PHYTOCHEMICAL SCREENING OF CERTAIN HERBAL ...VINAYAKA MISSIONS UNIVERSITY CERTIFICATE BY THE...
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PHYTOCHEMICAL SCREENING OF CERTAIN HERBAL DRUGS FOR ANTI-OXIDANT ACTIVITY Thesis submitted in Partial Fulfilment for the award of Degree of Doctor of Philosophy in Pharmaceutical Sciences By Mr. HAREESHBABU E. (Reg. No. M863600004) VINAYAKA MISSIONS UNIVERSITY SALEM, TAMILNADU, INDIA FEBRUARY 2015
Transcript of PHYTOCHEMICAL SCREENING OF CERTAIN HERBAL ...VINAYAKA MISSIONS UNIVERSITY CERTIFICATE BY THE...
PHYTOCHEMICAL SCREENING OF CERTAIN HERBAL DRUGS FOR ANTI-OXIDANT ACTIVITY
Thesis submitted in
Partial Fulfilment for the award of
Degree of Doctor of Philosophy
in Pharmaceutical Sciences
By
Mr. HAREESHBABU E.
(Reg. No. M863600004)
VINAYAKA MISSIONS UNIVERSITY
SALEM, TAMILNADU, INDIA
FEBRUARY 2015
VINAYAKA MISSIONS UNIVERSITY
CERTIFICATE BY THE GUIDE
I, Dr. (Sr.) MOLLY MATHEW certify that the thesis entitled
“Phytochemical Screening of Certain Herbal Drugs for Anti-oxidant Activity”
submitted for the Degree of Doctor of Philosophy by Mr. Hareeshbabu E., is
the record of research work carried out by him during the period from April
2008 to April 2013 under my guidance and supervision and that this work has
not formed the basis for the award of any degree, diploma, associate-ship,
fellowship or other titles in this University or any other University or Institution
of higher learning.
Place: Kasaragod
Date:
________________________ ___________ Dr. (Sr). Molly Mathew, B.Sc., M.Pharm, Ph.D. Principal, Malik Deenar College of Pharmacy, Seenthangoli, Bela Post, Kasaragod-671 321, Kerala, India
VINAYAKA MISSIONS UNIVERSITY
CERTIFICATE BY THE CO-GUIDE
I, Dr. V. Ganesan, certify that the thesis entitled “Phytochemical
Screening of Certain Herbal Drugs for Anti-oxidant Activity” submitted for the
Degree of Doctor of Philosophy by Mr. Hareeshbabu E., is the record of
research work carried out by him during the period from April 2008 to April
2013 under my co-guidance and supervision and that this work has not
formed the basis for the award of any degree, diploma, associate-ship,
fellowship or other titles in this University or any other University or Institution
of higher learning.
Place: Erode
Date:
___________________________________ Dr. V. Ganesan, M.Pharm, Ph.D. Principal, Erode College of Pharmacy, Perunduri Main Road, Erode-638 112, Tamil Nadu, India
VINAYAKA MISSIONS UNIVERSITY
DECLARATION
I, Mr. Hareeshbabu E declare that the thesis entitled “Phytochemical
Screening of Certain Herbal Drugs for Anti-oxidant Activity” submitted by me
for the Degree of Doctor of Philosophy is the record of work carried out by me
during the period from April 2008 to April 2013 under the guidance of
Dr. (Sr.) Molly Mathew and co-guidance of Dr. V. Ganesan and has not
formed the basis for the award of any degree, diploma, associate-ship,
fellowship, titles in this or any other University or other similar institutions of
higher learning.
Place: Chalakudy
Date:
___________________________________ Mr. Hareeshbabu E. Assistant Professor, St. James College of Pharmaceutical Sciences, St. James Medical Academy, Chalakudy, Thrissur (Dist.) - 680 307, Kerala, India
ACKNOWLEDGEMENTS
The completion of thesis is the cumulative efforts of a number of people
who have contributed directly and indirectly to my research work. I take the
opportunity to convey my regards to all those persons. First of all, with never
ending humbleness, I would like to thank the Almighty who bestowed me with
health and enough courage to complete this project.
It is my honour to be a part time Ph.D. student of Vinayaka Missions
University, Salem, India. Further, I express my deep sense of gratitude
originating from the innermost core of heart for my Mentor and Guide, Dr. (Sr)
Molly Mathew, Principal, Malik Deenar College of Pharmacy, Kasaragod,
Kerala, India for her guidance, valuable suggestions and moral support. Her
thorough guidance, meticulous methodology, critical assessment, constructive
criticism, and constant encouragement throughout the research period made it
possible to bring my work in its present shape. Working with her taught me
valuable things, about my subject and life, the thing which help me the way
through.
I express my sincere thanks to Prof. Dr. K. Rajendran, Dean
(Research), and members of research committee, for improving and
approving my thesis and recommending me to extend the fellowship.
