β cell
BHT - Butylated Hydroxyl Toluene
b.w - body weight
CAT - Catalase
cfu - colony forming unit
Chl - Chlorenchyma
Co - Cortex
Col - Colenchyma
CPCSEA - Committee for the Purpose of Control
and Supervision of Experiments on Animals
Cr - Crystal
DA - Dopamine
DNA - Deoxyribonucleic acid
DPPH - 1,1-diphenyl-2-picrylhydrazyl
EDTA - Ethylene Diamine Tetra Acetic acid
Ep - Epidermis
ESR - Erythrocyte Sedimentation Rate
GABA - Gamma-Aminobutyric Acid
GHS - Globalised Harmonized System
GPx - Glutathione Peroxidase
GSH - Reduced Glutathione
GSH-Rase - Glutathione Reductase
GSSG - Glutathione oxidized
GST - Glutathione –s-transferase
GT - Glandular Trichome
H - Head
HCl - Hydrochloric acid
H and E - Hematoxylin and Eosin stain
Hb - Haemoglobin
HDL - High-Density Lipoprotein
HLTE - Hind Limb Tonic Extension
HPTLC - High Performance Thin Layer
Chromatography
H2O2 - Hydrogen peroxide
Hy - Hypoderm
IAEC - Institutional Animal Ethics Committee
ICDRA - International Conference of Drug Regulatory
Authorities
i.p - intra peritoneal
IDDM - Insulin Dependent Diabetes Mellitus
La - Lamina
LB - Lateral Bundle
LC50 - Median Inhibition Concentration LD50 - Lethal Dose 50%
LDL - Low-Density Lipoprotein
LPO - Lipid Peroxidation
Lv - Lateral vein
IU/dl - International Units per deciliter
MB - Median Bundle
Mc - Mucilaginous cell
MDA - Malondialdehyde
MERR - Methanolic Extract of Rubus Racemosus
MES - Maximal electroshock
mg - milligram
mg/dl - milligram/deciliter
MIC - Minimal Inhibitory Concentration
min - minutes
ml - milliliter
mM - milimoles
MR - Midrib
NA - Noradrenaline
NaCl - Sodium chloride
NADP - Nicotinamide adenine Dinucleotide
Phosphate Reduced
NADPH - Nicotinamide Adenosine Dinucleotide
Phosphate
NGT - Non glandular trichome
Nm - Nanometer
NO - Nitric oxide
Ns - Non significant
OD - Optical Density
OECD - Organisation for Economic Co operation
and Development
OGTT - Oral Glucose Tolerance Test
OPT - O-phthalaldehyde
P - Parenchyma
Pa - Palisade tissue
PCV - Packed Cell Volume
Pf - Pericyclic fibre
Ph - Phloem
PHT - Phenytoin
Pi - Pith
PM - Palisade Mesophyll
po - per oral
RBC - Red Blood Cells
Rf - Relative factor
ROS - Reactive Oxygen Species
rpm - revolutions per minute
Sc - Sclerenchyma cells
SCMC - Sodium Carboxymethyl- Cellulose
SEM - Standard Error Mean
SGOT - Serum Glutamic-Oxaloacetic Transaminase
SGPT - Serum Glutamic-Pyruvate Transaminase
SI - Stomatal Index
SM - Spongy Mesophyll
SOD - Superoxide Dismutase
Sp - Spongy tissue
sq.mm - square millimeter
ST - Stalk cells
St - Stoma
STZ - Streptozotocin
T1DM - Type 1 Diabetes Mellitus
T2DM - Type 2 Diabetes Mellitus
TBA - Thio Barbituric Acid
TCA - Trichloro Acetic acid
TBARS - Thio Barbituric Acid Reactive Substances
TLC - Thin Layer Chromatography
Tr - Trichome
TRIS-HCL - Tris Hydrochloride
UV - Ultraviolet-Visible
V - Vessel
Vb - Vascular bundle
Vi - Vein islet
Vs - Vascular strand
Vt - Vein termination
WBC - White Blood Cells
W/V - Weight/Volume
WHO - World Health Organisation
Wi - Wing
Xy - Xylem
% - Percentage
µg - Microgram
µl - Microlitre
INTERNATIONALLY APPROVED NUMBER FOR STRAINS
NAME OF THE STRAIN
MICROBIAL TYPE OF CULTURE COLLECTION
AMERICAN TYPE OF CULTURE COLLECTION
Staphylococcus aureus 737 6538 p
Staphylococcus epidermides 435 155
Bacillus cereus 430 11778
Micrococcus luteus 106 4698
Klebsiella pneumoniae 618 29665
Pseudomonos aeruginosa 1688 9027
Escherichia coli 1687 8739
Aspergillus niger 1344 16404
Aspergillus fumigate 2550 13073
INTRODUCTION
CHAPTER - I
INTRODUCTION
Herbal Medicine sometimes referred as Herbalism or Botanical
Medicine is the use of herbs for their therapeutic or medicinal value. A herb is
a whole plant or plant part valued for its medicinal, aromatic or savory
qualities. Herbal plants contain a variety of chemical substances that act upon
the body.
Herbal medicine is a major component in all indigenous traditional
system of medicine and a common element in Ayurvedic, homeopathic,
naturopathic, traditional oriental and Native American Indian medicine. WHO
notes that of 119 plant-derived pharmaceutical medicines, about 74 percent are
used in modern medicine in ways that correlated directly with their traditional
uses as plant medicines by native cultures.
The World Health Organization (WHO) estimates that 4 billion people,
i.e. 80 percent of the world population, presently use herbal medicine for some
aspect of primary health care1.
In 20th century much of the pharmacopoeia of scientific medicine was
derived from the herbal folklore of native peoples. Many drugs commonly used
today are of the herbal origin. Indeed, about 25 percent of the prescribed drugs
dispensed in the United States contain at least one active ingredient derived
from plant material. Some are made from plant extracts, others are synthesized
to mimic a natural plant compound.
The use of medicinal plants for healing purpose predates human history
and forms the origin of much modern medicines. Many conventional drugs
originate from plant sources. A century ago most of the few effective drugs
were plants based. Examples include aspirin (from willow bark), Digoxin (from
fox glove), Quinine (from cinchona bark) and Morphine (from the opium
poppy).
The leads of CNS active medicinal plants which have emerged besides
Rawolfia serpentina, Mucuna pruriens for Parkinson’s disease, Ocim santum as
an antistress agent, Withania somnifera as anxiolytic. The study related to
epilepsy is focused towards the traditional medicine. The recent trends in the
pharmacological studies are based on the biochemical and molecular
mechanism which leads to the development of CNS active principles from the
herbal drugs2.
Traditional medicine3 has been practiced for millennia. Resulting in a
particularly long and rich heritage that continues to influence growing
acceptance of the efficacy and clinical use of traditional herbal medicines.
Practices of traditional medicine can be categorized into two sample groups.
One category constitutes highly evolved and disciplined applications of both
medical and pharmaceutical methodologies. Among some of the most
developed practices are Traditional Chinese Medicine and Traditional Indian
Medicine (such as Ayurveda and Sidda). Which engender distinct,
comprehensive and systematic principles as the foundation of their specific
therapeutic practices? Traditional Chinese Medicine has been widely adopted
in many neighbouring regions of China, where it has been customarily
modified by incorporating indigenous ingredients and/ or local practices; a few
specific examples include, Kampo Medicine (Japan), Han Medicine (Korea),
and North Medicine (Vietnam). Ayurveda is also popularly exercised
worldwide, largely advocated and exercised by Indian scholars and migrants.
Distinctly, Folk Medicine comprises another category of traditional
medicine varies considerably around the world. In Japan, the system of
traditional (Kampo) medicine was officially integrated into the healthcare
system in1976. Today many Kampo herbal medicines are commonly available
as OTC or prescription drugs. Japan’s pharmaceutical industry accounts for
sales of approximately 6.2 trillion yen annually, of which Kampo medicines
constitute around 2%, according to 1995 statistics. Nearly 65-70% of Japanese
medical doctors utilize Kampo medicines in clinical practice, either exclusively
or in combination with modern drugs.
The nation’s medical school curriculum is now being revised, to better
educate physicians on the theories and uses of Kampo medicines, while until
now Japanese doctors had been trained only in the applications of modern
drugs.
In Germany, herbal remedies are particularly prevalent, both as self-
medications and prescription drugs, constituting nearly 5.4% of all medical
prescriptions and 10% of the entire drug market. In the US, however the food
and drug administration has conventionally prohibited the lawful use of herbal
medicines. Nevertheless, the growing interest in herbal medicines throughout
the world can be ascribed to benefits of cost effectiveness, efficacy and
generally reduced side-effects, of many of these treatments. The US
government has recently classified herbal medicines and the products of
medicinal plants as dietary supplements. However, this half-step measure lacks
the regulation necessary to prevent the misuse of herbal medicines (designated
as dietary supplements) which can exhibit adverse effects, and chronic or acute
toxicity if over-ingested.
More recently, though, the US has substantially increased its funding
allocated to Complimentary and Alternative Medicine by millions of dollars,
while further establishing. The office of alternative medicine, in order to better
facilitate research and clinical trials. These are welcome measures towards the
development of more practical and effective regulatory policies concerning the
use of traditional herbal remedies and phytomedicines.
In the midst of the present boom in natural products and herbal
medicines, and intends to introduce some of the most current research on
traditional herbal medicines, and to depict potential and functional applications
of these agents in our modern healthcare systems, which continue to be
practiced most widely in Asia. The contributing studies also characterize state-
of –the-art pharmacological mechanisms and develop more effective
formulations of traditional herbal remedies, in concert with their potential roles
in our modern healthcare systems. The editors believe much more basic
pharmacological research into traditional herbal medicines is essential, and that
will assist continued research as an important reference.
During the past decade, traditional systems4 of medicine have become a
topic of global importance. Current estimates suggest that, in many developing
countries, a large proportion of the population relies heavily on traditional
practitioners and medicinal plants to meet primary health care needs. Although
modern medicine may be available in these countries, herbal medicines
(phytomedicines) have often maintained popularity for historical and cultural
reasons. Concurrently, many people in developed countries have begun to turn
to alternative or complementary therapies, including medicinal herbs.
Few plant species that provide medicinal herbs have been scientifically
evaluated for their possible medical application. Safety and efficacy data are
available for even fewer plants, their extracts and active ingredients, and the
preparations containing them. Furthermore, in most countries the herbal
medicines market is poorly regulated, and herbal products are often neither
registered nor controlled. Assurance of the safety, quality, and efficacy of
medicinal plants and herbal products has now become a key issue in
industrialized and in developing countries. Both the general consumer and
health-Care professionals need Up-to-date, authoritative information on the
safety and efficacy of medicinal plants.
During the fourth International Conference of Drug Regulatory
Authorities (ICDRA) held in Tokyo in 1986, WHO was requested to compile a
list of medicinal plants and to establish international specifications for the most
widely used medicinal plants and simple preparations. Guidelines for the
assessment of herbal medicines were subsequently prepared by WHO and
adopted by the sixth ICDRA in Ottawa, Canada, in 1991. As a result of
ICDRA’s recommendations and in response to requests from WHO’s member
states for assistance in providing safe and effective herbal medicines for use in
national health-care systems, WHO is now publishing this first volume of 28
monographs on selected medicinal plants; a second volume is in preparation.
Preparation of the monographs
The medicinal plants featured in this volume were selected by an
advisory group in Beijing in 1994. The plants selected are widely used and
important in all WHO regions, and for sufficient scientific information seemed
available to substantiate safety and efficacy. The monographs were drafted by
the WHO Collaborating Centre for Traditional Medicine at the University of
Illinois at Chicago, United States of America. The content was obtained by a
systematic review of scientific literature from 1975 until the end of 1995:
review articles: bibliographies in review articles; many pharmacopoeias-the
International, African, British, Chinese, Dutch, European, French, German,
Hungarian, Indian, and Japanese; as well as many other reference books.
Draft monographs were widely distributed, and some 100 experts in
more than 40 countries commented on them. Experts included members of
WHO’s expert Advisory panels on Traditional Medicine, on the International
pharmacopoeia and pharmaceutical preparations, and on Drug Evolution and
National Drug policies; and the drug regulatory authorities of 16 countries.
A WHO Consultation on selected Medicinal plants was held in Munich,
Germany, in 1996. Sixteen experts and drug regulatory authorities from
Member states participated. Following extensive discussion, 28 of 31 draft
monographs were approved. The monographs on one medicinal plant was
rejected because of the plant’s potential toxicity. Two others will be
reconsidered when more definitive data are available. At the subsequent eighth
ICDRA in Bahrain later in 1996, the 28 model monographs were further
reviewed and endorsed, and Member states requested WHO to prepare
additional model monographs.
Purpose and content of the monographs
Provide scientific information on the safety, efficacy, and quality
control/quality assurance of widely used medicinal plants, in order to facilitate
their appropriate use in Member states;
Provide models to assist member states in developing their own
monographs or formularies for these or other herbal medicines; and facilitate
information exchange among member states.
Readers will include members of regulatory authorities, practitioners of
orthodox and of traditional medicine, pharmacists, other health professionals,
manufactures of herbal products, and research scientists.
Each monograph contains two parts. The first part consists of
pharmacopoeial summaries for quality assurance: botanical features,
distribution, identity tests, purity requirements, chemical assays, and active or
major chemical constituents. The second part summarizes clinical applications,
pharmacology, contraindications, warnings, precautions, potential adverse
reactions, and posology.
In each pharmacopoeial summary, the Definition section provides the
Latin binomial pharmacopoeial name, the most important criterion in quality
assurance. Latin pharmacopoeial synonyms and vernacular names, listed in the
sections Synonyms and selected vernacular names, are those names used in
commerce or by local consumers. The monographs place outdated botanical
nomenclature in the synonyms category, based on the International Rules of
Nomenclature.
For example, Aloe barbadensis Mill is actually Aloe Vera (L.) Burm.
Cassia acutifolia Delile and Cassia angustifolia Vahl., often treated in separate
monographs, are now believed to be the same species, Cassia senna L.
Matricaria chamomile L., M.recutita L., and M.suaveolens L. have been used
for many years as the botanical name for chamomile. However, it is now
agreed that the name Chamomilla recutita (L.) Rauschert is the legitimate
name.
The vernacular names listed are a selection of names from individual
countries worldwide, in particular from areas where the medicinal plants in
common use. The lists are not complete, but reflect the names appearing in the
official monographs and reference books consulted during preparation of the
WHO monographs and in the Natural products Alert (NAPRALERT) database
(a database of literature from around the world on ethnomedical, biological and
chemical information on medicinal plants, fungi and marine organisms, located
at the WHO Collaborating Centre for Traditional Medicine at University of
Lillinois at Chicago).
A detailed botanical description (under Description) is intended for
quality assurance at the stages of production and collection, whereas the
detailed description of the drug material (under Plant material of interest) is for
the same purpose at the manufacturing and commerce stages. Geographical
distribution is not normally found in official compendia, but it is included here
to provide additional quality assurance information.
General identity tests, Purity tests, and chemical assays are all normal
compendial components included under those headings in these monographs.
Where purity tests do not specify accepted limits, those limits should be set in
accordance with national requirements by the appropriate Member State
authorities.
Each medicinal plant and the specific plant part used (the drug) contain
active or major chemical constituents with a characteristic profile that can be
used for chemical quality control and quality assurance. These constituents are
described in the section major chemical constituents.
The second part of each monograph begins with a list of Dosage forms
and of Medicinal uses categorized as those uses supported by clinical data,
those uses described in pharmacopoeias and in traditional systems of medicine,
and those uses described in folk medicine, not yet supported by experimental or
clinical data.
The first category includes medical indications that are well established
in some countries and that have been validated by clinical studies documented
in the world’s scientific literature. The clinical trials may have been controlled,
randomized, double-blind studies, open trials, or well-documented observations
of therapeutic applications. Experts at the Munich Consultation agreed to
include Folium and Fructus Sennae, Aloe, Rhizoma Rhei, and herba Ephedrae
in this category because they are widely used and their efficacy is well
documented in the standard medical literature.
The second category includes medicinal uses that are well established in
many countries and are included in official pharmacopoeias or national
monographs. Well-established uses having a plausible pharmacological basis
and supported by older studies that clearly need to be repeated are also
included. The references cited provide additional information useful in
evaluating specific herbal preparations. The uses described should be reviewed
by local experts and health workers for their applicability in the local situation.
The third category refers to indications described in unofficial
pharmacopoeias and other literature, and to traditional uses. The
appropriateness of these uses could not be assessed, owing to a lack of
scientific data to support the claims. The possible use of these remedies must
be carefully considered in the light of therapeutic alternatives.
The final sections of each monograph cover Pharmacology (both
experimental and clinical); Contraindications such as sensitivity or allergy;
Warnings; Precautions, including discussion of drug interactions,
carcinogenicity, teratogenicity and special groups such as children and nursing
mothers; Adverse reactions; and Posology.
Man has been using herbs and plants products for combating diseases
since times immemorial.
The Indian subcontinent is enriched by a variety of flora – both aromatic
and medicinal plants. This is due to the wide diversity of climate conditions in
India ranging from deserts to swamplands. Numerous types of herbs have been
well recognized and catalogued by botanists from the high ranges of the
Himalayan tract up to the sea-shore of Kanyakumari. This extensive flora has
been greatly utilized as a source of many drugs in the Indian traditional system
of medicine.
In India, the earliest mention of the use of medicinal plants is to be
found in the Rigveda which was written between 4500-1600 BC. A detailed
account of the world’s first symposium on medicinal plants is given in the first
chapter of Vrihat Samhita and since 1600 BC the amount of literature on this
subject is boundless. The traditional system of medicine is so engrained in our
culture that, even now 75% of the Indian population depend on this indigenous
system for relief. With such a huge section of an ever-increasing population
relying on herbal remedies, it is imperative that the plant products which have
been in use for such a long time be scientifically supported for their efficacy.
The World Health Organisation is now actively encouraging developing
countries to use herbal medicine which they have been traditionally used for
centuries. They have identified 3000 plants from the forests of India and other
tropical countries which can be used as medicine. The active ingredients from
these plants are worth nearly Rs.2000 crores of rupees for the US market alone
and nearly 8 times that for the world market. Only with the scientific
advancements in the fields of pharmacology and toxicology in the western
hemisphere, has drug development based on natural products gained intensity
in Europe and USA. The importance of such an investigation, in India was
realized long back and the first systematic study with these aims was started by
Sir. Ramanath Chopra at Calcutta about 45 years back.
In the early stages, the science of medicine developed around those
plants which had curative properties. A continued search for medicinal plants
during the last several centuries has given rise to a long list of plants which are
of great use in the treatment of diseases, and for promoting health. It can be
stated, more or less truthfully, that every disease has a cure in a plant growing
in nature. Recently, Moose has described a number of vegetable drugs that can
be used as single drug remedies.
Drug used in medicine today, are either obtained from nature or are of
synthetic origin. Natural drugs are those obtained from plants, animals,
microbes or minerals. Those obtained from plants and animals are called drugs
of biological origin and are produced in the living cells of plants or animals.
Until now, only 6000 plant constituents have been isolated and studied.
The flora on this earth, representing and inexhaustible source of medicinal
plants, remains incompletely explored. This unexplored world provides the
most challenging aspects of pharmaceutical and medical science to scientists in
search of new and more potent drugs with marked therapeutic virtues and
negligible side effects. During the last few decades, tremendous progress has
been made in the study of phytochemicals.
Natural products, as a basis for new drugs, have great promise and it is
gratifying to note that the World Health Organization have shown an abiding
interest in plant-derived medicines, described in the folklore of various
countries.
Plants have been one of the important sources of medicines since the
dawn of human civilization. For instance, the Chinese drug Mahung was in use
for over 5000 years for the treatment of different types of fever and respiratory
disorders. Cinchona sp was in use in Peru even in 1825, primarily for
controlling malaria. In spite of the tremendous development in the field of
synthetic drugs and antibiotics during the 21st century, plants still contribute
one of the major sources of drugs in modern as well as traditional medicine
throughout the world. One-third of the world’s population treat themselves
with traditional medicines. Some of the compounds now commonly used in
medicine were isolated from plant sources and used as early as in the 19th
century. Examples are morphine (1803), quinine (1812), atropine (1831),
papaverine (1848), cocaine (1860), digitoxin (1865), and pilocarpine (1875).
Examples of some important compounds isolated in the 20th century include
ergotamine (1518), labeline (1921), digoxin (1930), reserpine (1931),
tubocuraine (1935) diosganin, vincristine (1961) and vinblastine (1963).
Plants are the only economic source of a number of well-established and
important drugs. In addition, they are also the source of chemical intermediates
needed for the production of some drugs.
As stated before, about 75% of the Indian population relies heavily on
the use of herbal drugs for the treatment of diseases. The factory responsible
for the continued and extensive use of herbal remedies in India are their
effectiveness, easy availability, low cost, comparatively less toxic effect and
the shortage of practitioners of modern medicine in rural areas. There is a
growing appreciation in India, as in many other developing countries, of the
need to make greater use of traditional remedies in order to be able to provide
medicine for primary health care.
Although use of traditional remedies is advantageous, it does suffer
some limitations. The main limitation is the lack of standardization of raw
materials, of processing methods and of the final products, dosage formulation,
and the non-existence of criteria for quality control.