I express my sincere thanks to my Co-guide Dr. V. Ganesan, Principal,
Erode College of Pharmacy, Erode, for his valuable suggestion and support to
accomplish this research work.
I extend my warm regards to the Department of Research, Malik
Deenar College of Pharmacy, Kasaragod for creating the huge
infrastructure of facilities and making them available to carry out the research
work.
I am very thankful to the members of Pharmacology department,
KMCH, College of Pharmacy, Tamil Nadu for giving needful help to conduct
my animal studies.
I extend my sincere gratitude to the Management and Faculty of St.
James College of Pharmaceutical Sciences, Chalakudy for their valuable
support.
My father, mother, wife (Dr. Dia S.), son (Dev Rish E.) and all other
family members deserve special thanks for their immense love, inseparable
support and prayers that result into the successful completion my research
work.
I extend my acknowledgements to my friends for their love, support and
ever ready help. My sincere thanks to Dr. Aneesh S, Mr. Jithin T V, Mrs.
Sreeja E, Dr. Ethiraj, Mrs. Geetha Elias, Dr. Della Grace Thomas Parambi,
Dr. Unnikrishnan B, Mr. Ranjith P B, Mr. Srikanth S K, Mr. Arun Menon,
Mrs. Smitha K Nair, Mrs. Jaismy Jacob P, Mr. Baldwin Mathew, Dr.
Sreena Sujith, Mrs. Asa Samuel, Mrs. Ann Shine, Mr. Dipu, Mr. Preejith O
K, Mr. Prasanth (4 Line) and Mr. Sarath (4 Line) for their immense help for
the fulfilment of this thesis.
I would like to thank all, who contributed to the realization of my thesis
work and I also express my apology that I could not mention their names
personally one by one.
Hareeshbabu E
INDEX
Chapter No. TOPIC Page
No.
1 INTRODUCTION 2-28
2 REVIEW OF LITERATURE 30-38
3 NEED FOR THE STUDY 40
4 OBJECTIVE AND HYPOTHESES 42
5 METHODOLOGY 44-66
6 RESULTS AND DISCUSSION 68-157
7 CONCLUSIONS 159-163
8 REFERENCES 165-178
9 ANNEXURE 180-183
LIST OF TABLES
Table No. TITLE Page
No.
1.1 Various ROS and corresponding neutralizing antioxidants 13
5.1 Protocol of the Anti-inflammatory study using carrageenan induced paw oedema method 65
6.1 Data showing ash values of Thespesia populnea (leaves) and Strychnos potatorum (seeds) 68
6.2 Data showing extractive values of methanolic extract of Thespesia populnea (leaves) and Strychnos potatorum (seeds)
69
6.3 Results of methanolic maceration of Thespesia populnea (leaves) and Strychnos potatorum (seeds). 69
6.4 Results of preliminary phytochemical screening of methanolic extract of Thespesia populnea (leaves) and Strychnos potatorum (seeds)
70
6.5 Results of antioxidant activity of different fractions of Thespesia populnea and Strychnos potatorum by DPPH free radical scavenging assay
71
6.6(a) Results showing isolated compounds and their Rf values from ethyl acetate fraction of TP. 75
6.6(b) Results showing isolated compounds and their Rf values from aqueous fraction of SP. 76
6.7 Results showing DPPH radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid.
106
6.8 Results showing Nitric oxide radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid.
107
6.9 Results showing Super oxide radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid.
108
6.10 Results showing Hydroxyl radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid.
109
Table No. TITLE Page
No.
6.11 Results showing Inhibition of lipid peroxide formation of ethyl acetate extract of Thespesia populnea and α-Tocopherol
110
6.12 Results showing DPPH radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid.
112
6.13 Results showing Nitric oxide radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid.
113
6.14 Results showing Super oxide radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid.
114
6.15 Results showing Hydroxyl radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid.
115
6.16 Results showing Inhibition of lipid peroxide formation of aqueous extract of Strychnos potatorum and α-Tocopherol
116
6.17(i) Results showing DPPH free radical scavenging activity of isolated compounds from bio-active fraction of TP 118
6.17(ii) Results showing DPPH free radical scavenging activity of curcumin (standard). 118
6.18(i) Results showing nitric oxide radical scavenging activity of isolated compounds from bio-active fraction of TP 120
6.18(ii) Results showing nitric oxide radical scavenging activity of curcumin (standard). 120
6.19(i) Results showing super oxide radical scavenging activity of isolated compounds from bio-active fraction of TP 122
6.19(ii) Results showing super oxide radical scavenging activity of curcumin (standard). 122
6.20(i) Results showing hydroxyl radical scavenging activity of isolated compounds from bio-active fraction of TP 124
6.20(ii) Results showing hydroxyl radical scavenging activity of curcumin (standard). 124
Table No. TITLE Page
No.
6.21(i) Results showing inhibition of lipid peroxide formation by isolated compounds from bio-active fraction of TP 126
6.21(ii) Results showing inhibition of lipid peroxide formation by curcumin (standard). 126
6.22(i) Results showing DPPH free radical scavenging activity of isolated compounds from bio-active fraction of SP 128
6.22(ii) Results showing DPPH free radical scavenging activity of curcumin (standard). 128
6.23(i) Results showing nitric oxide radical scavenging activity of isolated compounds from bio-active fraction of SP 130
6.23(ii) Results showing nitric oxide radical scavenging activity of curcumin 130
6.24(i) Results showing super oxide radical scavenging activity of isolated compounds from bio-active fraction of SP 132
6.24(ii) Results showing superoxide radical scavenging activity of curcumin (standard). 132
6.25(i) Results showing hydroxyl radical scavenging activity of isolated compounds from bio-active fraction of SP 134
6.25(ii) Results showing hydroxyl radical scavenging activity of curcumin (standard). 134
6.26(i) Results showing inhibition of lipid peroxide formation by isolated compounds from bio-active fraction of SP 136
6.26(ii) Results showing inhibition of lipid peroxide formation by curcumin (standard). 136
6.27 Results showing IC50 values of isolated compounds from TP and SP 137
6.28
Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Malondialdehyde, Glutathione and Superoxide dismutase in liver homogenate of CCl4 treated Wistar rats.
140
6.28(a) Statistical analysis (One-Way ANOVA) of data in Table 6.28 140
Table No. TITLE Page
No.
6.29
Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Glutathione reductase, Glutathione peroxidase and Catalase in liver homogenate of CCl4 treated Wistar rats.
144
6.29(a) Statistical analysis (One-Way ANOVA) of data in Table 6.29 144
6.30
Results showing effect of aqueous extract of Strychnos potatorum on the activity of Malondialdehyde, Glutathione and Superoxide dismutase in liver homogenate of CCl4 treated Wistar rats.
148
6.30(a) Statistical analysis (One-Way ANOVA) of data in Table 6.30 148
6.31
Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione reductase, Glutathione peroxidase and Catalase in liver homogenate of CCl4 treated Wistar rats.
152
6.31(a) Statistical analysis (One-Way ANOVA) of data in Table 6.31 152
6.32 Effect of ethyl acetate extract of Thespesia populnea and aqueous extract of Strychnos potatorum on plantar oedema in Albino mice.
157
LIST OF FIGURES
Figure No. TOPIC Page
No.
1.1 Major cellular sources of ROS in living non-photosynthetic cell 7
1.2 Peroxidation of unsaturated lipids 9
1.3 Defence mechanism against damage by ROS 11
1.4 Picture showing different parts of Thespesia populnea 25
1.5 Picture showing different parts of Strychnos potatorum 28
6.1 Result showing TLC for ethyl acetate fraction of Thespesia populnea 73
6.2 Result showing TLC for aqueous fraction of Strychnos potatorum 73
6.3 LCMS of ethyl acetate fraction of Thespesia populnea 74
6.4 LCMS of aqueous fraction of Strychnos potatorum 74
6.5 IR spectrum of EaTP-1 77
6.6 1H NMR spectrum of EaTP-1 78
6.7 13C NMR spectrum of EaTP-1 79
6.8 Mass spectrum of EaTP-1 80
6.9 IR spectrum of EaTP-2 82
6.10 1H NMR spectrum of EaTP-2 83
6.11 13C NMR spectrum of EaTP-2 84
6.12 Mass spectrum of EaTP-2 85
6.13 IR spectrum of EaTP-3 87
6.14 1H NMR spectrum of EaTP-3 88
6.15 13C NMR spectrum of EaTP-3 89
6.16 Mass spectrum of EaTP-3 89
Figure No. TOPIC Page
No.
6.17 IR spectrum of EaTP-4 91
6.18 1H NMR spectrum of EaTP-4 92
6.19 13C NMR spectrum of EaTP-4 93
6.20 Mass spectrum of EaTP-4 94
6.21 IR spectrum of ASP-1 96
6.22 1H NMR spectrum of ASP-1 96
6.23 13C NMR spectrum of ASP-1 97
6.24 Mass spectrum of ASP-1 98
6.25 IR spectrum of ASP-2 100
6.26 1H NMR spectrum of ASP-2 101
6.27 13C NMR spectrum of ASP-2 102
6.28 Mass spectrum of ASP-2 103
6.28a Co-TLC for quercetin, gossypol, kaempferol and gallic acid 104
6.29 Results showing DPPH radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid
106
6.30 Results showing Nitric oxide radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid
107
6.31 Results showing Super oxide radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid
108
6.32 Results showing Hydroxyl radical scavenging activity of ethyl acetate extract of Thespesia populnea and Ascorbic acid
109
Figure No. TOPIC Page
No.