Research has to be directed to the use of modern scientific methodology
and techniques to standardize all these steps and for quality control.
Before Independence, the production of plant-based drugs in India was
confined mainly to cinchona and opium alkaloids, galenicals (i.e. medicine
extracted from plants) and tinctures. In the last three decades, bulk production
of plant drugs has become an important aspect of the India pharmaceutical
industry. Some of the drugs which are manufactured today include morphine,
codeine, papaverine, the baine, emetine, quinine, quinidine, digoxin, caffeine,
hyoscine, hyocyamine, atropine, xanthotoxin, sennosides, colchicines,
berberine, vinblastine, vincristine and ergot alkaloids, papaine, nicotine,
strychnine, brucine and pyrethroids.
In India, there are about 20 well-recognised manufacturers of herbal
drugs, 140 medium or small-scale manufacturers, and about 1200 licensed
small manufacturers on record, in addition to many vaidyas having small
manufacturing facilities. The estimated current annual production of herbal
drugs is around Rs. 100 crores. The demand for herbal remedies is ever-
increasing. Herbal medicines represent an estimated $60-billion a year global
market, about 20 percent of the overall drug market, according to the United
Nations agency. There are 1650 herbal formulations in the Indian market and
540 major plants involved in their formulations.
During the last two decades, over 3000 plants have been screened in
India for their biological activities. As a result, a number of new drugs have
been introduced in clinical practice and some are in the advance stages of
clinical development.
There are well-documented scientific data on a good number of
medicinal plants that have been investigated. In spite of all these efforts, very
few drugs of plant origin would reach stage I of a clinical trial or gain enough
creditability for clinical use by practitioners of modern medicine.
This is because herbal drugs are sometimes considered dubious and its
practitioners considered as quakes. The reasons are many. Doubts have been
raised on the use of herbal drugs for the following reasons:
1. Herbal of different origin are after known by the same popular name.
2. Plants growing in different climatic and seasonal conditions do not have
identical chemical constituents or therapeutic effect.
3. The process of collection (fresh, shade or sun-dried), extraction,
processing and storage of herbal medicines cause variation in potency
and safety.
4. The lack of specific standards for herbal medicines in suitable dosage
form creates difficulty in administration.
These shortcomings have delayed the integration of some of the better
known Ayurvedic and Unani principles with the modern system of medicine.
But things are looking up with the gradual acceptance of Ayurvedic
medicine. Further, detailed investigation on the mode of action of herbal drugs
has revealed that they are involved in enzymatic, endocrine and
immunomodulation functions. These have helped to widen their profile of
activity and opened new vistas of therapeutic applications.
1.1 AIMS AND OBJECTIVES OF THE STUDY
The Plant selected for the project work is Rubus racemosus family
Rosaceae.
Rubus species are known to provide extracts which have been used in
traditional medicine as astringent, emmenagogne, abortfacient, anti-microbial,
anti-convulsant, anti-diabetic, muscle relaxant, free radical scavenging agents5.
Decoction of the root is useful for relaxed bowel and dysentery6. Infusion of
leaves was administered to stop diarrhoea and some bleeding. Family Rosaceae
is known as a source of folk medicine for treatment to nervous disorders7.
Preliminary phytochemical screening of the plant Rubus racemosus
revealed the presence of flavanoids and phenolic compounds and tannins.
Flavanoids have been reported to exert multiple biological effects due to their
antidiabetic, antioxidant and free radical scavenging activity8.
But the literature review revealed no documentation of scientific work
on the aerial parts of Rubus racemosus. This prompted us to take up this
project.
In the present study, an attempt was made to isolate individual phyto-
constituent and extracts were subjected to anti-diabetic, anti-oxidant, anti-
convulsant activity and antimicrobial studies.
REVIEW OF
LITERATURE
CHAPTER - II
REVIEW OF LITERATURE
Rubus (Rosaceae)
Sharma.B.B. and Varshney M.D. et al (1986) screened the antifertility
activity of the plant Rubus Niveus on early and late pregnancy in albino rats9.
Bhakuni.R.S. et al (1987) reported chemical examination of the roots
of Rubus ellipticus10.
Rana.A.C. and Saluja.A.K. et al (1990) undertook pharmacological
screening of the alcoholic extract of the leaves of Rubus ellipticus and reported
the acute toxicity studies, anticonvulsant, analgesic, anti-inflammatory and
hypnotic activities11.
Pal.R. et al (1991) studied chemical examination of Rubus ellipticus
and reported the presence of saponins and glucosides12.
Costantino.L. et al (1992) reported antilipoperoxidant activity of
polyphenolic crude extracts of Rubus idaeus, Rubus fruticosus and Rubus
occidentalis fruits13.
Xiao-Hong Zhou et al (1992) reported the phytoconstituents of Rubus
species and reported the presence of holeanane and ursane glucosides14.
Emile M. Gaydou (1995) studied phytoconstituents and reported the
presence of long chain epoxide from stem of Rubus thibetanus15.
Robertson.G.W. et al (1995) observed changes in the chemical
composition of volatiles released by the flowers and fruits of the red raspberry
(Rubus idaeus) cultivar glen prosen16.
Durham et al (1996) reported the isolation of phytoconstituents from
the roots of Rubus pinfaensis17.
Wang.B.G. et al (1997) reported the isolation of phytoconstituents from
the aerial parts of Rubus pileatus18.
Nogueira.E. et al (1998) studied involvement of GABAA-
benzodiazepine receptor in the anxiolytic effect induced by hexanic fraction of
Rubus brasiliensis7.
Vassilieff.V.S. et al (1998) reported the anxiolytic effect of Rubus
brasilensis in rats and mice19.
Zhong-Jian Jia et al (1998) conducted chemical investigations of
Rubus Pungens camb. Var Oldham II and reported the presence of triterpenes
and triterpene glycosyl ester20.
Lien et al (1999) reported the isolation of phytoconstituents from
ethanolic and butanolic extracts of the leaves of Rubus cochinchinensis21.
Kim.T.G. et al (1999) studied the inhibitory effects of Rubus coreanus
on hepatitis B virus replication in Hep G2 2.2.15 cells22.
Kim S.Y. et al (1999) reported the phytoconstituents from the roots of
Rubus parvifoluis and evaluated anti-inflammatory activity using invivo mouse
ear edema test23.
Lemus.I. et al (1999) reported the hypoglycaemic activity in petroleum
ether extract of Rubus Ulmifolius24.
Shepherd.T et al (1999) studied the epicuticular wax ester and
triacylglycerol composition in relation to aphid infestation and resistance in red
raspberry25 (Rubus idaeus L.).
Gunter Adam et al (1999) reported the phytoconstituents of Rubus
colchinchinensis and reported the presence of triterpenes26.
Tom Shepherd et al (1999) studied epicuticular wax composition in
relation to aphid infestation and resistance in red raspberry27 (Rubus idaeus L.).
Yunes.R.A. et al (1999) identified phytoconstituents and evaluated
antinociceptic activity from the aerial parts of Rubus imperialis28.
Deighton.N. et al (2000) studied antioxidant properties from fruits of
Rubus occidentalis, Rubus idaeus and Rubus strigosus29.
Dhanabal.S.P. et al (2000) validated alcoholic extracts of leaves of
various species of Rubus, Rubus ellipticus, Rubus niveus, Rubus Racemosus
and Rubus rugosus (Rosaceae). They were tested for antifertility activity in
female Wistar albino rats. The results indicate decreased implantation sites and
increased resorption sites, which denote anti implantation and early
abortifacient activities of Rubus species. The results are in agreement with the
traditional use of this plant as abortifacient by the tribals of Nilgiris6.
Kim.N.D. et al (2000) studied the activity of Crude extract of Rubus
roots as crataegifolius roots as a potent apoptosis inducer and DNA
topoisomerase I inhibitor30.
Lin.H.S. et al (2000) has studied antioxidant activity in fruits and leaves
of blackberry, raspberry, and strawberry varies with cultivar and developmental
stage31.
Vassilieff.V.S. et al (2000) studied the hypnotic, anticonvulsant and
muscle relaxant effects of Rubus brasiliensis and involvement of GABAA-
System32.
Wang.B.G. et al (2000) reported the phytoconstituents from an ethanol
extract of the aerial parts of Rubus Pungens33.
Wynne Griffiths.D. et al (2000) made a comparitive study of the
composition of epicuticular wax from red raspberry (Rubus idaeus L.) and
hawthorn (Crataefus monogyna Jacq.) flowers34.
Derek Stewart et al (2001) made studies on ripening related changes in
raspberry cell wall composition and structure35.
Dugo.P. et al (2001) identified the anthocyanins in berries by narrow-
bore high-performance liquid chromatography with electrospray ionization
detection36.
Seeram.N.P. et al (2001) reported the cyclooxygenase inhibitory and
antioxidant cyanidin glycosides in cherries and berries37.
Wang.S.Y et al (2001) reported the changes in oxygen-scavenging
systems and membrance lipid peroxidation during maturation and ripening in
blackberry38.
Catalano.S. et al (2002) has studied the phytoconstituents and exhibited
the in vitro antimicrobial activity of Rubus ulmifolius39.
Cho.S.M. et al (2002) studied the phytoconstituents and evaluated
inhibitory effects in B16 mouse melanoma cells from the fruits of Rubus
Coreanum40.
Corao.G.M. et al (2002) reported hyaluronidase inhibitory activity from
the polyphenols in the fruit of Rubus fruticosus41.
Cui.C.B. et al (2002) identified phytoconstituents and evaluated new
cell-cycle inhibitors from air dried roots of Rubus aleaefolius42.
De Coroa et al (2002) reported antiviral activity from crude extract of
Rubus fruticosus43.
Gabriela Maria Konig et al (2002) reported that the methanolic extract
of Rubus rigidus inhibited the activity of both enzymes HIV1 reverse
transcriptase (HIV1-RT) and TKP5644.
Mohamed Eddouks et al (2002) reported the hypoglycaemic effect of
Rubus fructicosis L. and Globularia alypum L. in normal and streptozotocin-
induced diabetic rats45.
Mohamed Eddouks et al (2002) reported that the fruits of blackberry
exhibited significant hyaluronidase inhibitory activity from polyphenols46.
Moyer.R.A. and Wrolstad.R.E. et al (2002) reported the anthocyanins,
phenolics, and antioxidant capacity in diverse small fruits: Vaccinium and
Rubus47.
Nakatani.K. et al (2002) reported the inhibitions of histamine release
and prostaglandin E2 synthesis from aqueous of Rubus suavissimus48.
Patel.A.V. et al (2002) reported the uterine relaxant activity from
methanolic extract of Rubus idaeus49.
Wada.L. et al (2002) reported the antioxidant activity and phenolic
content from Rubus occidentalis50.
Alan Crozier et al (2003) analysed ellagitannins and conjugates of
ellagic acid and quercetin in raspberry fruits51.
Brian E. Ellis et al (2003) has studied the family of polyketide synthase
genes expressed in ripening from the fruits of Rubus idaeus52.
Hamill.F.A. et al (2003) has studied the physical characterization of
bioactive alkanols from Rubus apetalus53.
Mahmoud A M Nawwar et al (2003) has studied the phytoconstituents
of Rubus sanctus and reported the presence of caffeoyl sugar esters and
ellagitannin5.
Yesilada et al (2003) reported the anti-inflammatory and
antinociceptive activity assessment of plants used as remedy in ethanolic and
aqueous extracts of Rubus hirtus and Rubus sanctus54.
Meckes.M. et al (2004) reported the carrageenan induced rat paw
edema activity of methanolic extract of Rubus Coriifolius55.
Moon P.D. et al (2004) studied the inhibition of mast cell-mediated
anaphylactic- like reaction and tumor necrosis factor-alpha-secretion from the
methanolic extract of Rubus croceacanthus56.
Thiem.B. et al (2004) reported antibacterial activity of Rubus
chamaemorus leaf butanolic fraction of the methanolic extract was evaluated
against gram positive and gram negative bacteria57.
Ivanova.D. et al (2005) studied the polyphenols and antioxidant
capacity from the leaves of Rubus sp. Diversa58.
Liu.Z. et al (2005) reported the antiangiogenic activity in a novel
human tissue based in vitro fibrin clot angiogenesis assay from Rubus
occidentalis59.
Tomezyk.M. et al (2005) reported three phenolic compounds from
Rubus saxatilis60.
Brisht.A. et al (2006) made a review on ethanobotanical studies on
Rubus ellipticus and Rubus pedunuculosus61.
Venskutonis.P.R. et al (2007) reported that the radical scavenging
activity and composition of raspberry (Rubus idaeus) leaves from different
locations in Lithuania62.
Elizabeth Barbosa et al (2007) reported in vivo antigiardial activity of
three flavonoids isolated of some medicinal plants used in Rubus Coriifolius
for the treatment of diarrhea8.
A Perusal literature review on Rubus racemosus revealed that only one
pharmacological activity has been reported on the plant and no phytoconstiuent
has been isolated and characterized.
MATERIALS AND
METHODS
CHAPTER – III
MATERIAL AND METHODS
3.1 GLASSWARE AND CHEMICALS
For the entire project, Borosil glasswares were used. They were soaked
in chromic acid for 3 days, washed with tap water, rinsed with distilled water
and dried over hot air oven.
Analytical grade chemicals supplied by S.D.Fine Chemicals, Sigma
Chemicals Co and Qualigens Fine Chemicals were used. All chemical
solvents, enzyme kits used for this research work were of analytical reagent
grade.
3.2 PLANT PROFILE
The plant seen in Rubus racemosus
Vernacular Name: Tamil - Cheethi
Botanical information
Distribution:
The straggling shrub Rubus Racemosus belongs to the family Rosaceae.
It is indigenous to India, distributed in the Southern Western ghats, the Nilgiri
and Palani hills at an altitude of 1,880 metre.
Description
It occurs as decidious shrub; Subshrub; tender parts glandular; prickles
recurved. Leaves odd-pinnate, to 12(16) x 8 (10) cm, chartaceous; margin
serrate; petiole to 5 (7) cm; stipules adnate to petiole, to 6 mm, persistent;
terminal leaflet ovate, acute, to 8 x 6 cm, often sublobulate; laterals ovate-
lanceolate, 7 x 3.5 cm. Inflorescence axillary, a few-flowered; peduncle 2 cm.
Flowers 1 cm wide; pedicel to 1 cm; bracts subulate, 6mm. calyx-tube
shallowly cup-shaped, with glandular hairs; lobes 5, ovate-acuminate. Petals 5,
red, longer than sepals. Stamens α.Ovary glabrous; ovule 1. Fruits globose,
1 cm wide, purple.
Fig. 1 RUBUS RACEMOSUS - HABITAT
Fig. 1.1 Flower of Rubus Racemosus
Fig.1.2 Leaves of the plant
3.3 PLAN OF WORK
I. Pharmacognostical studies of the leaf
a) Macroscopy
b) Microscopy
c) Powder analysis
d) Fluorescence analysis
e) Quantitative microscopy
f) Determination of leaf constants
g) Determination of physiochemical constants
II. Phytochemical studies
a) Extractive values of different extracts
b) Preliminary phytochemical screening
c) Fluorescence analysis of extracts
d) Thin layer chromatography
e) Isolation of constituents by column chromatography
f) Spectral studies
g) HPTLC studies
III. Pharmacological and Biochemical Studies
a) Acute oral toxicity studies
b) Sub acute toxicity studies
c) Haematological Parameters
d) Antidiabetic activity
i. Effect of MERR on blood glucose level in normal rats
ii. Effect of MERR on blood glucose level on glucose fed
hyperglycemic rats
iii. Effect of acute treatment of MERR on blood glucose level
in streptozotocin induced diabetic rats
iv. Effect of sub-acute treatment of MERR on blood glucose
level in streptozotocin induced diabetic rats
v. Biochemical Estimation
a. Total bilirubin
b. Serum glutamate oxalocetate transminase
c. Serum glutamate pyruvate trasaminase
d. Serum total protein
e. Alkaline phosphatase
f. Serum total cholesterol
g. Serum HDL cholesterol
h. Serum triglyceride
i. Serum LDL-Cholesterol
vi. Antioxidant enzymes in liver homogenate
1. Superoxide dismutase
2. Catalase
3. Glutathione peroxidase
4. Glutathione reductase
5. Lipid peroxidation
vii. Histopathological studies on Liver and pancreas
e) In vitro anti-oxidant studies
i. Free radical scavenging activity by DPPH
ii. Nitric oxide scavenging activity
iii. Hydroxyl radicals scavenging activity
iv. Determination of reducing power
v. Determination of total phenolic compounds
f) Antiepileptic activity
i. Maximal electroshock induced convulstion
ii. Effect of MERR on neurotransmitter levels in MES
induced rats
1) Determination of the effect of Rubus racemosus and
standard on neurotransmitter concentrations in rat brain after
induction of epilepsy
IV. Antimicrobial Studies
a) Antibacterial Activity
b) Antifungal Activity
3.4 ANATOMICAL STUDIES
Collection of specimens
The plant specimens for the proposed study were collected from Nilgiri
Hills. Care was taken to select healthy plants and normal organs. The required
samples of different organs were cut and removed from the plant and fixed in
FAA (Formalin-5ml + Acetic acid – 5ml + 70% Ethyl alcohol – 90ml). After
24 hrs of fixing, the specimens were dehydrated with graded series of tertiary-
Butyl alcohol as per the schedule given by Sass, 1940. Infiltration of the
specimens was carried out by gradual addition of paraffin wax (melting point
58-600 C) until TBA solution attained supersaturation. The specimens were cast
into paraffin blocks63.
Sectioning
The paraffin embedded specimens were sectioned off with the help of
Rotary Microtome. The thickness of the section was 10-12 µm. Dewaxing the
sections was achieved by customary procedure64. The sections were stained
with Toluidine blue as per the method published by O’Brien et al., 1964.
Since Toluidine blue is a polychromatic stain, the staining results were
remarkably good and some Cytochemical reactions were also obtained. The
dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark
green to suberin, violet to the mucilage, blue to the protein bodies etc.
wherever necessary sections were also stained with safranin and Fast-green
and I+KI for Starch65.
Photomicrographs
Microscopic descriptions of tissues are supplemented with micrographs
wherever necessary. Photographs of different magnifications were taken with
Nikon Labphot 2 Microscopic Unit. For normal observations bright field was
used. For the study of crystals, starch grains and lignified cells, polarized
light was employed. Since these structures have birefringent property, under
polarized light they appear bright against dark back ground, Magnifications of
the figures are indicated by the Scale-bars.
Descriptive terms of the anatomical features are as given in the standard
Anatomy books66.
3.5 POWDER ANALYSIS
The Powdered aerial parts of Rubus Racemosus was passed through
sieve no.100 and subjected to microscopical features67
i. The powder was mounted in glycerine water and observed for
calcium oxalate crystals.
ii. The powder was stained with Iodine solution and observed for
starch grains.
iii. The powder was cleared with chloral hydrate and stained with
phloroglucinol and concentrated HCl and observed in a self-
illuminating compound microscope.
3.6 QUANTITATIVE MICROSCOPY68
Measurement of length and width of trichomes in powdered leaf of
Rubus racemosus
Powder of the leaf was observed under low power magnification.
Trichomes of simple unicellular, uniseriate type were observed and hence their
dimensions were determined.
The first step involved is the calibration of the eyepiece micrometer
using stage micrometer. To determine this calibration factor, the eyepiece was
replaced by eyepiece micrometer in the ridge. The stage micrometer was then
placed on the stage of the microscope and focused under high power with the
eyepiece scale. The calibration factor was calculated by applying the formula
given below after noting down the coincidence of micrometer division with
eyepiece.
No of division of stage micrometer Each division of eyepiece micrometer ________________________________ x 10 No. of divisions of eyepiece micrometer
The stage micrometer was replaced with the slide containing the
powdered drug. For the preparation of the slide a little quantity of powder was
first boiled with chloral hydrate solution. The cleared powder was taken in a
watch glass and stained with one drop each of phloroglucinol and concentrated
hydrochloric acid.
A little of this powder was then placed on a slide mounted in dilute
glycerin and observed under low power. The length and width of the trichomes
were measured by focusing them on the lines of the eyepiece micrometer.
The dimensions for about 40 trichomes were measured and multiplied
with the calibration factor to give the dimensions of the trichomes in microns.
Calibration factor
Seventh division of eyepiece coincides with tenth division of the stage
micrometer.
One smallest divisions of stage = 0.01mm (or) 10µm
No. of divisions of stage micrometer x 10 Each divisions of eyepiece micrometer = No. of divisions of eyepiece micrometer
= 10 x 10 7
= 14.4 µm
3.7 DETERMINATION OF LEAF CONSTANTS69
a) Determination of vein islet and vein termination numbers
Vein islet number is defined as the number of vein islets present per
square millimeter of the leaf surface midway between the mid rib and the
margin. It is constant and characteristic feature for a given species of the plant
and for used for differention from allied species.
Vein let termination number is defined as the number of vein let
termination present per square millimeter of the leaf surface midway between
the mid rib and the margin. A vein termination is the ultimate free termination
of vein let.