6.33 Results showing Inhibition of lipid peroxide formation of ethyl acetate extract of Thespesia populnea and α-Tocopherol
110
6.34 Results showing DPPH radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid
112
6.35 Results showing Nitric oxide radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid
113
6.36 Results showing Super oxide radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid
114
6.37 Results showing Hydroxyl radical scavenging activity of aqueous extract of Strychnos potatorum and Ascorbic acid
115
6.38 Results showing Inhibition of lipid peroxide formation of aqueous extract of Strychnos potatorum and α-Tocopherol
116
6.39 Results showing DPPH free radical scavenging activity of isolated compounds from TP 119
6.40 Results showing nitric oxide radical scavenging activity of isolated compounds from TP 121
6.41 Results showing super oxide radical scavenging activity of isolated compounds from TP 123
6.42 Results showing hydroxyl radical scavenging activity of isolated compounds from TP 125
6.43 Results showing inhibition of lipid peroxide formation by isolated compounds from TP 127
6.44 Results showing DPPH free radical scavenging activity of isolated compounds from SP 129
Figure No. TOPIC Page
No.
6.45 Results showing nitric oxide radical scavenging activity of isolated compounds from SP 131
6.46 Results showing super oxide radical scavenging activity of isolated compounds from SP 133
6.47 Results showing hydroxyl radical scavenging activity of isolated compounds from SP 135
6.48 Results showing inhibition of lipid peroxide formation by isolated compounds from TP 137
6.49 Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Malondialdehyde in liver homogenate of CCl4 treated Wistar rats
141
6.50 Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Glutathione in liver homogenate of CCl4 treated Wistar rats
142
6.51
Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Superoxide dismutase in liver homogenate of CCl4 treated Wistar rats
143
6.52 Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Glutathione reductase in liver homogenate of CCl4 treated Wistar rats
145
6.53 Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Glutathione peroxidase in liver homogenate of CCl4 treated Wistar rats
146
6.54 Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Catalase in liver homogenate of CCl4 treated Wistar rats
147
6.55 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Malondialdehyde in liver homogenate of CCl4 treated Wistar rats
149
Figure No. TOPIC Page
No.
6.56 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione in liver homogenate of CCl4 treated Wistar rats
150
6.57 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Superoxide dismutase in liver homogenate of CCl4 treated Wistar rats
151
6.58 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione reductase in liver homogenate of CCl4 treated Wistar rats
153
6.59 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione peroxidase in liver homogenate of CCl4 treated Wistar rats
154
6.60 Results showing effect of aqueous extract of Strychnos potatorum on the activity of Catalase in liver homogenate of CCl4 treated Wistar rats
155
6.61 Results showing effect of ethyl acetate extract of Thespesia populnea and aqueous extract of Strychnos potatorum on plantar oedema in Albino mice
157
LIST OF ABBREVIATIONS AND SYMBOLS
C - Degree Celsius
µg - Micro gram
µL - Micro litre
µm - Micro metre
µM - Micro molar
4-HDA - 4-hydroxyalkenals
ANOVA - Analysis of Variance
Aq. - Aqueous
CCl4 - Carbon tetrachloride
CHCl3 - Chloroform
cm - Centimetre
cm-1 - Per centimetre or wavenumber
DPPH - 1, 1- diphenyl-2-picryl hydrazyl
EDTA - Ethylenediaminetetraacetate
EtOAc - Ethyl acetate
EtOH - Ethanol
g - Gram
GPx - Glutathione peroxidase
GR - Glutathione reductase
GSH - Reduced Glutathione
GSSG - Glutathione disulfide
LIST OF ABBREVIATIONS AND SYMBOLS
hr - Hour
IC50 - Concentration exhibiting 50 % inhibition
IR - Infra red
KBr - Potassium bromide
Kg - Kilo gram
LCMS - Liquid chromatography-mass spectrometry
M - Molar
m/z - Mass to charge ratio
MDA - Malondialdehyde
mg - Milli gram
MHz - Mega Hertz
min - Minute
mL - Millilitre
mm - Millimetre
mM - Millimolar
mmol - Millimole
Mol - Molar
N - Normality
NBT - Nitroblue tetrazolium
nm - Nano metre
NMR - Nuclear magnetic resonance
PBS - Phosphate buffered saline
LIST OF ABBREVIATIONS AND SYMBOLS
ppm - Parts per million
PUFA - Polyunsaturated fatty acids
Rf - Retention factor
ROS - Reactive oxygen species
SD - Standard deviation
SOD - Superoxide dismutase
SP - Strychnos potatorum
TBA - Thiobarbituric acid
TBARS - Thiobarbituric acid reactive substance
TLC - Thin layer chromatography
TP - Thespesia populnea
UV - Ultra violet
WHO - World Health Organisation
μmol - Micromole
1
Chapter 1: INTRODUCTION
2
1 INTRODUCTION
Plants have been used for thousands of years, based on experience
and folk remedies. They continue to draw wide attention for their role in the
treatment of mild and chronic diseases. In recent times, focus on plant
research has increased the world over. A large body of evidence has been
accumulated to highlight the immense potential of medicinal plants in various
traditional systems of medicine1,2,3.