Procedure
Few leaves were boiled in chloral hydrate solution in a test tube placed in a
boiling water bath. The preparation was mounted in glycerin water. The camera
lucida was set up and the black board was divided into squares of 0.5 sq mm by
means of the stage micrometer. The stage micrometer was replaced by the
cleared leaf preparation and the veins were traced in sixteen continuous
squares. The vein islets and vein let terminations were traced by looking
through the microscope when a superimposed image of the leaf portion and
paper were seen at the same time. The number of vein islets and vein let
termination present within the square were counted by taking into consideration
incomplete vein islet on any two adjacent sides of the square. The value for 1
sq mm was calculated. 16 sets of such counts were taken. The observations
were recorded in the form of range and mean values.
b) Determination of stomatal number and stomatal index
Stomata is a minute epidermal opening covered by two kidney shaped
guard cells in dicot leaves. Those guard cells in turn are surrounded by
epidermal (subsidiary) cells. Stomata performs the functions of gaseous
exchange and transpiration in plants. The nature of the stomata as well as
stomatal index and stomatal number are important diagnostic characteristics of
dicot leaves.
Stomatal number is defined as the number of stomata present per sq mm
of epidermis of the leaf. The actual number of stomata per sq mm may vary for
the leaves of the same plant grown in different environments or under different
climatic conditions. It is however shown that the ratio of the number of
epidermal cells in a given area of epidermis is fairly constant for any age of the
plant under different climatic conditions.
Stomatal index is a percentage in which the number of stomata forms
the total number of epidermal cells, each stomata being counted as one cell.
Stomatal index can be calculated by using the following equation
S.I = S x 100 E+S
Where,
S.I. = Stomatal index
S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area
It is employed for the differentiation of closely related species of the
same genus in air dried as well as fresh condition.
Procedure
Fragments of the leaves from the middle of the lamina were cleared by
boiling with chloral hydrate solution. The upper and the lower epidermis were
peeled out separately by means of forceps. The mounts of the upper and lower
epidermis were separately prepared in glycerin water. A square of known
dimension was drawn by means of stage micrometer and camera lucida on
black board drawing paper. The stage micrometer was replaced by the cleared
leaf preparation focused under the same magnification and the epidermal cells
and stomata were traced by looking through the microscope when a super
imposed image of the leaf is seen at the same time. The number of epidermal
cells and stomata within the square were counted, a cell being counted if at
least half of its area lies within the square provided, two adjacent sides are
counted for the purpose of calculation. Successive adjacent fields were
examined until about hundred cells were counted and the stomatal number and
stomatal index were calculated.
3.8 DETERMINATION OF PHYSIOCHEMICAL
CONSTANTS
1. Determination of ash values70
Procedure
The ash content of a crude drug is generally taken to be the residue
remaining after incineration. It usually represents the inorganic salts naturally
occurring in the drug and adhering to it, but it may also involve the inorganic
matter added for the purpose of adulteration. There is a considerable difference
which varies within narrow limits in the case of some individual drug. Hence
an ash determination furnishes a basis for judging the identity and cleanliness
of a drug and gives information related to its adulteration with inorganic
matter. Ash standards have been established for a number of official drugs.
Usually these standards set a maximum limit on the total ash or on the acid
insoluble ash permitted, the total ash is the residue remaining after incineration.
The acid insoluble ash is a part of the total ash, which is insoluble in dilute
hydrochloric acid.
The ash or residue yielded by an organic chemical compound is a rule to
measure the amount of inorganic matter which is present as impurity. In most
cases the inorganic matter is present in small amounts which are difficult to
remove in the purification process and which are not objectionable if only
traces are present. Ash values are helpful in determining the quality and purity
of the crude drug in powdered form.
1a. Determination of total ash
About 3g of accurately weighed powdered drug was taken in tarred
silica cruicible previously ignited and weighed, the powdered drug was
scattered in a fine even layer at the bottom of the crucible and incinerated
gradually by increasing the temperature not exceeding dull red heat until free
from carbon, cooled and weighed. As, a carbon free form was not obtained the
charred mass was extracted with hot water and the residue was collected on an
ashless filter paper. The residue and the filter paper were incinerated and the
filtrate was added evaporated to dryness and ignited at low temperature. The
percentage of ash with reference to air-dried drug was calculated.
1b. Determination of acid insoluble ash
The above procedure was repeated to collect the total ash and was boiled
for 5 minutes with 25ml of conc. hydrochloric acid (AR grade) and the
insoluble matter was collected on an ashless filter paper wet with hot water
ignited and weighed. The percentage of acid insoluble ash was calculated with
reference to air dried drug.
1c. Determination of sulphated ash
The total ash was moistened with conc. sulphuric acid (AR grade)
ignited gently again moistened with sulphuric acid reignited cooled and
weighed. The percentage of sulphated ash was calculated with reference to air
dried drug.
1d. Determination of water soluble ash
The total ash was boiled for 5 minutes with 25ml of water and filtered.
The insoluble matter was collected on an ashless filter paper wet with hot water
and ignited to constant weight at a low temperature. The weight of insoluble
matter was subtracted from the weight of the ash. The difference in weight
represents the water soluble ash. The percentage of water soluble ash was
calculated with reference to air dried drug.
2. Determination of extractive values
Procedure
Extractive values of crude drugs are useful for their evaluation
especially when the constituents of the drug cannot be readily estimated by any
other means. Further, these values indicate the nature of the constituents
present in a crude drug.
2a. Alcohol soluble extractive
Macerated 5 g of dried coarse powder of Rubus racemosus with 100 ml
of 90% ethanol in a closed flask for 24 hours, shaking frequently during 6
hours and allowing to stand for 18 hours.
It was filtered immediately taking precaution against loss of alcohol and
25ml of filtrate was evaporated to dryness in a tarred flat bottomed shallow
dish and dried at 105o C and weighed. The percentage of alcohol soluble
extractive was calculated with reference to air dried drug.
2b. Water Soluble Extractive
Rubus racemosus weighing 5 g of powder was added to 50ml of water at
80oC in a stoppered flask. It was shaken well and allowed to stand for 10
minutes. It was Cooled to 15oC, 2g of kieselghur was added into it and filtered.
Transferred 5ml of the filterate to a tarred evaporating basin and evaporated on
a water bath and the residue was weighed. The percentage of water soluble
extractive was calculated with reference to air dried drug.
3. Determination of loss on drying
The loss on drying is the loss of weight in percentage w/w resulting
from water and volatile matter of any kind that can be driven of under specified
conditions. The test was carried out on well-mixed sample of the substance.
Glass stoppered shallow bottle was weighed that had been dried in the
same conditions to be employed in the determination. Transferred 1.0g of the
sample powder to the bottle. The loaded bottle was placed in a drying chamber.
The sample was dried at a temperature 1050 C to a constant weight. The drying
chamber was opened and bottle was allowed to cool. The bottle and contents
were weighed. The process was repeated until the successive weights differed
not more than 0.5mg.
3.9 PLANT MATERIAL AND EXTRACTION
The aerial parts of Rubus racemosus were collected from Nilgiri Hills in
the month of August in the year 2006. The plant was authenticated by
Dr.S.Rajan, Field Botanist, Survey of Medicinal Plants & Collection Unit,
(Central Council for Research in Homoeopathy), Department of AYUSH,
Ministry of Health & Family Welfare, Govt. of India, 112, Government Arts
College Campus, Udhagamandalam – 643 002.
The aerial parts were shade dried for seven days and then powdered by
means of a grinder and the powder was passed through the sieve no.60. Fine
powder was used for microscopical analysis and coarse powder was used for
phytochemical work. Powdered material was extracted successively with
petroleum ether (60-800), ethyl acetate (770C), chloroform (640C), methanol
(700C) and water. The residues were collected by evaporation of solvent under
reduced pressure by rotary evaporator.
a. Purification of Solvents
1. Petroleum ether
The petroleum ether was distilled and the fraction boiling between
60o-80oc was collected and used for extraction and chromatographic purposes.
2. Chloroform
The chloroform was shaken well with equal volume of distilled water
twice to remove water soluble impurities and separated using a separating
funnel. It was dried over anhydrous calcium chloride for 24 hours, filtered and
dried again over anhydrous potassium carbonate for 24 hours. This was
decanted, distilled and the fraction boiling at 64oc was collected and stored in a
dark brown bottle. Absolute alcohol of 1ml was added as preservative.
3. Ethyl acetate
Ethyl acetate was refluxed for 4 hours and distilled. The distillate was
shaken with sufficient amount of anhydrous potassium carbonate, filtered and
redistilled. The fraction boiling at 770C was collected and used.
4. Methanol
Methanol was lime distilled and used for extraction and
chromatographic purposes.
3.10 PRELIMINARY PHYTOCHEMICAL SCREENING71
Various extracts of the aerial parts of Rubus racemosus were subjected
to preliminary phytochemical screening.
1. Test for alkaloids
All the extracts were treated with dilute hydrochloric acid and filtered.
The filtrate was treated with alkaloidal reagents like Mayer’s reagent,
Dragendroff’s reagent, Hager’s reagent and Wagner’s reagent. There was no
characteristic colour change indicating the absence of alkaloids.
2. Test for carbohydrate (Reducing and Non Reducing Sugar)
a) The extracts on treatment with Molisch’s reagent showed violet ring at
the junction of two liquids suggesting the presence of the carbohydrate.
b) The extracts were treated with Fehling’s solution A and B and heated. A
reddish brown precipitate is formed indicating presence of reducing
sugar.
c) The extracts on treatment with Benedict’s reagent gave reddish orange
colour indicating the presence of reducing sugar.
d) The extracts, when treated with Barford’s reagent gave no characteristic
reaction confirming the presence of non-reducing sugar.
3. Test for steroids and sterols
a) Libermann Burchard test: The extracts were treated with concentrated
sulphuric acid, glacial acetic acid and acetic anhydride which did not
show green colour. This confirms the absence of steroids.
b) When the extracts treated with 5% potassium hydroxide, they did not
give pink colour which indicates the absence of sterols.
4. Test for Proteins
a) Biuret test: The extracts were treated with copper sulphate and sodium
hydroxide they did not show violet colour confirming the absence of
proteins.
b) Millon’s reagent: The extracts were treated with Millon’s reagent it did
not show pink colour which confirms of the absence of proteins.
5. Test for phenols
a) The extracts on treatment with ferric chloride produced violet colour
confirming the presence of phenols.
b) The extracts were treated with 10% sodium chloride solution and
showed cream colour. This confirms the presence of phenols.
6. Test for tannins
When the extracts treated with 10% lead acetate, 10% sodium chloride
and aqueous bromine solutions separately, produced white precipitate
indicating the presence of tannins.
7. Test for flavanoids
The extracts on treatment with amyl alcohol followed by sodium acetate
and ferric chloride produced a pink or blood red colour which shows the
presence of flavanoids.
8. Test for gums and mucilages
The extracts were treated with 25ml of absolute alcohol and filtered. The
filterate was examined for its swelling properties. No swelling was observed.
This shows the absence of gums and mucilage.
9. Test for glycosides
A pinch of the substance was dissolved in glacial acetic acid and few
drops of ferric choloride was added followed by concentrated sulphuric acid. A
red ring formed at the junction of two liquids. It shows the presence of
glycosides.
10. Test for Saponins
Foam test: All the dry extracts weighing 1g were treated with distilled
water and shaken well in a test tube. Formation of foam was observed in the
upper part of the test tube due to the presence of saponins.
11. Test for terpenes
Extracts were treated with tin and thionyl chloride produced pink colour.
Confirming the presence of terpenes.
3.11 THIN LAYER CHROMATOGRAPHY72
Preparation of the plate
About 40 gm of silica gel G was shaken to a homogenous suspension
with 100 ml of distilled water to form a slurry. This suspension was poured
onto a TLC plate of 0.25mm thickness of 20x5 cm. The plates were kept for air
drying until the transparency of the layer disappeared, dried in hot air oven at
121oC for 30 minutes for activation and stored in a dry atmosphere.
Application of the substance mixture for separation
The substance mixture was taken in a capillary tube and it was spotted
on TLC plate, 2cm above its bottom end. Most solutions for application were
between 0.1-1% strength. The start points were equally sized as far as possible
and a diameter ranging from 2-5mm was seen.
Development of chromatogram
The plates were developed in a chromatographic tank by using a range
of solvents from non polar to polar as mobile phase. The raise of the mobile
phase was allowed upto 3/4 the length and then removed. The solvent front
was marked immediately and the plates were allowed to dry in a dryer. The
spots were identified and their Rf values determined after spray reagents. The
results are furnished in Table no.4 to 6.
3.12 ISOLATION OF CONSTITUENTS BY COLUMN
CHROMATOGRAPHY73
As many chemical constituents show their presence in the methanolic
extract and due to its quantitative abundance, this extract had been chosen for
the isolation of the individual phytochemicals by means of column
chromatography.
A suitable column of 2.5 cm in diameter and 60cm in length was
selected, washed thoroughly with water, dried and rinsed with acetone and
dried completely. A little pure cotton was placed in to column with help of a
big glass rod upto neck to avoid the leakage of smaller particles of the
adsorbent. A piece of filter paper with suitable size was placed over the cotton.
The column was packed with silica gel 100-200 mesh upto 1/3 of its length by
carefully pouring the slurry through the funnel to prevent the formation of air
bubbles in the column. The sides of the column was tapped slowly in order to
facilitate the packing of the adsorbent. The prepared column was thoroughly
washed with petroleum ether and the liquid level was always kept above the
surface of the column to prevent the cracking. A round filter paper of suitable
size was placed above the packed column.
Package of the sample in the column
About 2 gms of the concentrated methanolic extract was mixed with
suitable quantity of silica gel (100-200 mesh) to ensure the free flow of the
extract along with adsorbent it was packed in the column through the funnel,
then petroleum ether was added through the column and kept aside over night.
The column was eluted with different organic solvents in increasing order of
polarity
1. Petroleum ether
2. Chloroform
3. Ethyl acetate
4. Ethyl acetate and isopropanol at different ratios
5. Isopropanol and ethyl alcohol at different ratios
6. Ethyl alcohol
7. Ethyl alcohol and methyl alcohol at different ratios
8. Methyl alcohol
The fraction 100ml each of the eluate from the column was collected
into series of 500ml glass beakers. The eluate was concentrated by evaporating
the solvent and the residues if any were identified by Thin Layer
Chromatography.
3.13 HPTLC STUDIES
Materials and Methods
Name of the instrument : Shimadzu
Plate material : HPTLC precoated plates silica gel MERCK -
60F254
Solvent : Ethylacetate: Hexane (4:6)
Detecting agent : Fluorescence Mode
Chloroform extract and isolated pure compound were applied on
silicagel in 0.2mm layer thickness precoated on aluminium sheets using
linomet IV sample applicator and determined their Rf values.
The mobile phase used for developing the plate under study is given
above. The plate was scanned using camag densitometer scanner equipped with
cats v 3.20 software. The chromatogram is furnished in spectrum no.3.2 and
3.3.
3.14 EXPERIMENTAL ANIMALS
Inbred adult wistar rats 150-200 gm of either sex were obtained from the
animal house of C.L.Baid Metha College of Pharmacy. The animals were
maintained in well ventilated rooms with 12:12 light/dark cycle in
polypropylene cages. Standard pelleted feed and drinking water were provided
ad libitum throughout the experimental period. Animals were acclimatized to
the laboratory conditions one week prior to the initiation of the project work.
The project has got the ethical committee clearance from IAEC of CPCSEA.
IAEC Ref: no: IAEC/XIII/17/CLBMCP/2007-2008 dated on 20.04.2007
3.15 TOXICOLOGICAL EVALUATION
Acute oral toxicity studies74
The procedure was followed according to the OECD guidelines 423
(Acute toxic class method). The acute toxic class method is a stepwise
procedure with 3 animals of single sex per group. Depending on the mortality
and or moribund status of the animals, on an average 2-4 steps may be
necessary to allow judgment on the acute toxicity of the testing substance.
According to this procedure minimum number of animals were to be used for
acceptable data band scientific conclusion. The method uses defined doses
(5, 50, 300, 2000 mg/kg body weight) and the results allow a substance to be
ranked and classified according to the globally harmonized system (GHS) for
the classification of chemical which cause acute toxicity.
Adult Male Wistar rats weighing between 150-200 g were used for the
study. The starting dose of Rubus racemosus was 2000 mg/kg body wt. as most
of the crude extracts possess LD50 value more than 2000 mg/kg body weight.
The dose was administered to over night fasted rats and food was withheld for
a further 3-4 hours after administration of the drug and observed for signs of
toxicity.
Body weight of the rats before and after treatment were noted and any
changes in the skin, eyes and mucous membranes, salivation, nasal discharge
urination, and behavioral (sedation, depression), neuromuscular (tremors,
convulsions), cardiovascular lethargy and sleep and coma were noted. The
onset of toxicity and signs of toxicity was also noted. The animals were kept
under observation for 14 days.
Sub acute toxicity studies
Adult male albino rats (120 – 150 g) were used for sub acute toxicity.
These animals were maintained in polypropylene cages under identical animal
house conditions and provided with standard pellet and water and libitum.
Six groups of rats were used in sub acute toxicity study of methanolic
extract consists of 6 rats each at the dose of 400 mg/kg were given orally for 28
days.
Animals were observed for signs and symptoms, alteration of
behaviours, food and water intake and body weight changes. Blood samples
were collected after 24hrs of the last dose of methanolic extracts for
haematological studies. A portion of liver, brain, kidney, spleen, heart, lungs,
testis and ovary were dissected out and kept in 10% formalin for
histopathalogical studies.
In these samples haematological parameters such as Hb, RBC and total
WBC and differential WBC were determined using routine methods.
Figure: 2
Haematological Parameters75
Hb (haemoglobin), RBC (red blood cell) count, total WBC (white blood
cell) differential count, ESR and PCV were determined in the blood.
Estimation of Haemoglobin
The haemoglobin content was estimated by the standard method using
Sahli’s haemoglobinometer.
Apparatus and reagents
1. Comparator
2. Haemoglobinometer
3. Haemoglobin tube
4. Haemoglobin pipette
5. 0.1N HCI
Procedure
A clean and dry Sahli’s tube was filled with 0.1N HCL up to the mark.
Blood was drawn in to the pipette up to the mark and pushed into Sahli’s tube
containing HCL solution. Contents were mixed well and compared after 10
min. Distilled water was added until the colour matches the standard. The
reading was taken from the upper meniscus. Values are expressed as g/dl.
Estimation of total red blood cell count
The total red cell count was determined by the standard method using
haemocytometer.
Apparatus and reagents
1. Haemocytometer
2. RBC pipette
3. Hayem’s fluid
4. Microscope
Procedure
Blood was taken up to the 0.5 ml in the RBC pipette followed by
Hayem’s fluid to the mark 101, thus achieving 1: 200 dilution of blood sample.
The counting chamber was charged with the fluid and cells were counted with
the aid of microscope.
Calculations
Total RBC count = cells counted x 5 (1/5 sq. cm) x 10 (depth) x 200 (dilution factor)
Values are expressed as million-cells/cu.mm of blood.
Estimation of total white blood cell count
The total and differential white cell count was estimated by the standard
method using haemocytometer
Apparatus and reagents
1. Haemocytometer
2. WBC pipette
3. Turk’s fluid
Procedure
Blood was taken up to the 0.5 ml in the RBC pipette followed by Turk’s
fluid to the mark 11, thus achieving 1: 20 dilution of blood sample. The
counting chamber was charged with the fluid and cells were counted with the
aid of microscope.
Calculations
Cells counted x 10 (depth) x 20 (dilution factor) Total WBC count = _______________________________________
4 (sq.mm counted)
Values are expressed as thousand cells/ cu.mm.
Differential WBC count
Apparatus and reagents
1. Microscope
2. Leishman’s stain
3. Pasteur pipette
Procedure
A thin film of blood was prepared on a clear glass slide and stained by
using Leishman’s stain for 2 min. The slide was washed with distilled water
and allowed to stand for 6 min. Then it was observed under microscope and
different WBC cells were identified and counted. Values are expressed as % of
cells.
3.16 ANTIDIABETIC ACTIVITY
Induction of diabetes mellitus in experimental rats
Adult inbred wistar albino rats [42 numbers] of either sex were over
night fasted, and subjected to a single intrapertoneal injection of freshly
prepared streptozotocin [50 mg/kg] dissolved in ice-cold citrate buffer and PH
4.5. After injection, the animal had free access to food and water and were
given 5 % glucose solution to drink overnight to counter the hypoglycemic
shock76.
The development of diabetes was confirmed after 48 hr of the
streptozotocin injection. The animals with fasting blood glucose level more
than 200mg/dl were selected for the experimentation. Out of the 42 animals
subjected for diabetes induction, 6 animals died before grouping and four
animals were omitted from the study, because of mild hyperglycemia. Of the
remaining 32 diabetic animals, four groups of eight animals each were formed
and used for the experimentation. In the present study, glibenclamide (0.5
mg/kg b.w) was used as the standard drug.
Collection of blood sample and blood glucose determination
Blood samples were collected by end tail vein cutting method and blood
glucose level was determined by one touch electronic glucometer using glucose
test strips. This method permits the measurement of blood glucose levels with
minimal injury to the animal and was previously validated by comparison with
glucose oxidase method.
Blood glucose in fasting rats
Effect of MERR treatment on blood glucose level in normo glycemic rats
The animals were divided into three groups of six rats each,
GROUP 1 Animals received normal control [1% SCMC 1ml/100gm/po/b.w
of rat].
GROUP 2 Animal received MERR [200mg/kg/po/b.w of rat in 1% SCMC].