The history of herbal medicines is as old as human civilization. Plants
were used medicinally in China, India, Egypt and Greece long before the
Christian era4. Medicinal plants are of great value in the field of treatment and
cure of disease. It is now a universally accepted fact that plant drugs and
remedies are far safer than synthetic medicines, for curing complex diseases
like Cancer and Aids. Modern developments in instrumental techniques of
analysis and chromatographical methodologies have added numerous
complex and rare natural products to the armoury of phyto-medicine5.
World Health Organization (WHO) estimated that 80% of the population
of developing countries rely on traditional medicines, mostly plant drugs for
their primary health care needs. Medicinal plants are the major components of
all indigenous or alternative systems of medicine. For example, they are
common elements in Ayurveda, Homeopathy, Naturopathy, Oriental and
Native American Indian medicine. Demand for herbal drugs is increasing
throughout the world due to growing recognition of natural plant-based
products. Being non-toxic, they have fewer side effects and are easily
3
available at affordable prices, at times the only source of health care available
to the poor. Hence, medicinal plant sector has traditionally occupied an
important position in the socio-cultural, spiritual, economic values of rural and
tribal lives of both developing and developed countries6.
1.1 Antioxidants
An antioxidant is a chemical that reduces the rate of a particular
oxidation reaction. The term antioxidant has many definitions7,8,
Chemical definition: “A substance that opposes oxidation or inhibits reaction
promoted by oxygen or peroxides”.
Biological definition: “Synthetic or natural substances that prevent or delay
deterioration of a product, or are capable of counteracting the damaging
effects of oxidation in animal tissue”.
Institute of Medicine definition: “A substance that significantly decreases
the adverse effects of reactive species such as ROS (reactive oxygen
species) or RNS (reactive nitrogen species) on normal physiological function
in humans”.
All living organisms utilize oxygen to metabolize and use the dietary
nutrients producing energy for survival. Oxygen thus is a vital component for
living. Although it is one of the most essential components, due to its high
reactivity, are capable of producing potentially damaging molecules commonly
called “free radicals”.
4
1.2 Free radicals
An atom or group that has at least one unpaired electron and is
therefore unstable and highly reactive are called free radicals. In our body it is
usually an oxygen molecule that has lost an electron and will stabilize itself by
stealing an electron from a nearby molecule. This stabilizes the free radical
but generates another in the process. Soon a chain reaction begins and
thousands of free radical reaction can occur within a few seconds of the
primary reaction. These free radicals can adversely alter lipids, proteins, DNA
and trigger a number of human diseases.
1.3 Reactive oxygen species (ROS)
Aerobic organisms, which derive their energy from the reduction of
oxygen, are susceptible to the damaging actions of small amounts of
superoxide anion radical (•O2-), hydroxyl radical (•OH) and hydrogen peroxide
(H2O2) that inevitably form during the metabolism of oxygen, especially in the
reduction of oxygen by the electron transfer system of mitochondria. These
three species, together with unstable intermediates in the peroxidation of
lipids, are referred to as Reactive Oxygen Species. During times of
environmental stress (e.g., UV or heat exposure), ROS levels can increase
dramatically, which may result in significant damage to cell structures. This is
known as oxidative stress. Many diseases are linked to damage from ROS as
a result of oxidative stress. Examples are Alzheimer disease, auto immune
5
diseases, myocardial infarction, radiation injury, atherosclerosis, emphysema,
Parkinson’s disease, sunburn and many more.
1.4 Sources of oxygen radicals
Cells generate energy aerobically by reducing molecular oxygen (O2) to
water. The cytochrome complex oxidase-catalyzed reaction involves transfer
of electrons to oxygen, in principle without intermediates, but, in fact, partially
reduced oxygen species are produced. Other enzymes, especially flavin
enzymes, also generate partially reduced oxygen species. One to two percent
of total oxygen consumption may, in fact, be converted to superoxide anion
radical (•O2-). Other sources of ROS include radiation (e.g., UV light), toxic
chemicals and drugs.
Formation of superoxide anion radical leads to a cascade of other
ROS9,10 (Fig. ). Superoxide dismutates to hydrogen peroxide (H2O2) and
oxygen. This reaction is spontaneous and fast, but the SOD-catalyzed
reaction is four orders of magnitude faster. Clearly, O2- is more toxic than
H2O2 and its rapid removal is important.
H2O2 is reduced by three general mechanisms,
1) It is the substrate for two enzymes, catalase and glutathione (GSH)
peroxidase11 that catalyze the conversion of H2O2 to H2O and O2; this
presumably is a detoxification mechanism.
6
2) H2O2 is converted by myeloperoxidase (MPO) in neutrophils to
hypochlorous acid (HOCl). This appears to be a mechanism for a
physiological toxic agent, since HOCl is a strong oxidant that acts as a
bactericidal agent in phagocytic cells. Reaction of HOCl with H2O2 yields
singlet oxygen (1O2) and water. The biological significance of singlet oxygen is
unclear.
3) H2O2 is converted in a spontaneous reaction catalyzed by Fe2+ (Fenton
reaction) to the highly reactive hydroxyl radical (•OH). The hydroxyl radical
reacts instantaneously with any biological molecule (RH) from which it can
abstract a hydrogen radical. The resulting free radical (R•) is more stable and
hence longer-lived than the hydroxyl radical.
HOCl1O2 +
H2O2 H2O
Hypochlorus acid Singlet oxygen
O2 H2O2
e Fe2+
Fe3+
MPO
SOD Hydrogenperoxide
Hydroxylradical
H2OO2
H2O
H.
OH .
Cl .
O2. -
O2. -
+ R-H H2O +OH . .
R
7
Figure No.1.1: Major cellular sources of ROS in living non-photosynthetic cell
8
1.5 Mechanism of Damage
Reactive oxygen species, in particular the hydroxyl radical, can react
with all biological macromolecules (lipids, proteins, nucleic acids and
carbohydrates). The initial reaction generates a second radical, which in turn
can react with a second macromolecule to continue the chain reaction. Among
the more susceptible targets are polyunsaturated fatty acids. Abstraction of a
hydrogen atom from a polyunsaturated fatty acid initiates the process of lipid
peroxidation (Figure No.1.2). In step 3 of Figure No.1.2, a hydrogen atom is
abstracted from a second lipid, leading to a new ROS. Numerous products are
formed, presenting special analytical problems. The choice is between simple,
non-specific assays for classes of lipid peroxidation products
(e.g., thiobarbituric acid reaction for aldehydes), more specific but less
sensitive assays (e.g., uv absorbance by conjugated dienes) or specific, highly
sensitive methods that require expensive instrumentation (e.g., mass spectral
analysis of hydroxy fatty acids). A sensitive and specific colorimetric assay
based on measurement of malondialdehyde and 4-hydroxyalkenals is a
frequent compromise12.
9
Figure No.1.2: Peroxidation of unsaturated lipids
The variety of lipids and the random nature of free radical reactions lead to
many products. These include 4-hydroxyalkenals (4-HDA) and, when there
are three or more unsaturated bonds, malondialdehyde (MDA). These can
serve as targets for the measurement of fatty acid peroxidation. The initiating
event can be reaction with another radical, uv light or radiation. Since a radical
is also produced in the process, it is a chain reaction.
Y X
O O H
Y .+
OO
MDA
+ X
O
C
H
R
OH
4-HDA
Polyunsaturated fatty acid
Y X
H
Y X
O O .
Y X
.
OH .
OH
1
O2
2
LH
L .
3
H
10
ROS modify both the structure and function of proteins. Metal-catalyzed
protein oxidation results in addition of carbonyl groups or cross-linking or
fragmentation of proteins. Lipid (peroxidation) aldehydes can react with
sulfhydryl (cysteine) or basic amino acids (histidine, lysine). Similarly,
modification of individual nucleotide bases, single-strand breaks and cross-
linking are the typical effects of reactive oxygen species on nucleic acids.
1.6 Defence Mechanisms
Mammalian cells possess elaborate defence mechanisms to detoxify
radicals (Figure 1.3). The key metabolic steps are SOD catalysis of the
dismutation of superoxide to hydrogen peroxide and oxygen and the
conversion of H2O2 to 2H2O by glutathione peroxidase or to O2 + H2O by
catalase. Since the reaction catalyzed by glutathione peroxidase requires
GSH as substrate and depends in part on the ratio of GSSG: GSH, the
concentrations of these reactants and their ratio, which is a reflection of the
redox state of the cell, are important to ROS detoxification. Similarly, the
concentration of redox-active metals such as iron, catalyze formation of some
ROS. This is minimized by keeping the concentrations of these metal ions
very low due to binding to storage and transport proteins (e.g., ferritin,
transferrin, lactoferrin), thereby minimizing •OH formation. Finally, radical-
scavenging antioxidants (e.g., vitamin E) interrupt the chain reactions by
capturing the radical; the vitamin E radical is relatively stable, and it can be
enzymatically converted back to its non-radical form. Radical scavengers thus
terminate the chain reaction of radical damage.
11
The potential significance of these ROS defence mechanisms is apparent
from considerations of the whole body and sub-cellular distribution of the
different components. Vitamin E, the enzymes (SOD, catalase and GSH-
peroxidase) and substrates (GSH) tend to be in higher concentration in
locations where ROS damage is more likely (e.g., in more highly oxygenated
locations) and potentially more damaging3.