GROUP 3 Animal received MERR [400mg/kg/po/b.w of rat in 1% SCMC].
In this study, the entire group of animals was overnight fasted prior to
the experimentation and administered with the respective drugs as per the
above mentioned dosage schedule. Blood samples were collected before
administration of the drugs and at 30, 60, 90 and 120th min after drug
administration to determine the blood glucose levels by using electronic
glucometer77.
*MERR – Methanolic Extract of Rubus Racemosus
Induced blood glucose level
Effect of MERR on blood glucose level on glucose fed hyperglycemic rats
Oral glucose tolerance test – [OGTT]
The animals were divided into four groups of six rats each.
GROUP 1 Animals received glucose solution at a dose of 2gm/kg/p.o.
GROUP 2 Animals received glibenclamide 0.5mg/kg and glucose solution at a
dose of 2 gm/kg/p.o
GROUP 3 Animals received MERR* 200mg/kg/b.w and glucose solution at a
dose of 2 gm/kg/oral.
GROUP 4 Animals received MERR 400mg/kg/b.w and glucose solution at a
dose of 2 gm/kg/p.o.
In this study, the entire group of animals was fasted and treated with
above dosage schedule only. The MERR and glibenclamide were administered
half an hour before administration of glucose solution. Blood samples were
collected before glucose administration and at 30,60,90 and 120th min after
glucose administration to determine the blood glucose level by using electronic
glucometer78.
*MERR – Methanolic Extract of Rubus Racemosus
Effect of acute treatment of MERR on blood glucose level in STZ induced
diabetic rats
The animals were divided into five groups. Group 1 consisted of 6
normal animals. The remaining 4 groups consisted of 6 STZ* induced diabetic
rats.
GROUP 1 Normal control animals received 1% SCMC 2ml/kg/p.o
GROUP 2 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received
1% scmc 2ml/kg/p.o
GROUP 3 Streptozotocin [50mg/kg/b.w] induced diabetic animals received
glibenclamide 0.5 mg/kg/p.o
GROUP 4 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received
MERR 200mg/kg/p.o
GROUP 5 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received
MERR 400mg/kg/p.o
In the single day acute study all the surviving diabetic animals and
normal animals were fasted overnight. Blood samples were collected from the
fasted animals prior to the treatment with above dosage schedule and after drug
administration at 1st, 3 rd and 5th hour to determine the blood glucose level by
using electronic glucometer79.
* STZ - Steptozotocin
Effect of sub acute treatment of MERR on blood glucose level in STZ
induced diabetic rats
GROUP 1 Normal control animals received 1% SCMC 2ml/kg/p.o for 28 days.
GROUP 2 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received 1% scmc 2ml/kg/p.o for 28 days
GROUP 3 Streptozotocin [50mg/kg/b.w] induced diabetic animals received glibenclamide 0.5 mg/kg/p.o for 28 days.
GROUP 4 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received MERR 200mg/kg/p.o for 28 days.
GROUP 5 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received MERR 400mg/kg/p.o for 28 days.
The above mentioned treatment schedule was followed for the
respective group of animals for 10 days. Blood samples were collected from
overnight fasted animals, in morning one hour after drug administration on the
1st, 7th, 14th, 21th and 28th day and the blood glucose levels were estimated using
electronic glucometer80.
At the end of the study, all the surviving animals of the respective
groups were anaesthetised by anaesthetic ether. Blood was collected by
bleeding carotid artery and serum was separated to study the biochemical
parameters. After exsanguination of the animals, the liver and pancreas were
removed immediately and washed with ice-cold saline. The liver and
pancreatic tissues were preserved in bovine fluid for histopathological studies.
3.17 BIOCHEMICAL ESTIMATIONS
All the animals were sacrificed at the end of the experiment under light
ether anesthesia, the blood samples were collected separately by carotid
bleeding into sterile dry centrifuge tubes and allowed to coagulate for 30 min at
370C the clear serum was separated at 2500 rpm for 10 min and biochemical
investigations were carried out to assess liver function Viz.,
• Total bilirubin
• Serum glutamate oxalocetate transaminase (SGOT)
• Serum glutamate pyruvate transaminase (SGPT)
• Total protein
• Alkaline phosphatase
• Total cholesterol
• HDL cholesterol
• Triglyceride
• LDL-Cholesterol
All the enzyme assays were carried out at using shimadzu
spectrophotometer, UV – 1601 model81.
Estimation of total bilirubin
The total bilirubin of the serum was estimated using the test kit based on
the Vadeberg’s method82 .
Estimation of glutamate oxaloacetate transaminase (GOT)
The enzyme activity was estimated based on test kit by Reitman and
Frankel method83.
Estimation of Glutamate pyruvate transaminase (GPT)
The enzyme activity was estimated in the serum by using GPT test kit83.
Estimation of Total protein
Total protein was estimated using total protein kit based on biuret
method84.
Estimation of Alkaline phosphatase (ALP)
Alkaline Phosphatase was estimated using Alkaline Phosphatase kit.
Based on para nitro phenyl phosphate method85.
Estimation of Total cholesterol
Total cholesterol was estimated in the serum by using test kit86.
Estimation of HDL-Cholesterol
HDL cholesterol was estimated in the serum by using test kit86.
Estimation of Triglycerides
Triglyceride levels were estimated by using test kit86.
Estimation of LDL-Cholesterol
LDL-cholesterol was calculated by using the formula
LDL-cholesterol = Total cholesterol – [HDL-C + (triglycerides/5)
3.18 ESTIMATION OF ANTIOXIDANT ENZYME LEVELS
IN VARIOUS TISSUES
Preparation of liver homogenate
Immediately after the sacrifice, the liver was excised devoid of blood
and washed in icecold physiological saline. Small pieces of liver was collected
in 10% formalin solution for histopathological examination. The remaining
portion of the liver was weighed and homogenate prepared in Tris HCI buffer
(0.5 M pH 7.4) at 40C. The homogenate was centrifuged and the supernatant
was used for the assay of total protein and cytoprotective enzymes namely
glutathione peroxidase (GPX), superoxide dismutase (SOD), Catalase (CAT),
glutathione reductase (GR), lipid peroxidation (LPO). All the enzymatic assays
were carried out using Shimadzu Spectrometer UV 16.1 model87.
Superoxide dismutase (SOD)
Superoxide dismutase activity was measured by inhibition of pyrogallol
autoxidation at 420 nm for 10 min88
Reagents
1. Tris-HCI buffer
2. Pyrogallol
3. HCL
Procedure
The assay mixture consisted of 1.8 ml of 50mM Tris-HCL buffer
(containing 10mM EDTA), 0.1 ml of 6.0 mM pyrogallol and the diluted tissue
supernatant to a final volume of 2 ml. The reaction was stopped by adding
0.05ml of 1N HC1. The enzyme activity was expressed as Unit/mg protein,
where one unit of superoxide dismutase is the amount of enzyme required to
bring about 50% inhibition of autoxidation of pyrogallol.
Estimation of catalase activity
Catalase activity was determined89.
Reagents
1. Phosphate buffer (pH 7.0)
2. H2O2 freshly prepared
Procedure
The incubation mixture consisted of 0.05 ml of 10% liver homogenate,
1.95 ml of 50 mM phosphate buffer (pH 7.0), to this 1 ml of freshly prepared
30 mM H2O2 was added. The rate of decomposition of H2O2 was measured
spectrophotometrically at 240 nm for 1 min. The result for catalase activity was
expressed as n moles of H2O2. The catalase activity is expressed as n moles of
H2O2 utilized /min/mg protein in tissue homogenate.
Glutathione peroxidase
The activity of glutathione peroxidase was determined90.
Reagents:
1. Phosphate buffer
2. Glutathione
3. Glutathione reductase
4. EDTA
5. NADPH
6. H2O2
Procedure
The assay mixture consisted of 2.0 ml of 10% liver homogenate, 2 ml of
75 mM Phosphate buffer (pH 7.0), 50 ml of 60 mM glutathione, 0.1 ml of 30
units/ml glutathione reductase, 0.1ml of 15mM EDTA, 0.1 ml of 3mM
NADPH and made up a final volume of 3 ml. The reaction was started by the
addition of 0.1 ml of 7.5 mM H2O2. The rate of change of absorbance during
the conversion of NADPH to NADP+ was recorded spectrophotometrically at
340 nm for 3 min. GPx activity for tissues was expressed as µM of NADPH
oxidised to NADP+/min/mg/protein.
Glutathione reductase
Glutathione reductase activity was assayed by measuring the decrease in
absorbance at 340 nm91.
Reagents
1. Sodium phosphate buffer pH 7.5)
2. EDTA
3. GSSG
4. NADPH
Procedure
The reaction mixture containing 50mM sodium phosphate buffer
(pH 7.5), 10 mM EDTA, 0.67 nM glutathione oxidized, and 0.1 mM NADPH
was made upto 3 ml with water. The change in the optical density was
monitored after adding 0.1ml liver homogenate at 340 nm for 3 minutes at 30
seconds interval. The enzyme activity is expressed as n moles of GSSG
utilized/ min/mg protein in tissue homogenate.
Lipid peroxidation
Lipid peroxidation was assayed by the measurement of malondialdehyde
(MDA) levels on the basis of reaction with thiobarbituric acid92.
Reagent:
1. Sodium dodecyl Sulphate AR
2. Acetic acid AR
3. TBA
4. n-butanol AR
Procedure
The incubation mixture consisted of 0.2 ml of 10% liver homogenate, to
this added 0.2 ml of 8.1% Sodium dodecyl sulphate, 1.5 ml of 20% acetic acid
(pH 3.5) and 1.5 ml of 0.8% TBA. The mixture was made upto 4.0 ml with
distilled water and heated in a water bath at 900C for 60 min. After cooling
with tap water, 1.0 ml of distilled water and 5.0 ml of n – butanol were added
and shaken vigorously and centrifuged at 4000 rpm for 10 min. The upper
butanol layer was taken and its absorbance at 532 nm was measured. The lipid
peroxide concentration was expressed as n moles of MDA liberated/min/mg
protein in liver homogenate.
Histopathology
After draining the blood, liver samples were excised, washed with
normal saline and proceeded separately for histological observations. The
materials were fixed in 10% buffered neutral formalin for 48 hrs. Paraffins
sections were taken at 5µm thickness, processed in alcohol-xylene series and
were stained with haematoxylin-eosin dye. The sections were examined
microscopically for histopathological changes93.
Statistical analysis
The statistical analysis was carried out using analysis of variance
(ANOVA) followed by Dunnet’s ‘t’ test. p values <0.05 were considered as
significant.
3.19 ANTIOXIDANT STUDIES
IN VITRO EXPERIMENTS
DPPH radical scavenging activity: 94
To a methanolic solutions of DPPH (2.95 ml of 100 µM), 0.05 ml of the
test compounds dissolved in methanol were added at different concentrations
(25 µg/ml to 1000 µg/ml). Equal amount of methanol was added to the control.
Absorbance was recorded at 517 nm at regular intervals of 30 sec for 5 min.
% Inhibition = [(control – test)] x 100 Control
Nitric oxide scavenging activity: 95
Nitric oxide scavenging activity was measured by spectrophotometry.
Sodium nitroprusside (4ml of 5 mM) in phosphate buffered saline was mixed
with different concentrations of the extracts (25µg/ml to 1000 µg/ml) dissolved
in 10ml of methanol and incubated at 250 C for 30 min along with a control.
After 30 min, 1.5 ml of the incubated solution was diluted with 1.5 ml of Greiss
Reagent (1% sulphanilamide, 2% phosphoric acid and 1% N-1-
napthylethylene diamine hydrochloride in water) and mixed well. The
absorbance of the chromophore formed during diazotization of the
sulphanilamide and subsequent coupling with N-1 napthylethylene diamine
was measured at 546 nm.
% Inhibition = [(control – test)] x 100 Control
Scavenging of hydroxyl radicals92
The reaction mixture contained deoxyribose (2.8 mM), FeCl3 (0.1 mM),
EDTA (0.1 mM) H2O2 (1 mM), ascorbic acid (0.1 mM), KH2 PO4-KOH buffer
(20 mM, pH 7.4) and various concentrations of the extracts (25 µg/ml to 1000
µg/ml) to a final volume of 1 ml, was incubated for 1 hr. at 370C. Deoxyribose
degradation was measured as TBARS. The percentage inhibition was
determined by comparing the results of test and control.
% Inhibition = [(control – test)] x 100 Control
Determination of reducing power97
The reducing power of Rubus racemosus was determined according to
the following method. Mixed 10 mg of methanolic extract of Rubus racemosus
in 1 ml of distilled water with phosphate buffer (PH 6.8) and potassium
ferricyanide [K3 Fe (CN)6] (2.5 ml, 1 %). The mixture was incubated at 500 C
for 20 min. A portion (2.5 ml) of trichloroacetic acid (15%) was added to the
mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer
of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric
chloride (0.5 ml, 0.1%), and the absorbance was measured at 700nm. Increased
absorbance of the reaction mixture indicates increased reducing power.
Determination of total phenolic compounds98
Total phenolic compounds in the methanolic extract of Rubus racemosus
were determined with folin-ciotalteu reagent according to the method of
Slinkard using pyrocatechol as a standard. To 0.1 ml of extract solution
containing 1000 µg of drug extract in a 50ml volumetric flask diluted with
distilled water. Added 1 ml of folin-ciotalteu reagent and the contents of the
flask mixed thoroughly. After 3 min, 3 ml of 2% Sodium carbonate was added,
then the mixture was allowed to stand for 2 hrs with intermittent shaking and
made up to the volume with distilled water. The absorbance was measured at
760 nm. The phenolic compound in Rubus racemosus was determined as
catechol by interpolation from the calibration curve. The equation is given
below:
Absorbance = 0.001 x pyrocathecol (µg) + 0.0033
3.20 ANTIEPILEPTIC ACTIVITY
a.Maximal electroshock induced convulsion99
Procedure
Seizures are induced to all the groups by using an
electroconvulsiometer. Maximal electroshock seizures were elicited by a 60 Hz
alternating current of 150 mA intensity for 0.2 sec. A drop of electrolyte
solution (0.9% NaCl) with lignocaine was applied to the corneal electrodes
prior to application to the rats. This increased the contact and reduced the
incidence of facilities. Different doses of the MERR were administered for 14
days before induction of seizures. The duration of various phases of epilepsy
were observed. The percentage protection was estimated by observing the
number of animals showing abolition of Hind Limb Tonic Extension100
(HLTE).
Group I Animals treated with 1% SCMC,1ml/100g
Group II Animals treated with phenytion (25mg/kg) suspended in 1% w/v
SCMC
Group III Animals treated with MERR (200mg/kg) suspended in 1% w/v
SCMC
Group IV Animals treated with MERR (400mg/kg) suspended in 1% w/v
SCMC
b.Determination of the effect of Rubus racemosus and on neurotransmitter
concentrations in rat brain after induction of epilepsy
The various biogenic amines in discrete regions of the rat brain were
estimated by spectroflurorimetric method.
Reagents
1) HCl-Butanol – 0.85ml of 37% HCl was added to one liter of
butanol to get HCl-butanol solution
2) Heptane
3) 0.4M HCl
4) EDTA (pH 6.9)
5) 0.1 M Iodine
6) Sodum thiosulphate solution
7) 5 M Sodium hydroxide
8) 10 M acetic acid
9) Dopamine Standard
10) Nor-adrenaline Standard
11) 0.1M HCl
12) o-pthaldialdehyde reagent
PROCEDURE
Preparation of Tissue Extracts100
Dissected frozen rat brains were first cut on a cooled microtome (-200)
in to frontal slices (about 1mm thick) at pre determined antero posterior levels.
The frontal slices were subsequently placed on the cooled stage (-200) of a
punching apparatus where cylindrical tissue samples (usually 1 mm in
diameter, same thickness as the slice) were punched out of selected brain areas
with a glass tube. The x and y co-ordinates of the center of the area were
adjusted is a stereo microscope, ocular of which contained crossline that were
concentric with the center of the glass tube. For weight determination the tissue
pieces were transferred immediately to pre-cooled microhomogenizers which
were closed with glass stored at –250.
Extraction
The tissue was homogenized in 0.1ml HCl-Butanol for 1 minute in a
glass homogenizer made from a small centrifuge tube (vol. 1.5ml) the total
volume was considered to give 0.105ml, taking account of the tissue volume
(1mg = 0.001ml) the sample was then centrifuged for 10 min at 2000 rpm.An
aliquot of the supernatant phase (0.8ml) was added to an eppendroff reagent
tube containing 0.2ml heptane (for spectroscopy) and 0.025ml HCl 0.1M. After
10 min of vigorous shaking, the tube was centrifuged under the same condition
as above in order to separate the two phases and the overlaying organic phase
was discarded, the aqueous phase (0.02ml) was then taken either for a 5-HT or
NA and DA assay. All steps carried at 00C.
Serotonin Assay101
As mentioned earlier some modifications in reagent concentration
became necessary together with changes in proportions of the solvent, in order
to obtain in a good fluorescence yield with reduced volume for 5-HT
determination, when o-pthaldialdehyde (OPT) method was employed. Added
0.025ml of OPT reagent to 0.02ml of the HCl extract. The fluorophore was
developed by heating to 100oC for 10min. After the samples reached
equilibrium with the ambient temperature, excitation and emission spectra of
intensity reading at 360 and 470nm were recorded.
Nor-Adrenaline and Dopamine assay100
To 0.02ml of HCl phase, 0.005ml 0.4M HCl and 0.01ml EDTA/Sodium
acetatebuffer (pH 6.9) were added, followed by 0.01ml iodine solution for
oxidation. The reaction was stopped after two minutes by the addition of
0.01ml sodium thiosulphate in 5 M sodium hydroxide and 10 M acetic acid was
added 1.5 minutes latter. The solution was then heated to 1000C for 6 minutes.
When the sample again reached room temperature, excitation and emission
spectra were read (330 and 375 nm for Dopamine and 395 – 485 nm for Nor-
adrenaline) in a spectrofluorimeter compared the tissue values (fluorescence of
tissue extract minus fluorescence of tissue blank) with an internal reagent
standard (fluorescence of internal reagent standard minus fluorescence of
internal reagent blank). Tissue blanks for the assay were prepared by adding
the reagents of the oxidation step in reversed order (sodium thiosulphate before
iodine). Internal reagent standards were obtained by adding 0.005ml distilled
water and 0.1 ml HCl Butanol to 20 ng of dopamine and Nor-adrenaline
standard.
3.21 ANTIMICROBIAL METHODS
DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION
Agar streak dilution method102
MIC of the extract was determined by agar streak dilution method. A
stock solution of the extract (100 µg mL-1) in respective solvent was prepared
and incorporated in specified quantity of molten sterile agar (nutrient agar for
anti-bacterial activity and sabourand dextrose agar medium for anti-fungal
activity). A specified quantity of the medium (40 – 500C) containing the extract
was poured into a petridish to give a depth of 3-4 mm and allowed to solidify.
Suspension of the microorganism were prepared to contain approximately 105
cfu mL-1 (colony forming unit per milliliter) and applied to plates with serially
diluted extracts in respective solvent and incubated at 370C for 24 hr and 48 hr
for bacteria and fungi, respectively. The MIC was considered to be the lowest
concentration of the test substance exhibiting no visible growth of bacteria of
fungi on the plate.
a. Antibacterial Activity103
The antibacterial activity of different extracts were studied by Disc
Diffusion method against the following organisms.
• Staphylococcus aureus (gram positive)
• Staphylococcus epidermides (gram positive)
• Bacillus cereus (gram positive)
• Micrococcus luteus (gram positive)
• Klebsiella pneumoniae (gram negative)
• Pseudomonos aeruginosa (gram negative)
• Escherichia coli (gram negative)
Extracts were used in the concentrations of 25, 50 and 100 µl, using
their respective solvents comparing with Ciproflaxacin (5 mcg/disc) as
standard. The disc diffusion method was employed for the screening of
antibacterial activity.
Media Used
Soyabean casein digest agar media gm/lit
Casein enzymic hydrolysate 15.0
Papain digest of soyabean meal 5.0
Sodium chloride 5.0
Agar 15.0
Final pH at 250C 7.3 + 0.2
Disc Diffusion Method104
A suspension of Micrococcus luteus was added to sterile soyabean
casein digest agar media at 450C. The mixture was transferred to sterile petri
dishes and allowed to solidify. Sterile discs of 5mm in diameter (made from
whatmann filter paper previously sterilized by U.V.lamp) dipped in solutions of
the different extracts, standard and a blank were placed on the surface of agar
plates.
The plates were allowed to stand for 1 hour at room temperature as a
period of preincubation diffusion to minimize the effects of variation in time
between the application of the different solutions. Then the plates were
incubated at 370C for 18 hours and observed for antibacterial activity. The
diameters of the zones of inhibition was observed and measured.
The average area of zone of inhibition was calculated and compared
with that of the standards.
A similar procedure was carried out for the study of antibacterial activity
of other extracts against Staphylococcus aureus, Staphylococcus epidermides,
Bacillus cereus, Kl.pneumoniae, Pseudomonos aeruginosa and E.coli.
b. Antifungal activity
The anti fungal activity of the extracts were studied by disc diffusion
method against the following organisms.