Figure No.1.3: Defence mechanism against damage by ROS
Superoxide dismutase (SOD) plus catalase or glutathione peroxidase
(GPx) eliminate many damaging oxygen species. Lactoferrin (binds iron) and
radical scavengers such as vitamin E, further limit damage.
Harmful effects of reactive oxygen species on the cell include,
1. damage of DNA.
2. oxidations of polyunsaturated fatty acids in lipids (lipid peroxidation).
3. oxidations of amino acids in proteins.
H2O + O2
H2O2 OH .
2 H2O
Fe2+
Fe3+
SODLipid peroxidation
GSH
GSSG
LactoferrinRadical
scavengers
X X
GPx
CatalaseO2. -
12
4. oxidatively inactivate specific enzymes by oxidation of co-factors.
1.7 Classification of antioxidants
To protect the cells and organ system of the body against reactive
oxygen species, humans have evolved a highly sophisticated and complex
antioxidant protection system. It involves a variety of components, both
endogenous and exogenous in origin, that function interactively and
synergistically to neutralize free radicals13. These components include:
Nutrient derived antioxidants like ascorbic acid (vitamin C), tocopherols
and tocotrienols (vitamin E) carotenoids and other low molecular weight
compounds such as glutathione and lipoic acid.
Antioxidant enzymes, e.g., superoxide dismutase, glutathione
peroxidase and glutathione reductase, which catalyze free radical
quenching reactions.
Metal binding proteins, such as ferritin, Lactoferrin, albumin and
ceruloplasmin that sequester iron and copper ions that are capable of
catalyzing oxidative reactions.
Numerous antioxidant phyto nutrients present in a wide variety of plant
foods.
13
Table No.1.1: Various ROS and corresponding neutralizing antioxidants
ROS Neutralizing antioxidants
Hydroxyl radical Vitamin C, glutathione, flavonoids, lipoic acid.
Superoxide radical Vitamin C, glutathione, flavonoids, lipoic acid,
SOD.
Hydrogen peroxide Vitamin C, glutathione, β-carotene, vitamin E,
CoQ 10, flavonoids, lipoic acid.
Lipid peroxides β-carotene, vitamin E, ubiquinone, flavonoids,
glutathione peroxidase.
Vitamin C is capable of neutralizing ROS in the aqueous phase before
lipid peroxidation is initiated. Vitamin E protects membrane fatty acids from
lipid peroxidation. β-carotene and other carotenoids also provide antioxidant
protection to lipid-rich tissue.
Phytochemicals are now becoming increasingly known as antioxidants.
Phenolic compounds such as flavonoids have been demonstrated to have
anti-inflammatory, anti-allergic, anti-viral, anti-aging and anti-carcinogenic
activity14-17. In addition to an antioxidant effect, flavonoids have ability to
protect from heart diseases16.
The antioxidant enzymes (glutathione peroxidase, catalase and
superoxide dismutase), metabolize oxidative toxic intermediates and require
micronutrient cofactors such as selenium, iron, copper, zinc and manganese
14
for optimum catalytic activity. Research indicates that consumption and
absorption of these important trace minerals may decrease with aging18.
Glutathione directly quenches ROS such as lipid peroxides, and also
plays a major role in xenobiotics metabolism. Research suggests that,
glutathione and vitamin C work interactively to quench free radicals and that
they have a sparing effect upon each other13. Lipoic acid is capable of
quenching free radicals in both lipid and aqueous domains and as such has
been called a “universal antioxidant”. Lipoic acid may also exert its antioxidant
effect by chelating with pro-oxidant metals19.
1.8 In vitro antioxidant screening methods
Antioxidant activity cannot be measured directly but rather by the effect
of the antioxidant in controlling extend of oxidation. The features of an
oxidation process are a substrate, an oxidant and an initiator, intermediates
and final products. Measurement of any one of these can be used to assess
antioxidant activity.
Most test procedures use accelerated oxidation involving an initiator to
manipulate one or more variable in the test system. Initiator include,
o increased temperature and partial pressure of oxygen,
o addition of transition metal catalysts,
o exposure to light to promote photosensitized oxidation by singlet
oxygen,
15
o variable shaking to enhance reactant contact and free radical
sources.
The effect of substrate can be attributed to the strong influence of the
unsaturation type and degree of the lipid system on the kinetics and
mechanism of the antioxidant action. After the substrate is oxidized under
standard conditions, either extends of oxidation or rate of oxidation is
measured by chemical, instrumental or sensory methods.
Various chemical and physico-procedures are used to monitor oxidation
processes. We can examine directly the free radical production and its
inhibition by antioxidants, or indirectly measure the inhibition of various
intermediates or final reaction products of oxidation. Individual measurement
of the antioxidant activity of all components in a sample is possible, but this
can be time consuming and expensive. The desirable features of an
antioxidant screening are the use of a substrate, conditions in the test that
mimic the real situation and the ability to quantify the result by reference to a
suitable standard20.