1. Aspergillus niger
2. Aspergillus fumigates
Extracts were used in the concentrations of 25, 50 and 100 µ/l using
their respective solvents. The standard used was ketaconazole (50 mcg/disc)
against both the organisms. The disc diffustion method was employed for the
screening of anti fungal activity.
Media used
Sabouraud Dextrose Agar Medium gm/lit.
Mycological peptone 10.0
Dextrose 40.0
Agar 15.0
Final pH at 250C 5.6 + 0.2
Disc Diffusion Method
A suspension of Aspergillus niger was added to Sabouraud Dextrose
Agar Media at 450C. The mixture was transferred to sterile petridishes and
allowed to solidify. Sterile discs 5mm in diameter (made from whatmann filter
paper previously sterilized in U.V.lamp) dipped in solutions of the different
extracts, standard and a blank were placed on the surface of agar plates.
The plates were allowed to stand for 1 hour at room temperature as a
period of preincubation diffusion to minimize the effects of variation in time
between the application of the different solutions.
Then the plates were incubated at 370C for 18 hours and observed for
antifungal activity. The diameters of the zones of inhibition was measured for
the plates in which the zone of inhibition was observed. The average area of
zone of inhibition was calculated and compared with that of the standards.
A similar procedure was carried out for the study of antifungal activity
of other extracts against Aspergillus fumigates.
RESULTS
CHAPTER – IV
RESULTS
4.1 PHARMACOGNOSTICAL STUDIES
4.1.1 Macroscopy
Colour : Green
Odour : Characteristic
Taste : Characteristic
Size : 3 – 5 cm in length
1.5 – 2.5 cm in width
Shape : Oval
Petiole : A small petiole present
Margin : Serrate
Apex : Acute
Base : Symmetrical
Veins : 6 – 8 veins on each side
Fig: 3 Cross Sectional View of young folded Leaf
[GT – Glandular trichome; La – Lamina; LV – Lateral vein;
MR – Midrib; NGT – non glandular trichome; VB – Vascular bundle].
Fig:4 Anatomy of the Leaf
4.1 Transverse Section of Leaf through midrib with lamina
4.2 & 4.3 Transverse Section of Midrib & Lamina
[AbE – Abaxial epidermis ; Abs – Abaxial side; Ads – Adaxial side;
Ep – Epidermis; GT – Ground tissue; La - Lamina; LV – Lateral vein;
MR – Midrib; Ph – Phloem; PM – Palisade mesophyll; SM – Spongy
mesophyll; VB – Vascular bundle; X - Xylem].
Fig:5 Anatomy of the midrib and Trichomes
5.1 Transverse section of young leaf showing midrib and lateral vein
5.2 Glandular and Non-glandular trichomes in sectional view
5.3 Glandular and Non-glandular trichomes enlarged
[B – Body of the trichome; Ep – Epidermis; GT – Ground tissue;
LV – Lateral vein; MR – Midrib; NGT – Non-glandular trichome; Ph –
Phloem; St – Stalk cells; X - Xylem].
Fig :6 Anatomy of the Petiole
6.1 Transverse Section of petiole entire view [Distal region]
6.2 Transverse Section of petiole ground Plane [Proximal region]
[AdG – Adaxial groove; Col – Collenchyma; Ep – Epidermis; LB – Lateral
bundle; AB – Abaxial bundle; MB – Median bundle; Pa – Parenchyma cells;
Ph – Phloem; Sc – Sclerenchyma cells; Tr – Trichome; X – Xylem].
Fig :7 Structure of the Petiole – Proximal region
[AB – Adaxial bundle; AdG – Adaxial groove; Col – Collenchyma;
Ep – Epidermis; LB – Lateral bundle; MB – Median bundle; Pa – Parenchyma
cells; Ph – Phloem; Sc – Sclerenchyma cells; X – Xylem].
Fig :8 Anatomy of the Young Stem
8.1 Transverse Section of Stem under low magnification
8.2 Transverse Section of Stem under high magnification
[Co – Cortex; Col –Collenchyma cells; Ep – Epidermis;
MR – Medullary – ray; Ph – Phloem; Pi – Pith; Sc – Sclerenchyma; St – Stele;
X - Xylem].
Fig: 9 Glandular trichome of the Stem
9.1 Transverse Section of Stem showing a glandular trichome
9.2 Glandular trichome enlarged
[Co – Cortex; Col – Collenchyma; H – Head; Pi – Pith; St – Stalk].
Fig: 10 Trichome Morphology
10.1 Non-glandular trichome stained with Toluidine Blue
10.2 Non-glandular trichome stained with Safranin
[NGT – Non-glandular trichome].
Anatomy of the leaf
The leaf has prominent midrib and lateral veins and their lamina (Fig.3).
The leaf has dense epidermal trichomes on the adaxial side and smooth on the
abaxial side. Young leaves are plicate longitudinally and the mature leaves are
flat (Fig.4.1).
4.1.2 Microscopy
Midrib (Fig.4.1, 4): The midrib has concave adaxial side and broad and thick
abaxial part. It has the length of 1.6mm vertically and 1.75 mm horizontally.
The epidermal layer is thin, made up of small, thick walled cells. Two or three
layers inner to the epidermis are collenchymatous, rest of the ground tissue has
thin walled, circular, compact parenchyma cells. The vascular bundle is single,
broad and bowl shaped. It consists of several long, radial rows of xylem
elements and several small groups of phloem elements.
Lamina (Fig.4.3): The lamina is 120 µm thick. The adaxial side is even and
smooth; the abaxial side is undulate and hairy. The adaxial epidermis is thick
with large squarish cells and thick cuticle; the cells are 20 µm thick. The
abaxial epidermis is thin comprising of narrow, rectangular cells. The
mesophyll tissue consists of two layers of short, thin palisade cells and 6-8
layers of small, lobed spongy parenchyma cells. The vascular bundles of the
veinlets are small, surrounded by a layer of dilated hyaline bundle-sheath cells
and narrow adaxial and abaxial extensions.
Trichomes: The leaf has dense glandular and nonglandular trichomes. The non
glandular trichomes are unicellular, unbranched, thick walled and pointed at the
tip (Fig5.2,3). They arise from a pedestal of a group of cells raised above the
level of the epidermis (Fig.5.2). The non glandular trichomes are 250-650 µm
long.
The glandular trichomes are more complex in structure. They occur on
the leaf (Fig.5.1 – 3) and the petiole or stem (Fig.9.1,2).The glandular
trichomes have a long filamentous stalk and prominent, spherical head. The
stalk consists of a single vertical row of cells or multiseriate elongated cells
(Fig.9). The head of the gland has darkly staining compact mass of cells. The
gland varies in length from 120 µm to 1.3 mm. The head is 50-120 µm thick.
Petiole: The petiole is circular in sectional outline and has shallow adaxial
groove (Fig.6.1, 2). It is 1.75mm in diameter. The upper part (distal part) of the
petiole has a broad main, medianly placed vascular bundle and three accessory
lateral bundles on either side, which diminish in size towards adaxial part
(Fig.6.1). The lower part (proximal part) has wider median lervelle, two
prominent lateral bundles and two smaller less prominent bundles (one set on
either side of the adaxial part) (Fig.6.2,7). All the bundles are collateral with
thick band of sclerenchyma cells abutting the phloem tissue. The petiole has
their epidermis with small thick walled cells. Inner to the epidermis is a narrow
zone of two or three layers of collenchyma cells. The remaining ground tissue
is homogeneous and parenchymatous (Fig.7).
Stem (Fig.8.1.2): The stem is uneven in outline with ridges and furrows due to
the presence of thick epidermal trichomes. The stem has a thin layer of
epidermis comprising of small thick walled cells. Epidermis is followed by
about five layers of collenchyma cells. Inner to the collenchyma zone is a
narrow cortical zone of parenchyma cells, some of them having chloroplasts.
The vascular cylinder is thin and continuous and consisting of several
wedge-shaped vascular bundles placed close to each other. The vascular
bundles are collateral and have thick mass of sclerenchyma caps, wide zone of
phloem and radial files of xylem. The sclerenchyma cells are thin walled and
wide lumened. Xylem elements are circular and thick walled. The pith is wide
and parenchymatous.
Trichome Morphology (Fig.10)
The powdered sample of the leaf shows the trichomes and epidermal
fragments. The non glandular trichomes are seen randomly distributed on the
fragments of the epidermis. They are long, whiplike, thickwalled with smooth
surface; they are tapering at the ends. The glandular trichomes are long stalked
with spherical head. The cells of the head portion are darkly staining.
4.1.3 Powder analysis
Sensory Characters
Appearance Coarse
Colour Green
Odour Characteristic
Taste Characteristic
Microscopical Characters
Trichomes Unicellular, Non glandular trichomes
Stomata Anisocytic
Epidermal cells Cells are thin and wavy in nature in lower surface
and straight walls in upper surface
Mesophyll Palisade and spongy parenchyma with epidermis
Vessels They are spiral in nature
Calcium oxalate Prismatic type
Crystals
4.1.4 Flourescence analysis
The powder of Rubus racemosus was treated with various reagents and
visualized under ultra violet radiations. The observations are furnished in Table
No.1.
Table: 1
Drug Fluorescent colours
Powder Green
Powder + Water Green
Powder + Conc.H2SO4 Dark green
Powder + Conc.HNO3 Pale green
Powder +Conc.HCl Dark green
Powder + dil.H2SO4 Light green
Powder + dil.HNO3 Light green
Powder + dil.HCl Green
Powder +1N NaOH Blackish green
Powder + Ac.anhydride Emerald green
Powder + Acetone Dark green
4.1.5 Quantitative microscopy
The length of trichomes of Rubus racemosus is
Range 165µ 320.18µ 622.5µ
Minimum Average Maximum
The width of trichomes of Rubus racemosus is
Range 12.7µ 34.82µ 41.4µ
Minimum Average Maximum
4.1.6 Determination of Leaf Constants
The range for the vein islet number of Rubus racemosus is
Range 2 3 4
Minimum Average Maximum
The vein let termination number of Rubus racemosus is
Range 5 7 9
Minimum Average Maximum
The range for the stomatal number of Rubus racemosus is adaxial surface
Range 4 6 8
Minimum Average Maximum
The stomatal index of of Rubus racemosus in adaxial surface
Range 6.7 11.2 14
Minimum Average Maximum
The range of stomatal number of Rubus racemosus in abaxial surface
Range 2.5 3.75 5
Minimum Average Maximum
The range for stomatal index of of Rubus racemosus in abaxial surface
Range 5 3.7 2.2
Minimum Average Maximum
4.1.7 Determination of physiochemical constant
The ash values of Rubus racemosus is
1. Total ash - 2.97% w/w
2. Acid insoluble ash - 1.80% w/w
3. Sulphated ash - 2.65% w/w
4. Water soluble ash - 0.18% w/w
The extractive values of Rubus racemosus is
1. Alcohol soluble extractive value is 8.35% w/w
2. Water soluble extractive value is 4.21% w/w
The loss on drying of Rubus racemosus at 1050C is 7.84% w/w
4.2 PHYTOCHEMICAL STUDIES
4.2.1 Extractive Values of different extracts
The successive solvent extractive values of the aerial parts of Rubus
racemosus
Petroleum ether : 12.87% w/w
Ethyl acetate : 10.22% w/w
Chloroform : 12.52% w/w
Methanol : 23.76% w/w
Aqueous : 14.32% w/w
4.2.2 Preliminary phytochemical screening
Preliminary phytochemical analysis of methanolic extract of Rubus
racemosus
The result of preliminary phytochemical analysis of Rubus Racemosus
extracts is shown in table 2. All the extracts showed the presence of various
phytochemical constituents like terpenes, tannins, flavanoids, saponins,
carbohydrates, glycosides and phenols. However the extracts showed negative
results for proteins, steroid, sterols, alkaloids, gum and mucilages.
Table: 2
Phytoconstituents Petroleum
Ether Ethyl
AcetateChloroform Methanol Aqueous
Alkaloids
Mayer’s - - - - -
Hager’s - - - - -
Wagner’s - - - - -
Dragendroff’s - - - - -
Carbohydrates
Molisch + + + + +
Fehling’s + + + + +
Benedict’s + + + + +
Barford’s + + + + +
Steroids
Liberman - - - - -
Burchard Test - - - - -
Sterols
5% potassium hydroxide
- - - - -
Proteins
Biuret - - - - -
Millon’s - - - - -
Phenols
Ferric chloride + + + + +
10% sodium Chloride
+ + + + +
Tannins
10% lead acetate + + + + +
Phytoconstituents Petroleum
Ether Ethyl
AcetateChloroform Methanol Aqueous
!0 % sodium chloride
+ + + + +
Aqueous bromine Solution
+ + + + +
Flavanoids
Amylalcohol + Sodium acetate + Ferric chloride
+ + + + +
Gums and Mucilage
- - - - -
Glycosides
Glacial acetic acid + ferric chloride + conc. H2SO4
+
+
+
+
+
Saponins
Foam test + + + + +
Terpenes
Tin + Thionyl Chloride
+ + + + +
+ = Showed colour reactions
– = Did not show colour reaction
4.2.3 Flourscence analysis of different extracts
The different extracts were observed under ultra violet light and the
findings are furnished in Table no.3.
Table: 3
Extracts Fluorescent colours
Petroleum ether Dark green
Ethyl acetate Pale green
Chloroform Green
Methanol Emerald green
Aqueous Dark green
4.2.4 Thin layer chromatography
Petroleum ether, Ethyl acetate, Chloroform, Methanol and Aqueous
extracts of Rubus Racemosus were subjected to thin layer chromatography for
flavanoids, glycoside and tannins with respective solvent system. Their Rf
values were determined and furnished in Table 4,5 and 6.
TLC for flavanoids
Table: 4
Extracts Solvent System Detecting agent Rf Petroleum ether UV radiation 0.35 Ethyl acetate 0.48 Chloroform 0.56 Methanol 0.59 Aqueous
Chloroform: Ethyl acetate 60:40
0.35
TLC for glycoside
Table: 5
Extracts Solvent System Detecting agent Rf
Petroleum ether UV radiation 0.52
Ethyl acetate 0.62
Chloroform 0.51
Methanol 0.32
Aqueous
Chloroform: Methanol: Water 30:25:10
0.31
TLC for tannins
Table: 6
Extracts Solvent System Detecting agent Rf
Petroleum ether UV radiation 0.55
Ethyl acetate 0.65
Chloroform 0.34
Methanol 0.45
Aqueous
n-butanol: Glacial acetic acid: Water 14:1:5
-
4.2.5 Isolation of Constituents by column Chromatography
Examination of eluates
Table: 7
Eluent % of solvent Fractions Compound
Petroleum ether 100% 1-100 Wax
Chloroform 100% 101-149
Ethyl acetate 100% 150-154
Ethyl acetate : Isopropanol 90:10 155-159
Ethyl acetate : Isopropanol 80:20 160-164
Ethyl acetate : Isopropanol 70:30 165-169
Ethyl acetate : Isopropanol 60:40 170-174 Compound I
Ethyl acetate : Isopropanol 50:50 175-179 Compound I
Ethyl acetate : Isopropanol 40:60 180-184
Ethyl acetate : Isopropanol 30:70 185-189
Ethyl acetate : Isopropanol 20:80 190-194
Ethyl acetate : Isopropanol 10:90 195-199
Isopropanol 100% 200-204
Isopropanol : Ethyl alcohol 90:10 205-209
Isopropanol : Ethyl alcohol 80:20 210-214
Isopropanol: Ethyl alcohol 70:30 215-219
Isopropanol : Ethyl alcohol 60:40 220-224
Isopropanol : Ethyl alcohol 50:50 225-229
Isopropanol : Ethyl alcohol 40:60 230-234
Isopropanol : Ethyl alcohol 30:70 235-239
Isopropanol : Ethyl alcohol 20:80 240-244
Isopropanol : Ethyl alcohol 10:90 245-249
Ethyl alcohol 100% 250-254 Compound II
Ethyl alcohol : Methyl alcohol 90:10 255-259 Compound II
Ethyl alcohol : Methyl alcohol 80:20 260-264
Eluent % of solvent Fractions Compound
Ethyl alcohol : Methyl alcohol 70:30 265-269
Ethyl alcohol : Methyl alcohol 60:40 270-274
Ethyl alcohol : Methyl alcohol 50:50 275-279
Ethyl alcohol : Methyl alcohol 40:60 280-284
Ethyl alcohol : Methyl alcohol 30:70 285-289
Ethyl alcohol : Methyl alcohol 20:80 290-294
Ethyl alcohol : Methyl alcohol 10:90 295-299
Methyl alcohol 100% 300-304
Eluates (170-179), (250-259) showed residues with similar Rf values
with respect to thin layer chromatography. Hence all the residues were mixed
together and recrystallized from methanol: water (1:1). The substance was
examined for further process. The isolated compounds are yellow in colour and
highly hygroscopic in nature.
TLC for isolated compound
Table: 8
Compound Solvent System Detecting agent Rf
Compound I Compound II
Ethyl acetate : Isopropanol Ethyl alcohol : Methyl alcohol
UV radiation 0.62 0.59
4.2.6 Spectral studies
4.2.6.1 Compound I
The IR spectrum of the isolated compound I from Rubus racemosus
shows characteristic absorption bands at 3411cm-1 for –OH group and 1651 cm-
1 for α, β – unsaturated carbonyl group.
The 1HNMR spectrum revealed three distinct patterns of proton
resonances.
a) The first is typical for esterified p-hydrozy benzoic acid and contained
two signals at δ6.85 (d, 2H) and δ7.11 (d, 2H).
b) The second pattern is characteristic for sugar protons and contained four
anomeric resonances at δ5.3, δ5.2, δ4.91 and δ4.92. The chemical shift
values of these doublet signals indicated esterification of the anomoric
hydroxyl group and four sugar units were present. Other sugar protons
resonate at chemical shift values between δ3.04 to δ3.84.
c) The third pattern of H-NMR signals is indicative of a long chain epoxide
and contained signals at δ0.84 (t, 3H, CH3), a strong singlet at δ1.22 for
long chain methylene protons, two multiplets at δ2.83 and δ2.69 for
epoxide hydrogens and a signal at δ1.57 for the α – methylene protons to
the epoxide ring.
The above datas were strongly supported by the 13C-NMR spectral
datas. It exhibited a signal at δ174.99 for ester carbonyl group and at 156.46
(C-1), 143.36 (C-4), 114 (C-2, C-6) and 127.89 (C-3, C-5) for aromatic ring
carbons. The downfield signal at δ156.46 is due to the substitution with a –OH
group in that position.
The spectrum also indicated the presence of four anomeric carbon
signals at δ102.44, δ98.49, δ97.35 and δ92.68. The other carbon signals of the
sugar moiety appeared between δ63.38 to δ82.35. Further the 13C-NMR
spectral data exhibited the signals at δ14.39 (CH3), δ61.69 and δ63.38 (epoxide
ring carbons), 26.00 and 27.00 for α – carbons to the epoxide ring.
The presence of fragment ions at m/z 309 and m/z 153 arising from
fragmentations adjacent to the oxygen bearing carbons suggest that the epoxide
was located on C-20 and C-21 of the chain. All the other fragments are closely
related to the fragments of the compound II suggesting the same moiety may be
present in compound I.
On alkaline hydrolysis, compound II gave D-glucose as the sugar
component which was identified by direct comparison with an authentic
sample. Considering the above datas into consideration the following structure
is proposed for the compound I (1-[20,21 – dodeacylnanone] – α-1→6-D-
glucotetraose-6’’’(P-hydroxy) benzoate).
Spectrum 1.1
Specturm 1.2
Spectrum 1.3
Spectrum 1.4
Compound I
Considering the above facts the structure is arrived as follows.
OHO
HO OHO
1
2
3
45
6
G
OH1
2
3
C
O
O4
5
6
OHO
HO OH O
6'
G'1'
OHO
HO OH O
6"
G"1''
OHO
HO OH O
6"'
G'''1'''
O29
22
21
20
191
2
m/z 309
3 5 7
4.2.6.2 Compound II
The molecular formulae of the isolated compound II is found to be
C29 H58 O from the E1 mass spectrum [MH]+ m/z 423. The ‘H and 13C NMR
spectra indicated that II was a linear hydrocarbon with an epoxide ring (δH,
2.90; δc, 58.19). The 200MHz 1H-NMR data further showed two very close
methyl resonances at δ0.84 and δ 0.88. The methylene protons ά to the epoxide
ring resonated at δ1.47 and gave the chemical shifts of the corresponding
carbons at δc 27.42 and 28.65. The strong singlet at δH 2.25 and δc 30.52
corresponds to the long chain methylene protons and carbons respectively. The
epoxide position in the aliphatic chain was determined using mass
spectrometry. The presence of two fragments at m/z 155(10%) and m/z 309
(5%) arising from fragmentations adjacent to the oxygen bearing carbons,
suggested that the epoxide was located on C-9 and C-10 of the chain.
A search in the literature reveals that the compound was earlier isolated
from Rubus thibetanus peak by peak comparison was made. Comparison of 1H-
NMR and 13C-NMR data of compound II with the values reported in the
literature are furnished in Table no.9.
Spectrum 2.1
Spectrum 2.2
Spectrum 2.3
Spectrum 2.4
Spectrum 2.5
Compound II
Considering the above facts the structure is arrived as follows.
Om/z 155
m/z 309m/z 144m/z57
m/z43
29
10 9 1
O
10 9 1
29
Comparision of the spectra of isolated compound II with the reported are
furnished in Table no.9
Table: 9
Proton δH of II Literature value
Carbon position δc of II Literature
value
1-CH3 29-CH3
8&11-CH2
9&10, CH CH2
0.84 0.88 1.47 2.90
2.25
0.860 0.861 1.48 2.88
-
1&29 9,10
8 11
CH2
14.92 58.19 27.42 27.92 30.52
32.75 23.50
14.2 57.4 26.70 28.65 29.50 29.30 29.80 29.70 32.00 22.80
The compound II may be 9,10 epoxynonacosane.
4.2.7. HPTLC studies
HPTLC Studies of the extract of Rubus racemosus
The Chloroform extract was subjected to HPTLC studies. The sample showed 8 peaks scanning 254 nm and their Rf values were furnished in Table no.10 and Spectrum no. 3.1.
Table: 10
Track Peak Rf values Area Percentage 1 0.07 11.45%
2 0.08 24.33%
3 0.27 6.15%
4 0.39 13.63%
5 0.54 9.48%
6 0.59 4.99%
7 0.67 26.02%
8 0.81 3.94%
Specturm 3.1
Spectrum 3.2
HPTLC fingerprint of the isolated compound showed single peak with
Rf value 0.39.
Spectrum 3.3
4.3 PHARMACOLOGICAL STUDIES
4.3.1 Acute oral toxicity studies
The acute oral toxicity study was carried out according to the OECD
guidelines 423 (acute toxic class method) as represented in Table 11. The
starting dose of 2000 mg/kg body weight / p.o of the one methanolic extract of
Rubus racemosus was administered to 3 male rats and observed for three days.
There was no considerable weight change in body weight before and after
treatment and no signs of toxicity was observed. When the experiment was
repeated again with the same dose level 2000 mg/kg body weight / p.o of the
methanolic extract of Rubus racemous for three more days and observed for
fourteen days no change was observed from the first set of experiments. LD50
cut off mg/kg was observed as class X (unclassified) and Globally Harmonized
system (GHS).
4.3.2 Sub Acute toxicity Studies
The Methanolic extract of Rubus Racemosus at a dose of 400 g/kg b.w
p.o was administered for 28 days. The changes in body weight, food and water
intake was observed for entire study. No significant decrease in body weight
was observed. The MERR treated rats did not show any significant changes in
haematological parameters like Hb, RBC, WBC, neutrophils, monocytes,
eosinophil and lymphocyte, ECR, PCV values when compared with normal
control animal. Histopathological examination of internal organs like liver,
kidney, spleen, heart, lungs, testis, ovary and brain showed no change in their
normal architecture suggesting no damage caused by MERR treatment.
4.3.3 Haematological Parameters
The methanolic extract of Rubus racemosus did not show any
remarkable changes in Haemoglobin, RBC, WBC, ESR, PCV and Neutrophils.
The Results are shown in Table.12.
4.3.4 Antidiabetic Activity
4.3.4.1 Effect of MERR on blood glucose level in normal rats
The MERR at a dose level of 200mg/kg p.o did not exhibit any
significant hypoglycemic effect in the fasted normal rats after 60 min and 120
min of oral administration, when compared with the initial blood glucose levels
before administration of the extracts. A higher dose of 400 mg/kg, also did not
exhibit significant hypoglycemic action at the end of 60,90 and 120 min after
oral administration in the normal fasted rats. Results are shown in table 14 and
figure 12.
4.3.4.2 Effect of MERR on blood glucose level on glucose fed
hyperglycemic rats
The MERR at a dose level of 200mg/kg and high dose 400mg/kg/p.o
reduced the raised blood glucose level [hyperglycemic due to glucose load
2 gm/kg/p.o at 4 ml/kg] significantly [p<0.001] after 120 min of oral
administration when compared to control group. This standard drug
glibenclamide also produced significant [p<0.001] drop in blood glucose level.
Results are shown in table 15 and figure 13.
4.3.4.3 Effect of acute treatment of MERR on blood glucose level in STZ
induced diabetic rats
The effect of MERR was evaluated at a double dose administration of
MERR [low dose 200 mg/kg/p.o and high dose of 400mg/kg/p.o] a double dose
of extracts did not produce significant reduction in the blood glucose levels in
STZ induced diabetic rats.
Treatment with glibenclamide also did not reduce the blood glucose
levels significantly. Results are shown in table 16 and figure 14.
4.3.4.4 Effect of sub acute treatment of MERR on blood glucose level in
STZ induced diabetic rats
In the sub acute study STZ induced diabetic rats were treated with
MERR 200 mg/kg/p.o and 400mg/kg/p.o for duration of 28 days. Treatment
with MERR significantly [p<0.001] decreased the blood glucose level after 21
days and concomitantly after that in diabetic rats. Treatment with MERR
produced a significant drop in blood glucose level from 21st day onwards upto
28 days. Treatment with gilbenclamide produced significant [p<0.001]
decrease in blood glucose level steadily after the 14th day or oral administration
and thereafter. Results are shown in table 17 and figure 15.
4.3.4.5 Biochemical Estimation
4.3.4.5.1 Total bilirubin
The diabetic control animals showed significant [p<0.001] increase in
serum total bilirubin level when compared to control animals. The serum total
bilirubin levels in MERR (200mg and 400 mg/kg b.wt) treated diabetic rats
showed significant increase [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)
treatment showed significant [p<0.001] decrease when compared to STZ
induced diabetic animals. Results are shown in Table 18 and figure 16.
4.3.4.5.2 Serum glutamate oxalocetate transminase (SGOT)
The SGOT level was significantly [p<0.01] increased in STZ induced
diabetic rats when compared to control animals. SGOT levels of diabetic rats
treated with MERR (200mg and 400 mg/kg b.wt) showed significant decrease
[p<0.01] and [p<0.001] respectively. However glibenclamide (0.5 mg/kg
b.w/p.o) treatment showed significant [p<0.001] decrease when compared to
STZ induced diabetic rats. Results are shown in Table 19 and figure 17.
4.3.4.5.3 Serum glutamate pyruvate trasaminase (SGPT)
The SGPT level was significantly [p<0.01] increased in STZ induced
diabetic rats when compared to control animals. SGPT levels of diabetic rats
treated with MERR (400 mg/kg b.w p.o) and glibenclamide (0.5 mg/kg
b.w/p.o) showed significant (p<0.01) decrease when compared to STZ induced
diabetic rats. Results are shown in Table 20 and figure 18.
4.3.4.5.4 Serum total protein
The diabetic control animals showed significant [p<0.001] decrease in
serum total protein level when compared to control animals. The serum total
protein levels in MERR (200mg and 400 mg/kg b.wt) treated diabetic rats
showed significant increase [p<0.001]. Glibenclamide (0.5 mg/kg/p.o) showed
significant increase [p<0.001] when compared to STZ induced diabetic rats.
Results are shown in table 21 and figure 19.
4.3.4.5.5 Alkaline phosphatase (ALP)
The ALP level in serum was significantly [p<0.001] increased in STZ
induced diabetic rats when compared to control. ALP levels of diabetic rats
treated with MERR (400 mg/kg b.w p.o) and glibenclamide (0.5 mg/kg
b.w/p.o) showed significant (p<0.001 and p<0.001, respectively) decrease
when compared to STZ induced diabetic rats. Results are shown in Table 22
and figure 20.
4.3.4.5.6 Serum total cholesterol
The total cholesterol level significantly [p<0.001] increased in STZ
induced diabetic rats when compared to control rats. Serum total cholesterol
levels of diabetic animals treated with MERR (200 and 400 mg/kg b.w/p.o)
showed significant [p<0.01] decrease in cholesterol level when compared to
STZ induced diabetic animals. Results are shown in Table 23 and figure 21.
4.3.4.5.7 Serum HDL-Cholesterol
The serum HDL-cholesterol level was significantly decreased [p<0.001]
in STZ induced diabetic rats when compared to control rats. HDL-cholesterol
level of diabetic rats treated with MERR (200 and 400 mg/kg b.wt/p.o) showed
significant increase [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o) treatment
showed significant [p<0.001] increase HDL-cholesterol when compared to
STZ induced diabetic animals. Results are shown in Table 24 and figure 22.
4.3.4.5.8 Serum triglycerides
The serum triglyceride level was significantly [p<0.001] increased in
STZ induced diabetic rats when compared to control rats. Triglyceride level of
diabetic rats treated with MERR (200 and 400mg/kg b.wt/p.o) showed
significant decrease [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)
treatment showed significant [p<0.001] decrease when compared to STZ
induced diabetic animals. Results are shown in Table 25 and figure 23.
4.3.4.5.9 Serum LDL-Cholestrol
The serum LDL-cholesterol level was significantly [p<0.001] increased
in STZ induced diabetic rats when compared to control rats. LDL-cholesterol
level of diabetic rats treated with MERR (200 and 400 mg/kg b.wt/p.o) was
showed significant decrease [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)
treatment showed significant [p<0.001] decrease when compared to STZ
induced diabetic animals. Results are shown in Table 26 and figure 24.
4.3.4.6 Antioxidant enzymes in liver Homogenate
4.3.4.6.1 Superoxide Dismutase
A significant [p<0.01] decrease in the liver SOD was observed in STZ
induced diabetic animals when compared to control animals. The liver SOD
levels of diabetic animals treated with double dose of MERR and
glibenclamide showed significant [p<0.01] increase when compared to STZ
induced diabetic animals. Results are shown in table 27 and figure 25.
4.3.4.6.2 Catalase
A significant [p<0.001] decrease in the liver CAT was observed in STZ
induced diabetic animals when compared to control animals. The liver CAT
level of diabetic rats treated with double dose of MERR was significantly
[p<0.001] increased. Glibenclamide treatment showed slight [p<0.01] increase
in CAT when compared to STZ induced diabetic rats. Results are shown in
table 28 and figure 26.
4.3.4.6.3 Glutathione Peroxidase
The GSH PX lelvel in lever was significantly [p<0.001] decreased in
STZ induced diabetic animals when compared to control animals. Double dose
of MERR significantly [p<0.001] increased the GSH-PX level when compared
to STZ induced diabetic animals. Glibenclamide treatment showed [p<0.001]
increase in GSH-PX when compared to STZ induced diabetic rats. Results are
shown in table 29 and figure 27.
4.3.4.6.4 Glutathione Reductase
The GSH Rase level in and liver was significantly [p<0.001] decreased
in STZ induced diabetic rats when compared to control. The liver GSH Rase
level of diabetic rats treated with double dose of MERR significantly [p<0.01]
increased and glibenclamide also showed significant [p<0.05] increase when
compared to STZ induced diabetic animals. Results are shown in table 30 and
figure 28.
4.3.4.6.5 Lipid Peroxidation (LPO)
A significant [p<0.001] increase in the liver lipid peroxidation was
observed in STZ induced diabetic animals when compared to control animals.
Lipid peroxidation level of animals treated with double dose of MERR showed
significant [p<0.001] decrease and glibenclamide treatment showed significant
[p<0.001] decrease when compared to STZ induced diabetic animals. Results
are shown in table 31 and figure 29.
4.3.5 In vitro Antioxidants Studies
4.3.5.1 Free radical scavenging activity by DPPH reduction
Reduction of the DPPH radicals can be observed by the decrease in the
absorbance at 517 nm. The scavenging capacity of the MERR was found to be
86.81% with IC50 being 348.87 µg/ml. Results are shown in Table 34 and
figure 32.
4.3.5.2 Nitric oxide scavenging activity
The scavenging of nitric oxide by MERR was concentration dependent
and the IC50 value was found to be 219.53µg/ml. The percentage scavenging of
Nitric oxide was as high as 84.26% at the concentration of 1000 µg/ml. Results
are shown in Table 35 and figure 33.
4.3.5.3 Hydroxyl radical scavenging activity
The MERR scavenged the hydroxyl radicals generated by the
EDTA/H2O2 system, when compared with that of control. The percentage
scavenging of the Hydroxyl radicals by MERR increased in a dose dependent
manner and was found to the 83.42% at 1000 µg/ml concentration. The IC50
value was found to be 520.32 µg/ml. Results are shown in Table 36 and figure
34.
4.3.5.4 Determination of reducing power
The reducing power of MERR with increasing concentration of MERR
showed significant increase in activities than control. Results were comparable
with the standard (BHT). Results are shown in Table 37 and figure 35.
4.3.5.5 Determination of total phenolic compounds
Phenols are very important plant constituents because of their
scavenging ability due to their hydroxyl groups. In the MERR (1mg),
137±0.078 µg pyrocatechol equivalent of phenols was detected.
4.3.6 Antiepileptic activity
4.3.6.1 Maximal electroshock induced convulsion
Effects of MERR on MES induced Epilepsy
Phenytoin treated animals have shown 100% protection against MES
induced seizures where as MERR 200mg/kg and 400 mg/kg have shown
54.35% and 67.31% protection respectively against MES induced seizures.
The MERR at both doses and standard treated rats did not show any
significant change in duration of tonic flexion and clonic convulsions.
MERR 200 mg/kg and 400 mg/kg had shown a significant decrease in
the duration of tonic extensor phase and comparable p<0.001 with the standard.
Results are shown in Table 38 and figure 36.
4.3.6.2 Effect of MERR on neurotransmitter levels in MES induced rats
4.3.6.2.1 Serotonin
A significant p<0.001 & p<0.001 decrease in brain Serotonin levels was
observed in forebrain of epileptic control animals. MERR 200mg/kg and 400
mg/kg treated rats have shown a significant p<0.001, p<0.001 increase in
Serotonin levels in forebrain. The results are shown in Table 39 and figure 37.
4.3.6.2.2 Noradrenaline
A significant p<0.001 decrease is observed in forebrain in epileptic
control animals. MERR 200mg/kg and 400 mg/kg and PHT treated animals
showed a significant p<0.05 & p<0.001 increase in Noradrenaline levels in
forebrain of MERR treated animals. Results are shown in Table 40 and
figure 38.
4.3.6.2.3 Dopamine
A significant p<0.001 decrease in the dopamine levels is observed in
forebrain in epileptic control animals and a significant p<0.001 increase is
observed in forebrain on MERR 200mg/kg and 400 mg/kg treated rats, PHT
treated animals showed a significant p<0.001 increase forebrain. Results are
shown in Table 41 and figure 39.
4.4 ANTIMICROBIAL STUDIES
4.4.1 Antibacterial Activity
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Staphylococcus aureus. The Results were shown in Table 42 and figure 40.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Staphylococcus epidermidis. The Results were shown in Table 43 and figure
41.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against Bacillus
cereus. The Results were shown in Table 44 and figure 42.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Micrococcus luteus. The Results were shown in Table 45 and figure 43.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Klebsiella pneumoniae. The Results were shown in Table 46 and figure 44.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Pseudomonos aeruginosa. The Results were shown in Table 47 and figure 45.
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Escherichia coli. The Results were shown in Table 48 and figure 46.
4.4.2 Antifungal Activity
Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited
significant activity at the concentration of 100µg/ml whereas it showed
moderate activity at the concentration of 25µg/ml and 50µg/ml against
Aspergillus Niger and Aspergillus fumigatus. The Results were shown in
Tables 49 , 50 and figures 47 and 48.
The minimum inhibitory concentration values of all the extracts were
determined.
Acute toxicity class method OECD guidelines 423
Table: 11
Weight Of Animal Group
S. No
Group Dose Before Test
(on 1st day)
After Test
(on 4th day)
Signs Of toxicity
Onset of toxicity
Reversible Of
Irreversible Duration
1
MERR
2000mg
158
155
No sign of
toxicity
Nil
Nil
14 days
2
MERR
2000mg
162
159
No sign of
toxicity
Nil
Nil
14 days
3
MERR
2000mg
160
159
No sign of
toxicity
Nil
Nil
14 days
As no toxicity or death was observed for these dose levels the same dose level was tried again
1
MERR
2000mg
160
157
No sign of
toxicity
Nil
Nil
14 days
2
MERR
2000mg
163
161
No sign of
toxicity
Nil
Nil
14 days
3
MERR
2000mg
162
159
No sign of
toxicity
Nil
Nil
14 days
MERR: Methanolic Extract of Rubus Racemosus
121
Haematological parameters of MERR treatment on sub-acute toxicity study
Table: 12
Groups Hb% RBC
X106/mm3
WBC
Cells/mm3 ESR PCV
%
Neutrophil
%
Eosonophil
%
Lympocytes
%
Monocytes
Control 13.86±.76 4.7±.58 8.3±.1 3.4 ±1 40.2 ± 2.83 24±.8 4.38±.7 75.2±.4 2.15±.4
MERR 13.52±.68ns 4.57±.52ns 7.92±.26ns 3.2 ±.6 45.25 ± 3.56 21.1±.7ns 3.21±.92ns 69.1±1.6ns 1.3±.4ns
Values are expressed in mean ± SEM. Each group consists of 6 rats.
Statistical significance test for comparison was done by Student ‘t’ test.
ns – non significant
Histopathology report of various organs on MERR
treatment on sub-acute toxicity study
Table: 13
Report
S. No
Organ Control I Group II
1.
Liver
Shows normal liver with central vein with cords of hepatocytes
Shows normal liver with central vein with cords of hepatocytes
2.
Kidney
Normal kidney with glomeruli and tubules
Normal kidney with glomeruli and tubules
3.
Heart
Normal cardiac fibre
Normal cardiac fibre
4.
Testis
Shows normal testicular tubules with normal spermatogenesis
Shows normal testicular tubules with normal spermatogenesis
5.
Lung
Shows normal lung tissue with bronchi and alveoli
Shows normal lung tissue with bronchi and alvcoli
6.
Brain
Shows normal brain tissues with astrocytes and nerve fibres.
Shows normal brain tissues with astrocytes and nerve fibres.
7.
Spleen
Spleen with follicles and hyaline septa
Spleen with follicles and hyaline septa
8.
Ovary
Ovary with maturing follicles
Ovary with maturing follicles
Group I – Rats received 1% SCMC (2ml/kg) orally for 28 days.
Group II – rats received MERR (400mg/kg) orally for 28 days.
Figure.11
Histopathology of various organs in toxicity studies
ANTIDIABETIC ACTIVITY
Effect of Rubus Racemosus treatment on blood glucose level in normoglycaemic rats
Table: 14
Groups Initial 30min 60min 90min 120min
I 75.56 ± 0.87 87.85 ± 0.59 112.05 ± 1.07 105.03 ± 0.37 97.16 ±0.71
II 76.1 ± 0.47 111.8 ± 0.76 117.16 ± 0.78 85.33 ± 0.77 72.73 ± 1.49
III 76.08 ± 0.41 95.16 ± 1.05 104 ± 0.93 91.66 ± 0.64 80.8 ± 0.36
The values are expressed as mean ± SEM. Each groups having six animals.
Statistical significant test for comparison was done by ANOVA, followed by
Dunnet’s “t” test. The blood glucose values of group II and III are compared
with control animal values.
Figure.12
Effect of Rubus Racemosus of blood glucose level in normoglycaemic rats
0
20
40
60
80
100
120
140
Int ial 30min 60min 90min 120min
mg/
dl I
II
III
Effect of Rubus Racemosus on blood glucose fed hyperglycaemic rats
Table: 15
Groups Initial 30min 60min 90min 120min I 76.28 ± 0.7 74.8 ± 0.64 70.71 ± 1.00 69.66 ± 0.79 72.08 ± 0.69 II 79.71 ± 1.24a*** 126.5 ± 0.84a*** 129 ± 0.52a*** 113.46 ± 1.27a*** 80.33 ± 0.52a*** III 77.81 ± 0.79b*** 130 ± 0.93b*** 117.5 ± 1.05b*** 95.66 ± 0.91b*** 82.33 ± 0.60b*** IV 80 ± 0.78b*** 138.1 ± 0.52b*** 114.98 ± 0.65b*** 98.7 ± 0.64b*** 88.56 ± 0.8b***
The values are expressed as mean + SEM. Each group having six animals.
Statistical significant test for comparison done by ANOVA, followed by
Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III and IV are compared with group II values.
The 60th, 90th and 120th min values are compared with initial values: non
significant.
***p<0.001
Figure.13
Effect of Rubus Racemosus on blood glucose fed hyperglycaemic rats
0
20
40
60
80
100
120
140
160
Intial 30min 60min 90min 120min
mg/
dl
I
II
III
IV
Effect of acute treatment of Rubus Racemosus on
blood glucose in STZ induced diabetic rats
Table: 16
Groups Initial 1hr 3hr 5hr
I 86.28 ± 0.4 95.8 ± 0.53 95 ± 0.66 86.05 ± 0.57
II 274.33 ± 2.26ns 286 ± 1.07ns 297.83 ± 0.9ns 294.1 ± 0.61ns
III 270.16 ± 1.66ns 279 ± 2.18 ns 273.7 ± 1.94 ns 256.8 ±0.52ns
IV 245.83 ± 0.52ns 277.67 ± 2.07ns 250.5 ± 1.50ns 236.7 ± 0.52ns
V 234.33 ± 0.74ns 247.33 ± 1.38ns 256 ± 1.30ns 230 ± 3.34ns
The values are expressed as mean + SEM. Each groups having six animals.
Statistical significant test for comparison done by ANOVA, followed by
Dunnet’s “t” test. The values 1st, 3rd and 5rd hour are compared with initial
values. ns – non significant
Figure.14
Effect of acute treatment of Rubus Racemosus on blood glucose in STZ induced diabetic rats
0
50
100
150
200
250
300
350
Intial 1hr 3hr 5hr
mg/
dl
I
II
III
IV
V
Effect of sub-acute treatment of Rubus Racemosus on Blood glucose in STZ induced diabetic rats
Table: 17
Groups Initial 1 day 7 day 14 day 21 day 28 day
I 67.66 ± 0.53 75.33 ± 0.84 67.9 ± 0.54 71.5 ± 0.52 67.8 ± 0.37 69.33 ± 1.36
II 257.5 ± 0.96 277.66 ± 1.05 a*** 297.73 ± 0.63 a*** 334.5 ± 4.06a*** 356.33 ± 1.84 a*** 365.16 ± 1.20a***
III 255.16 ± 0.9 276.66 ± 0.39b** 278.83 ± 1.35b** 242.5 ± 3.28b** 218.16 ± 1.94b*** 191.66 ± 2.64b***
IV 248.66 ± 1.38 267.66 ± 0.90b** 271.26 ± 1.50b** 255.3 ± 1.12b** 228.5 ± 1.86b*** 163.16 ± 8.31b***
V 248.66 ± 1.08 264.83 ± 1.37 b** 268.66 ± 3.56 b** 191.66 ± 1.12b*** 168.33 ± 2.02b*** 135.3 ± 1.2b***
The values are expressed as mean ± SEM. Each groups having six animals.
Statistical significant test for comparison was done by ANOVA, followed by
Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001;** p<0.01
Figure.15
Effect of Sub-acute treatment on Rubus Racemosus on blood glucose in STZ induced diabetic rats
0
50
100
150
200
250
300
350
400
1 2 3 4 5 6
mg/
dl
Series1Series2Series3Series4Series5
BIOCHEMICAL PARAMETERS
Effect of Methanolic extract of Rubus Racemosus on Serum Total Bilirubin in STZ induced diabetic rats
Table: 18
Group Treatment Total Bilirubin (mg/dl)
I (n=6) Control 0.17 ± 0.006
II (n=6) Diabetic Control 0.38 ± 0.007a***
III (n=6) Gilbenclamide 0.25 ± 0.01b***
IV (n=6) MERR 200mg 0.19 ± 0.006b***
V (n=6) MERR 400mg 0.23 ± 0.004b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. ***p<0.001
Figure.16
Effect of Rubus Racemosus on serum Total bilirubin of STZ induced diabetic rats
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
I II III IV V
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on SGOT in STZ induced diabetic rats
Table: 19
Group Treatment SGOT (IU/dl)
I (n=6) Control 36.9 ± 0.46
II (n=6) Diabetic Control 92.57±0.22a**
III (n=6) Gilbenclamide 63.46±0.23b**
IV (n=6) MERR 200mg 42.01±0.75b**
V (n=6) MERR 400mg 45.62±0.06b*** The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. ***p<0.001; ** p<0.01
Figure.17
Effect of Rubus Racemosus on serum SGOT of STZ induced diabetic rats
0
20
40
60
80
100
I II III IV V
Groups
IU/d
l
Effect of Methanolic extract of Rubus Racemosus on SGPT in STZ induced diabetic rats
Table: 20
Group Treatment SGPT (IU/dl)
I (n=6) Control 42.8 ± 0.55
II (n=6) Diabetic Control 95.36±1.1a**
III (n=6) Gilbenclamide 64.18±0.51b**
IV (n=6) MERR 200mg 55.58±0.24b**
V (n=6) MERR 400mg 58.35±0.22b**
The values are expressed as mean ± SEM. Each groups having six animals. n= number of animals in each group. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s “t” test. (a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. **p<0.01
Figure.18
Effect of Rubus Racemosus on serum SGPT of STZ induced diabetic rats
0
20
40
60
80
100
120
I II III IV V
Groups
IU/d
l
Effect of Methanolic extract of Rubus Racemosus on Serum Total Protein in STZ induced diabetic rats
Table: 21
Group Treatment Total protein (mg/dl)
I (n=6) Control 7.3 ± 0.11
II (n=6) Diabetic Control 3.55 ± 0.14a***
III (n=6) Gilbenclamide 5.75 ± 0.13b***
IV (n=6) MERR 200mg 6.65 ± 0.07b***
V (n=6) MERR 400mg 6.16 ±0.03b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values (b) Group III, IVand V are compared with group II values. ***p<0.001
Figure.19
Effect of Rubus Racemosus on serum Total Protein of STZ induced diabetic rats
0
1
2
3
4
5
6
7
8
I II III IV V
Groups
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on Serum Alkaline Phosphatase in STZ induced diabetic rats
Table: 22
Group Treatment Alkaline
Phosphatase (mg/dl)
I (n=6) Control 68.1 ± 0.31
II (n=6) Diabetic Control 155.9 ± 0.58a***
III (n=6) Gilbenclamide 118.2 ± 0.22b***
IV (n=6) MERR 200mg 96.1 ± 0.71b***
V (n=6) MERR 400mg 104.8 ± 0.34b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values (b) Group III, IVand V are compared with group II values. ***p<0.001
Figure.20
Effect of Rubus Racemosus on serum Alkaline phosphatase of STZ induced diabetic rats
0
20
40
60
80
100
120
140
160
180
I II III IV V
Groups
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on Serum Total Cholesterol in STZ induced diabetic rats
Table: 23
Group Treatment Total Cholesterol
(mg/dl)
I (n=6) Control 64.46 ± 0.15
II (n=6) Diabetic Control 125.5 ± 0.76a***
III (n=6) Gilbenclamide 86.88 ± 0.23b***
IV (n=6) MERR 200mg 74.75 ± 0.28b***
V (n=6) MERR 400mg 68.5 ± 0.32b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IVand V are compared with group II values.
***p<0.001
Figure.21
Effect of Rubus Racemosus on serum Total Cholesterol of STZ induced diabetic rats
0
20
40
60
80
100
120
140
I II III IV V
Groups
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on Serum HDL-Cholesterol in STZ induced diabetic rats
Table: 24
Group Treatment HDL- Cholesterol
( mg/dl)
I (n=6) Control 48.13 ± 0.26
II (n=6) Diabetic Control 18.85 ± 0.18a***
III (n=6) Gilbenclamide 40.37 ± 0.38b***
IV (n=6) MERR 200mg 45.2 ± 0.3b***
V (n=6) MERR 400mg 46.13 ± 0.13b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group.Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001
Figure.22
Effect of Rubus Racemosus on serum HDL-Cholesterol of STZ induced diabetic rats
0
10
20
30
40
50
60
I II III IV V
Groups
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on Serum Triglyceride in STZ induced diabetic rats
Table: 25
Group Treatment Triglyceride (mg/dl)
I (n=6) Control 43.93 ± 0.26 II (n=6) Diabetic Control 64.9 ± 0.13a*** III (n=6) Gilbenclamide 56.9 ± 0.18b*** IV (n=6) MERR 200mg 58.1 ± 0.12b***
V (n=6) MERR 400mg 55.6 ± 0.11b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001
Figure.23
Effect of Rubus Racemosus on serum Triglyceride of STZ induced diabetic rats
0
10
20
30
40
50
60
70
I II III IV V
Groups
mg/
dl
Effect of Methanolic extract of Rubus Racemosus on Serum LDL-Cholesterol in STZ induced diabetic rats
Table: 26
Group Treatment LDL- Cholesterol ( mg/dl)
I (n=6) Control 18.6 ± 0.40 II (n=6) Diabetic Control 40.13 ± 0.78a*** III (n=6) Gilbenclamide 16.35 ± 0.19b*** IV (n=6) MERR 200 mg 14.55 ± 0.07b***
V (n=6) MERR 400 mg 13.92 ± 0.09b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001
Figure.24
Effect of Rubus Racemosus on serum LDL-Cholesterol of STZ induced diabetic rats
0
5
10
15
20
25
30
35
40
45
I II III IV V
Groups
mg/
dl
INVIVO – ANTIOXIDANT STUDIES
Effect of Methanolic Extract on Superoxide dimutase in STZ induced diabetic rats
Table: 27
Groups Treatment SOD (mg/dl)
I (n=6) Control 7.48 ± 0.11 II (n=6) Diabetic Control 4.47 ± 0.17a** III (n=6) Gilbenclamide 5.98 ± 0.096b** IV (n=6) MERR 200 mg 6.8 ± 0.06 b**
V (n=6) MERR 400 mg 7.58 ± 0.16b**
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. **p<0.01 SOD Units – Units/mg protein 1Units = The amount of enzyme required bring about 50% of inhibition of auto oxidation of pyrogallol
Figure.25
Effect of Rubus Racemosus extract on SOD levels in STZ induced diabetic rats
0
1
2
3
4
5
6
7
8
9
I II III IV V
Groups
U/m
g of
pro
tein
Effect of Methanolic Extract on Catalase in STZ induced diabetic rats
Table: 28
Groups Treatment Catalase (mg/dl)
I (n=6) Control 85.31 ± 0.34
II (n=6) Diabetic Control 76.4 ± 0.39 a***
III (n=6) Gilbenclamide 78.5 ± 0.56b**
IV (n=6) MERR 200 mg 80.8 ± 0.28b***
V (n=6) MERR 400 mg 82.8 ± 0.26b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001;**p<0.01
Figure.26
Effect of Rubus Racemosus extract on Catalase in STZ induced diabetic rats
70
72
74
76
78
80
82
84
86
88
I II III IV V
Groups
n m
oles
of h
ydro
gen
pero
xide
de
com
pose
d/m
in/m
g pr
otei
n
Effect of Methanolic Extract on Glutathione Peroxidase in STZ induced diabetic rats
Table: 29
Group Treatment Glutathione Peroxidase (mg/dl)
I (n=6) Control 9.18 ± 0.17 II (n=6) Diabetic Control 4.7 ± 0.18a*** III (n=6) Gilbenclamide 5.7 ± 0.27b*** IV (n=6) MERR 200 mg 7.4 ± 0.14b***
V (n=6) MERR 400 mg 8.6 ± 0.13b***
The values are expressed as mean ± SEM. Each groups having six animals. n= number of animals in each group. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001
Units – n moles of hydrogen peroxide decomposed/min/mg protein.
Figure.27
Effect of Rubus Racemosus extract on Glutathione Peroxidase in STZ induced diabetic rats
0
1
2
3
4
5
6
7
8
9
10
I II III IV V
Groups
mol
es/m
in/m
g of
pro
tein
Effect of Methanolic Extract on Glutathione Reductase in STZ induced diabetic rats
Table: 30
Group Treatment Glutathione Reductase (mg/dl)
I (n=6) Control 1.29 ± 0.05 II (n=6) Diabetic Control 0.68 ± 0.01a*** III (n=6) Gilbenclamide 0.81 ± 0.01b* IV (n=6) MERR 200 mg 0.87 ± 0.02b** V (n=6) MERR 400 mg 1.04 ± 0.06b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV, V are compared with group II values.
***p<0.001; **p<0.01; *p<0.05
Units – n moles of GSSG utilized/min/mg protein.
Figure.28
Effect of Rubus Racemosus extract on Glutathione Reductase in STZ induced diabetic rats
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
I II III IV V
Groups
n m
oles
of G
SSG
util
ized
m
in/m
g pr
otei
n
Effect of Methanolic Extract on Lipid Peroxidation in STZ induced diabetic rats
Table: 31
Groups Treatment Lipid Peroxidation
(mg/dl) I (n=6) Control 0.58 ± 0.01 II (n=6) Diabetic Control 0.92 ± 0.02a*** III (n=6) Gilbenclamide 0.83 ± 0.01b*** IV (n=6) MERR 200 mg 0.75 ± 0.01b*** V (n=6) MERR 400 mg 0.66 ± 0.02b***
The values are expressed as mean ± SEM. Each groups having six animals.
n= number of animals in each group. Statistical significant test for comparison
was done by ANOVA, followed by Dunnet’s “t” test.
(a) Group II is compared with group I values
(b) Group III, IV and V are compared with group II values.
***p<0.001
Figure.29
Effect of Rubus Racemosus extract on Lipid Peroxidation in STZ induced diabetic rats
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
I II III IV V
Groups
n m
oles
of M
DA
libe
rate
/min
/mg
prot
ein
Histopathological studies of the Liver
Table: 32
Sl.No.
Group
Observation
1.
Group I
H & E section shows normal liver with central vein surrounded by hepatocytes
2.
Group II
Liver shows vacuolated hepatocytes
3.
Group III
Liver shows almost normal hepatocytes (minimum vacuolation)
4.
Group IV
Liver shows almost normal liver parenchyma with minimal vacuolation
Figure.30
Histopathology of the liver section
Histopathological Studies of the Pancreas
Table: 33
Sl.No.
Group
Observation
1.
Group I
The pancreas section showed normal acini with islet of β-cells
2.
Group II
Pancreas shows atrophic acini and reduction islet β-cell size.
3.
Group III
Pancreas shows markedly proliferative (hyperplastic) islets β-cells
4.
Group IV
Pancreas shows preserved islet cells.
Figure.31
Histopathology of the pancreatic section
Free radical scavenging activity of MERR by DPPH reduction
Table: 34
SL. NO.
Concentration in MERR (µg/ml)
% Inhibition
IC50 value
(µg/ml) 1. 25 10.35±0.19
2. 50 29.42±2.01 3. 100 39.14±0.22 4. 200 54.54±0.20
5. 400 60.65±0.29 6. 800 71.13±1.19 7. 1000 86.81±0.19
348.87
Values are mean ± SEM of 6 parallel measurements.
MERR: Methanolic extract of Rubus Racemosus
Figure.32
0
20
40
60
80
100
25 50 100 200 400 800 1000
Concentration in mcg/ml
% in
hibi
tion
Nitric oxide scavenging activity of MERR
Table: 35
SL. NO. Concentration
of MERR (µg/ml)
% Inhibition IC50 value (µg/ml)
1. 25 22.01 ± 0.28
2. 50 32.28 ± 0.51
3. 100 53.87 ± 0.59
4. 200 53.78 ± 0.71
5. 400 77.03 ± 0.39
6. 800 82.22 ± 0.37
7. 1000 84.26 ± 0.40
219.53
Values are mean ± SEM of 6parallel measurements.
MERR: Methanolic extract of Rubus Racemosus
Figure.33
0
20
40
60
80
100
25 50 100 200 400 800 1000
Concentration in mcg/ml
% in
hibi
tion
Hydroxyl radical scavenging activity of MERR
Table: 36
SL. NO. Concentration
of MERR (µg/ml)% Inhibition IC50 value
(µg/ml)
1. 25 52.53±0.41
2. 50 62.82±0.15
3. 100 67.92±0.26
4. 200 74.65±0.31
5. 400 82.57±0.22
6. 800 84.15±0.20
7. 1000 83.42±0.32
520.32
Values are mean ± SEM of parallel measurements.
MERR: Methanolic extract of Rubus Racemosus
Figure.34
0
20
40
60
80
100
25 50 100 200 400 800 1000
Concentration in mcg/ml
% in
hibi
tion
Effect of MERR and BHT on reducing power
Table: 37
SL.NO. Concentration
of MERR (µg/ml)
Absorbance (OD) of MERR
Absorbance (OD) of BHT
1. Control 0.094
2. 25 0.086 ± 0.002 0.268±0.004
3. 50 0.108 ± 0.003 0.287±0.004
4. 100 0.194 ± 0.005 0.316±0.002
5. 200 0.213 ± 0.002 0.340±0.002
6. 400 0.967 ± 0.003 0.361±0.002
7. 800 1.016 ± 0.002 0.382±0.003
8. 1000 1.130±0.002 0.389±0.098
Values are mean ± SEM of 6 parallel measurements.
MERR: Methanolic extract of Rubus Racemosus
Figure.35
0
0.2
0.4
0.6
0.8
1
1.2
25 50 100 200 400 800 1000
Concentration in mcg/ml
Abs
orba
nce
MERRBHT
Effect of MERR on MES induced epilepsy Table: 38
Groups %
Protection Flexion Extensor Clonus Stupor Recovery
I. Control (CMC)
0 6.87±0.41 13.89±0.81 9.89±0.52 16±0.32 190.82
II. PHT 100 5.27±0.38 0 9±0.32** 12.17±0.58*** 109.34
III. MERR
200 51.69 6.18±0.35ns 6.28±0.61*** 10±0.43ns 15.98±0.47ns 147.35
IV.MERR 400
61 5.87±0.37*** 5.07±0.37*** 8.78±0.55ns 14.68±0.57ns 122.81
Values are expressed as mean± SEM of six observations. Comparison between Group I Vs Group II, Group II Vs Group II & Group IV. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test *p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Values expressed in seconds.
Figure.36
0
2.5
5
7.5
10
12.5
15
17.5
20
Flexion Extensor Clonus Stupor
Tim
e in
sec
onds Control (CMC)
PHTMERR 200MERR 400
Effect of MERR on Serotonin levels in MES induced epilepsy
Table: 39
Groups Drug used Serotonin (ng/mg)
I Control 181.33 ± 1.84
II MES 77.83 ± 1.34a***
III PHT 98.16 ± 0.9b***
IV MERR 200 84.5 ± 1.73b***
V MERR 400 88.83 ± 1.02b***
Values are expressed as mean ± SEM of six observations. Comparison between
Group I Vs Group II, Group III Vs Group IV & Group V. Statistical significant
test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test
*p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet
tissue
Figure.37
Effect of MERR on Serotonin levels in MES induced epilepsy
0
20
40
60
80
100
120
140
160
180
200
I II III IV V
ng/m
g w
et ti
ssue I
IIIIIIVV
Effect of MERR on Non adrenaline levels in MES induced epilepsy
Table: 40
Groups Drug used Nor adrenaline (ng/mg)
I Control 718.3 ± 2.50
II MES 440.16 ± 1.02a***
III PHT 597.66 ± 1.90b***
IV MERR 200 490.83 ± 3.17b***
V MERR 400 520.3 ± 2.52b***
Values are expressed as mean± SEM of six observations. Comparison between
Group I Vs Group II, Group III Vs Group IV & Group V. Statistical significant
test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test.
*p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet
tissue
Figure.38
Effect of MERR on Non adrenaline levels in MES induced epilepsy
0
100
200
300
400
500
600
700
800
I II III IV V
ng/mg wet tissue IIIIIIIVV
Effect of MERR on Dopamine levels in MES induced epilepsy
Table: 41
Groups Drug used Dopamine
(ng/mg)
I Control 813.3 ± 0.84
II MES 478.16 ± 1.29a***
III PHT 810 ± 1.12b***
IV MERR 200 626.66 ± 3.45b***
V MERR 400 710.3 ± 2.71b***
Values are expressed as mean± SEM of six observations. Comparison between
Group I Vs Group II, Group III Vs Group IV & Group V. Statistical
significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’
test. *p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet
tissue.
Figure.39
Effect of MERR on Dopamine levels in MES induced epilepsy
0
100
200
300
400
500
600
700
800
900
I II III IV V
ng/m
g w
et ti
ssue I
IIIIIIVV
RESULTS OF ANTIMICROBIAL STUDIES
In Vitro evaluation of Anti-microbial activity of different
extracts of Rubus Racemosus on Staphylococcus aureus
Table: 42
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 30 0 16 19 25 15
Chloroform 30 0 17 24 27 14
Petroleum ether 30 0 18 20 24 13
Aqueous
Staphylococcus
aureus
29 0 17 19 22 14
Figure.40
[S.aureus - Staphylococcus aureus; STD – Standard; C – Control; M.ex –
Methanol Extract; P.E – Petroleum ether Extract; Chlor. – Chloroform Extract;
Aque. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5mcg/disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Staphylococcus epidermidis
Table: 43
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 31 0 18 21 24 17
Chloroform 30 0 17 19 21 16
Petroleum ether 30 0 16 18 20 15
Aqueous
Staphylococcus epidermidis
30 0 15 17 20 14
Figure.41
[S.epi - Staphylococcus epidermidis; STD – Standard; CON – Control; Me.ext
– Methanol Extract; Pe.ex. – Petroleum ether Extract; Ch. ex. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5 mcg/disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Bacillus cereus
Table: 44
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 31 0 16 19 26 15
Chloroform 30 0 17 20 27 14
Petroleum ether 30 0 16 19 21 15
Aqueous
Bacillus cereus
30 0 14 18 22 13
Figure.42
[B.Cereus – Bacillus cereus; STD – Standard; CON – Control; Met.ext –
Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ext. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5 mcg /disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Micrococcus luteus
Table: 45
Zone of Inhibition (mm) Extract
Micro-
Organism Std. Control 25
(µl) 50
(µl) 100 (µl)
MIC Values
(µl)
Methanol 30 0 17 22 25 16
Chloroform 31 0 15 21 24 14
Petroleum ether 31 0 16 20 23 15
Aqueous
Micrococcus luteus
30 0 17 21 24 16
Figure.43
[M.luteus – Micrococcus luteus; STD – Standard; C – Control; Me.ex. –
Methanol Extract; P.E. – Petroleum ether Extract; Ch. ex. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5mcg/disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Klebsiella pneumoniae
Table: 46
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 30 0 17 19 22 14
Chloroform 29 0 16 20 23 15
Petroleum ether 28 0 14 17 22 13
Aqueous
Klebsiella
pneumoniae
28 0 14 16 21 12
Figure.44
[Kl.Pne – Klebsiella pneumonia; STD – Standard; C – Control; Me.ex. –
Methanol Extract; Pe.ex. – Petroleum ether Extract; Ch. ex. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5 mcg/disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Pseudomonos aeruginosa
Table: 47
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 31 0 18 20 22 16
Chloroform 30 0 18 20 23 15
Petroleum ether 32 0 15 18 21 13
Aqueous
Pseudomonos
aeruginosa
32 0 14 19 23 12
Figure.45
[Ps.aeru – Pseudomonos aeruginosa; STD – Standard; CON – Control; Me.ex.
– Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5 mcg /disc
In vitro evaluation of Anti-microbial activity of different
extracts of Rubus racemosus on Escherichia coli
Table: 48
Zone of Inhibition (mm) Extract Micro-
Organism Std. Control 25 (µl) 50 (µl) 100
(µl)
MIC Values
(µl)
Methanol 30 0 15 19 23 14
Chloroform 32 0 18 22 26 17
Petroleum ether 31 0 16 21 25 15
Aqueous
Escherichia coli
30 0 15 19 24 14
Figure.46
[E.coli – Escherichia coli; STD – Standard; CON – Control; Me.ex. – Methanol
Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. – Chloroform Extract; AQ.
ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ciprofloxacin → 5 mcg/disc
In Vitro evaluation of Anti-fungal activity of different
extracts of Rubus Racemosus on Aspergillus niger
Table: 49
Zone of Inhibition (mm) Extract Micro-
Organism Std Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 31 0 15 19 22 14
Chloroform 30 0 14 18 23 13
Petroleum ether 30 0 15 19 22 14
Aqueous
Aspergillus
niger
30 0 13 17 21 12
Figure.47
[A.niger – Aspergillus Niger ; STD – Standard; C – Control; Me.ex. –
Methanol Extract; P.E.. – Petroleum ether Extract; Ch. ex. – Chloroform
Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ketoconazole → 50 mcg /disc
In Vitro evaluation of Anti-fungal activity of different
extracts of Rubus Racemosus on Aspergillus fumigatus
Table: 50
Zone of Inhibition (mm) Extract Micro-
Organism Std Control 25 (µl)
50 (µl)
100 (µl)
MIC Values
(µl)
Methanol 31 0 17 20 23 16
Chloroform 30 0 16 21 24 15
Petroleum ether 31 0 17 22 25 14
Aqueous
Aspergillus fumigatus
32 0 15 18 21 13
Figure.48
[A.fumigatus – Aspergillus fumigatus; STD – Standard; CON – Control;
Me.ex. – Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. –
Chloroform Extract; AQ. ex. – Aqueous Extract]
25, 50, 100 – Concentration of the extracts in mcg/ml.
STD Used – Ketoconazole → 50 mcg /disc
DISCUSSION
CHAPTER - V
DISCUSSION
The aerial parts of Rubus racemosus were identified, collected,
authenticated and were subjected to organoleptic, microscopical and physical
studies.
Plant anatomical studies may have a crucial role in plant identification
and standardization.
In the midrib region, inner epidermis are collenchymatous and the rest
of the ground tissue parenchyma. The vascular bundle consists of radial rows
of xylem and phloem elements.
In the lamina region adaxial epidermis is thick with large squarish cells
and thick cuticle whereas abaxial epidermis shows narrow rectangular cells.
The mesophyll tissue consists of two layers of short, thin palisade cells and 6-8
layers of small, lobed spongy parenchyma cells.
The leaf has dense glandular and non-glandular trichomes. The non
glandular trichomes are unicellular, unbranched, thick walled and pointed at the
tip. They arise from a pedestal of a group of cells raised above the level of the
epidermis.
The upper part (distal) of the petiole has three accessory lateral bundles,
whereas lower part (proximal) has 2 prominent lateral bundles. All the bundles
are collateral with thick band of sclerenchyma cells abutting the phloem tissue.
Next to the epidermis, 2 to 3 layers of collenchyma cells are seen.
The Stem has a thin layer of epidermis comprising of small thick walled
cells. The vascular cylinder is thin and continuous consisting of several wedge
shaped vascular bundles. The vascular bundles are collateral. The
sclerenchyma cells are thin walled and wide lumened. Xylem elements are
circular thick walled. The pith is parenchymatous.
The powdered sample of the leaf shows non glandular trichomes, they
are long whip like thick walled with smooth surface. The glandular trichomes
are long stalked with spherical head.
Powder microscopical character of the plant Rubus racemosus showed
unicellular, non-glandular trichomes, anisocytic type of stomata and prismatic
type of calcium oxalate crystals.
Powdered material was extracted successively with petroleum ether
ethyl acetate, chloroform, methanol and water. The extracts were subjected to
preliminary phytochemical screening to find the chemical constituents present.
It was found that flavonoids were present in petroleum ether, ethyl acetate,
chloroform, methanol and aqueous extracts of hot percolation type which was
confirmed by treatment of extracts with different respective reagents.
Alkaloids, Sterols and Proteins were absent in Petroleum ether, ethylacetate,
chloroform, methanol and aqueous extracts of hot percolation. Phenols,
tannins, carbohydrates, glycosides, Saponins and terpenes were observed in
pertoleum ether, ethyl acetate, chloroform, methanol and aqueous extracts of
hot percolation and confirmed by their respective reagents. Gums and mucilage
is absent in all the extracts.
All the extracts were subjected to thin layer chromatography and their Rf
values were determined.
The compounds isolated from methanolic extract of Rubus
racemosus was found to be Compound I - 1-[20, 21 – dodeacylnanone] – α-
1→6-D-glucotetraose-6’’’(P-hydroxy) benzoate. Compound II-9, 10
epoxynonacosane. The Rf values of the isolated compounds were found to be
0.62, 0.59.
The chloroform extract of Rubus racemosus subjected to HPTLC
studies. It showed 8 peaks with Rf values 0.07, 0.08, 0.27, 0.39, 0.54, 0.59, 0.67
and 0.81. HPTLC fingerprint of the isolated compound showed single peak
with Rf value 0.39.
Acute toxicity studies revealed that MERR is relatively nontoxic up to
2000 mg/kg b.w. p.o. indirectly pronouncing the safety profile of the extract.
The subacute toxicity also supports that the MERR is nontoxic, predicted by
there is no remarkable changes in the both haematological and biochemical
parameters in the blood and histopathological parameters of tissues.
The MERR of a dose of (200 mg/kg/b.w/p.o) did not significantly
suppress blood glucose levels in over night fast normal animals. The same
effect was observed at a higher dose level of 400 mg/kg/b.w/p.o of the MERR
in over night fasted normal animals after 60, 90, 120 min of oral
administration, when compared with control group of animals.
MERR showed significant improvement in glucose tolerance in glucose
fed hyperglycemic rats. Such an effect may be accounted for, in part, by a
decrease in the rates of intestinal glucose absorption, achieved by an extra
pancreatic action including the stimulation of peripheral glucose utilization or
enhancing glycolytic and glycogenic process with concomitant decrease in
glycogenolysis and gluconeogenesis105. However the effect was less significant
when compared standard drug glibenclimide.
Sterptozotocin is the most commonly employed agent for the induction
of experimental diabetic animal models of human insulin dependent diabetes
mellitus106. There is increasing evidence that streptozotocin causes diabetes by
rapid depletion of β cells, by DNA alkylation and accumulation of cytotoxic
free radicals that is suggested to result from initial islet inflammation, followed
by infiltration of activated macrophages and lymphocyte in the inflammatory
focus. It leads to a reduction in insulin release thereby a drastic reduction in
plasma insulin concentration leading to stable hyperglycemic states. In this
study significant hyperglycemia was achieved within 48 hours after
streptozotocin (50 mg/kg/b.w/i.p) injection. Streptozotocin induced diabetic
rats with more 200 mg/dl of blood glucose were considered to the diabetic and
used for the study.
A double dose of MERR did not bring about any hypoglycemic action.
In the sub-acute study, glibenclamide treatment brought down the blood sugar
levels from the 14th day of treatment MERR (high dose) treatment produced
and a steady decrease in blood glucose levels from 21st day of treatment and a
steady decrease was observed there after. At the end of the study, a marked anti
hyperglycemic effect was observed in the plant extract treatment. The activity
may be due to the presence of phytochemicals like flavanoids, glycoside,
phenolics etc.
Sulphonylureas produces a hypoglycemic effect by stimulating
endogenous insulin secretion from β cells after binding to their receptors on the
plasma membrane and enhancing tissue sensitivity to insulin. The other
mechanism is extra pancreatic, action mainly upon liver, muscle and adipose
tissue.
Histopathological studies showed prominent islet cell hyperplasia and
regeneration of islet cells shows a proof for the possible antidiabetic property
of MERR.
Lipids play an important role in the pathogenesis of diabetes mellitus.
One level of serum lipids is usually raised in diabetic condition and such an
elevation posses to be a risk factor for cardiovascular diseases like coronary
heart disease and a two to four fold risk for the atherosclerosis which
constitutes the main cause of morbidity and mortality in diabetes mellitus. The
abnormal high concentration of serum lipids in diabetes is mainly due to
increased mobilization of free fatty acids from the peripheral depots, since
insulin inhibits the hormone sensitive lipase107. There hyperlipemia in diabetic
state may be regarded as a consequence of uninhibited actions of lipytic
hormones on fat depots108. In the present study, streptozotocin diabetic rats
clear cut abnormalities in the lipid profile were evidence by elevated serum
total cholesterol and reduced serum HDL cholesterol. The glibenclamide
treatment MERR in diabetic animals produced beneficial improvement in the
lipid profile by a reduction in the total cholesterol levels and increase in HDL-
Cholestrol level, which may not only be due to better glycemic control but
could also be due to the drug’s direct action on lipid metabolic pathways. Such
a biochemical state is desirable for preventing the progression of diabetes
related cardiovascular problems.
Free radicals can be defined as chemical species possessing an unpaired
electron, which is formed by homolytic cleavage of a covalent bond of a
molecule, by the loss of a single electron from a normal molecule or by
addition of a single electron to a normal molecule. Cells are equipped with both
enzymatic and non-enzymatic defence mechanisms to minimize cellular
damage resulting form interaction between cellular constituents and ROS. The
enzymatic antioxidant defence mechanism contains various forms of
superoxide dismutases, catalase, glutathione peroxides as well as enzymes
involved in the recycling of oxidized gluthione such as glutathione reductase
and glutathione-S-tranferases. Despite the presence of such delicate cellular
antioxidant systems, an over production of ROS in both intra and extracellular
spaces often occur upon exposure of cells to certain chemicals like
streptozotocin that yields to the pathogenesis of diabetes mellitus in
experimental animals. In the present study, we have examined the oxidative
stress pathway markers in streptozotocin diabetic rats. Superoxide dismutase
and catalase are the most important enzymes that scavenge toxic free radicals
and forms the superoxide anion, which initiates peroxidation of unsaturated
lipids.
In the biological system, it reduces the potential for hydroxyl radical
generation by catalyzing the reduction of superoxide radical to form hydrogen
peroxide, thereby diminishing the toxic effect of the free radicals. Catalase a
heme protein is the major determinant of hepatic antioxidant status. The
enzyme is localized in the cellular peroxisomes and micro-peroxisomes. It
catalyses the decomposition of hydrogen peroxide to water and oxygen, thus
protecting the cell from oxidative damage109. Superoxide dismutase and
catalase activity in the diabetic control animals was significantly low due to
increased oxidative stress when compared to the normal animals. However a
significant increase in the enzyme activity was observed in the glibenclamide
and MERR.
GSH is mainly involved in the synthesis of important macrolecules and
in protection against ROS. It is also essential for the maintenance of thiols of
proteins and components of antioxidants like ascorbates Tocopherol110. GSH-
Px is a selenium containing enzyme, that is active in the reduce form. This
enzyme catalyses the oxidation of GSH to GSSG at the expense of hydrogen
peroxide. A marked decrease in the hepatic GSH was observed in
streptozotocin diabetic rats. Such a decrease contributes to the pathogenesis of
complications associated with chronic diabetic state109. Glibenclamide, MERR
treated animals showed a marked increase in the hepatic glutathione peroxidase
antioxidant level significantly. This indicates that both the extracts have potent
properties to inhibit oxidative damage to tissues.
Impairment of enzymatic antioxidant system due to reduce levels of
insulin in diabetic state increases fatty acyl-coA oxidase and initates β
oxidation of fatty acids favours accumulation of free radials resulting in lipid
peroxidation. Increased lipid peroxidation impairs membrane function by
decreasing the membrane fluidity and changing the activity of membrane
bound enzymes and receptors111.
An elevated TBARS level in diabetic rats suggests the extent of
peroxidative injury, indicative of the development of diabetic complications. In
the present study, it was found that streptozotocin induction caused a
significant increase in TBARS in the hepatic tissue. But glibenclamide, MERR
exerted a protective effect against peroxide damage to the tissues.
Free radical scavenging activity of the extracts was measured in an
invitro chemical system by DPPH radical scavenging activity, Nitric oxide
scavenging activity, scavenging of Hydroxyl radical, Determination of
reducing power, Determination of total phenolic compounds methods while for
the antiperoxidative activity, a biological system comprising of hepatic tissue
homogenates were employed.
MERR is tested by their ability to bleach the stable radical DPPH. This
assay provided information on the reactivity of the compounds with a stable
free radical. Because of the odd electron, DPPH shows a strong absorption
band at 517nm in visible spectroscopy (deep violet color) as this electron
becomes paired off in the presence of a free radical scavenger, the absorption
vanishes and the resulting decolourisation is stoichiometric with respect to the
number of electrons taken up.
Nitric oxide is an important chemical mediator generated by the
endothelial cells, marophages, neurons, etc and involved in the regulation of
various physiological processes. Oxygen reacts with excess nitric oxide to
generate nirite and peroxynitrite anions, which act as free radicals. In this study
the extracts competes with oxygen to react with nitric oxide and thus inhibits
the generation of anions.
The hydroxyl radical scavenging activity in MERR is to inhibit hydroxyl
radical mediated deoxyribose degradation in a reaction mixture. The relative
extents of inhibition of deoxyribose degradation will give an induction of
scavenging and in the presence of EDTA, and mannitol, a classical hydroxyl
scavenger, significantly inhibited deoxyribose degradation in a concentrated
dependent manner112.
Incubation of sodium nitroprusside in phosphate buffer saline at 250C
for 2 hrs resulted in the time dependent nitric production which was reduced by
MERR.
The reducing capacity of a compound may serve as a significant
inductor of its potential antioxidant activity113. However the antioxidant activity
of putative antioxidants have been attributed to various mechanisms, among
which are prevention of chain initiation, binding of transition metal ion
catalysis, decomposition of peroxides, prevention of continuous hydrogen
abstraction and radical scavenging114&115. The reducing power of MERR
increased with increasing amount of the sample.
Phenols are very important plant constituents because of their
scavenging ability due to their hydroxyl groups116. The phenolic compounds
may contribute directly to antioxidative action. It is suggested that
polyphenolic compounds have inhibitory effects on necrosis in humans, this
made us to investigate the amount of total phenolic compound present in
MERR. In the MERR (1 mg), 129 ± 0.998 µg pyrocatechol equivalent of
phenols was detected.
In epilepsy, normal pattern of neuronal activity becomes disturbed
briefly when the nerves in the brain “Fire” spontaneously causing strange
sensations, emotions, behaviours and often times seizures with muscle spasms
as well as loss of consciousness117.
It has been reported to increase the brain levels of Dopamine and
Noradrenaline which causes an inhibition of seizure activity118.
MES induced epilepsy was altering the levels of monoamines like
noradrenaline, serotonin, dopamine119.
It is found that treatment with MERR on rats significantly reduces in
tonic hind limb extensor stage in MES induced epilepsy. Methanolic Extract of
Rubus Racemosus markedly protects epilepsy induced by MES which are
mediated by levels of monoamines.
Petroleum ether, Ethyl acetate, Chloroform, Methanol and Aqueous
extract of Rubus Racemosus were subjected to antibacterial and antifungal
against following organisms. Staphylococcus aureus, Staphylococcus
epidermides, Bacillus cereus, Micrococcus luteus, Kl.pneumoniae,
Pseudomonos aeruginosa, E.coli, Aspergillus fumigates, Aspergillus niger
respectively.
Petroleum ether extract exhibited significant activity against all the
tested organisms at the concentration of 100 µg/ml whereas it showed moderate
activity at the concentration of 25µg/ml and 50µg/ml when compared with
standard drug.
Methanolic extract exhibited significant activity against all the tested
organisms at the concentration of 100 µg/ml whereas it showed moderate
activity at the concentration of 25µg/ml and 50µg/ml when compared with
standard drug.
Chloroform extract exhibited significant activity against all the tested
organisms at the concentration of 100 µg/ml whereas it showed moderate
activity at the concentration of 25µg/ml and 50µg/ml when compared with
standard drug.
Aqueous extract exhibited significant activity against all the tested
organisms at the concentration of 100 µg/ml whereas it showed moderate
activity at the concentration of 25µg/ml and 50µg/ml when compared with
standard drug.
The Minimum Inhibitory concentration values of all the extracts
were determined.
From the above studies it reveals that all the extracts were active against
tested bacterial and fungal organisms.
SUMMARY AND
CONCLUSION
CHAPTER - VI
SUMMARY & CONCLUSION
The plant Rubus racemosus belongs to the family Rosaceae was
selected. A detailed pharmacognostical, toxicological and pharmacological was
studied on the plant. The aerial parts of the plant Rubus racemosus were
anatomically studied and reported first time. A detailed anatomical description
of the anatomical structures were studied. The midrib contains 2-3 layers of
epidermis, vascular bundle. Lamina contains mesophyll tissue consists of
palisade cells and spongy parenchyma. The vascular bundles are surrounded by
dilated hyaline bundle sheath cells. The type of trichomes present is glandular
and non-glandular trichomes. All the bundles in petiole are collateral with thick
band of sclerenchyma cells abutting the phloem tissues. Stem contains wedge
shaped vascular bundles. Sclerenchyma cells are thin walled and wide
lumened. The pith is parenchymatous.
The powder microscopy of the plant shows unicellular, non-glandular,
trichomes, anisocytic type of stomata, parenchyma cells and prismatic type of
calcium oxalate crystals. Length and Width of trichomes, leaf constants, physio
chemical constants, loss on drying and extractive values were determined.
Preliminary phytoconstituent screening of the plant Rubus Racemosus
showed the presence of various phytochemical constituents like flavanoids,
glycosides, phenols, carbohydrates, terpenes, tannins, saponins and the
compounds were isolated from the methanolic extract of Rubus racemosus. All
the extracts were subjected to thin layer chromatography, HPTLC and their Rf
values were determined. Both acute and Sub acute toxicity studies revealed that
MERR did not produce any mortality or sign of toxicity at the dose of
200mg/kg/b.w/p.o in the experimental rats. Treatment of MERR did not exhibit
any significant hypoglycemic effect on normal animals.
Significant improvement in glucose tolerance was observed with
methanolic extract treatment on glucose fed hyperglycemic rats. Experimental
diabetes in rats was induced by STZ (50 mg/kg/i.p) and animals with blood
glucose level more than 200 ml/dl were considered as diabetic and used for this
study. No significant reduction of blood glucose level was observed on the
acute treatment of MERR in STZ induced diabetic rats. In the sub acute study a
steady decrease in blood glucose level was observed on MERR treatment, in
STZ induced diabetic rats. The treatment of MERR showed marked increase in
the Total protein and HDL Cholesterol in serum of STZ induced diabetic
animals.
At the same time significant decrease in Total bilirubin, SGOT, SGPT,
ALP, Total Cholesterol, Triglycerides, LDL Cholesterol levels in serum of
diabetic animals were observed. The hepatic antioxidant enzyme levels
superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase
are significantly decreased in STZ induced diabetic animals with a high degree
of lipid peroxidation. The enzyme levels increased significantly on treatment
with MERR. Further, the antioxidant activity is confirmed by free radical
scavenging activity by DPPH, Nitric oxide, Reducing power, Hydroxyl radical
activity by MERR.
The antiepileptic activity of MERR was assessed by MES induced
convulsion.
In MES induced epilepsy, MERR at both 200 mg/kg and 400 mg/kg exhibits
significant antiepileptic activity particularly tonic hind limb extensor stage.
Neurochemical estimations of dopamine, nonadrenaline, and serotonin
in forbrain of MES induced rats reveals that MERR significantly increases
dopamine levels in forebrain. A significant increase in nonadrenaline levels
was observed in forebrain and also serotonin levels are shown to rise in
forebrain of MERR treated rats.
From the observations of the studies performed it could be predicted that
the MERR at both 200 mg/kg and 400 mg/kg exhibited significant anti-
epileptic activity.
Petroleum ether, Ethyl acetate, Chloroform, Methanol and Aqueous
extracts of Rubus racemosus have showed significant and moderate activity
against anti-bacterial and anti-fungal organisms.
Based on the results obtained and observation we can infer that the plant
under study, Rubus racemosus could be used for the supportive treatment of
diabetes mellitus, as the plant also offers effective protection against the attack
of free radicals that forms the basis for the development of diabetic
complications. And it also showed significant anti-epileptic, anti-bacterial and
anti-fungal activity.
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