In vitro antioxidant methods21 include,
1) Oxygen radical absorbance capacity (ORAC)
2) Lipid peroxidation inhibition capacity (LPIC)
3) Total radical trapping antioxidant parameter (TRAP)
4) Nitric oxide radical scavenging activity
5) DPPH free radical scavenging assay
6) Ferric reducing antioxidant power (FRAP)
16
7) Trolox equivalent antioxidant capacity (TEAC)
8) Total phenols by Folin-Ciocalteu
9) N,N-dimethyl-p-phenylenediamine (DMPD) assay
10) Hydroxyl radical scavenging activity
11) ABTS radical scavenging method
12) Superoxide radical scavenging assay
13) H2O2 radical scavenging method
14) Total oxidant scavenging capacity (TOSC) etc.
In the present study, the methods adopted are, DPPH free radical
scavenging assay, Nitric oxide radical scavenging activity, Superoxide radical
scavenging assay, Hydroxyl radical scavenging activity and Lipid peroxidation
inhibition capacity.
1.8.1 DPPH free radical scavenging assay22
The scavenging reaction between DPPH (1,1-diphenyl-2-picryl
hydrazyl) radical and an antioxidant (H-A) can be written as,
Antioxidant reacts with DPPH, which is a stable free radical and is
reduced to the DPPH-H and as consequence, the absorbance decreased from
the DPPH radical to the DPPH-H form. The degree of decolouration indicates
the scavenging potential of the antioxidant compound or extract. The values of
absorbance are measured at 517 nm.
NN
NO2
O2N
NO2 .+ H A + A.NNH
NO2
O2N
NO2
YellowPurple
17
1.8.2 Nitric oxide radical scavenging assay23
Sodium nitroprusside in aqueous solution at physiological pH
spontaneously generates nitric oxide, which interacts with oxygen to produce
nitric ions that can be estimated by use of Griess reagent. Scavenger of nitric
oxide competes with oxygen leading to reduced production of nitric oxide.
Sodium nitroprusside (10 mM) in phosphate-buffered saline (PBS) is mixed
with 3 ml of different concentrations (10-100μg/ml) of the drugs dissolved in
the suitable solvent systems and incubated at 250C for 150 min.
The samples from the above are reacted with Griess reagent (1%
sulphanilamide, 2% H3PO4 and 0.1% naphthylethylenediamine hydrochloride).
The absorbance of the chromophore formed during the diazotization of the
nitrite with sulphanilamide and subsequent coupling with
naphthylethylenediamine is read at 546 nm.
1.8.3 Superoxide radical scavenging activity8
The assay for superoxide radical scavenging activity is based on the
capacity of the sample to inhibit blue formazan formation by scavenging the
superoxide radicals generated in riboflavin-light-NBT system.
18
Fe2+
-EDTA + O2 Fe
3+-EDTA + O2
-
2O2- + 2H
+ H2O2 + O2
Fe2+
-EDTA + H2O2 OH- + OH
.+ Fe
3+-EDTA
OH . + deoxyribose
heat TBA plus acidfragments MDA
2 TBA + MDA chromogen
1.8.4 Hydroxyl radical scavenging method
Hydroxyl radicals are produced by Fenton reaction, which can be
described by the following scheme,
The values of absorbance are measured at 532 nm.
1.8.5 Inhibition of lipid peroxidation24
Effect on the inhibition of lipid peroxidation can be determined by the
thiobarbituric acid method. Different concentrations of the plant extract are
incubated at 370C with rat liver homogenate (25%) (0.1ml) containing 30mM
KCl, Tris-HCl buffer (0.04M; pH7), ascorbic acid (0.06mM) and ferrous iron
(0.16mM) (total volume was 0.5ml) for 1 hour. At the end of 1 hour,
thiobarbituric acid reactive substance (TBARS) is measured and percentage
of inhibition calculated from the control where no test extract was added.
The initiation of lipid peroxidation can be induced by OH radical and
metal-ion-free radical complexes (such as perferryl and ferryl). In propagation
19
step, the lipid radical (L ) reacts with oxygen molecule (O2) to form lipid
peroxyl radicals. The induction of lipid peroxidation is shown below,
LH + OH . H2O + L..
L + O2 LOO.
LOO + LH.
LOOH + L.
inert productLOO + LOO. .
inert productL + L. .
inert product LOO + L. .
Chain initiation step
Chain propagation step
Chain termination step
Lipid peroxidation may be prevented at the initiation stage by free
radical scavengers, while propagation reaction can be intercepted by peroxy-
radical scavengers such as phenolic antioxidants (A-OH).
LOO / L / LO + A - OH. ..
LOOH / LH / LOH + AO .
(Phenoxy
radical)
1.9 In vivo antioxidant screening
The important antioxidant enzymes within the body are superoxide
dismutase (SOD), catalase and glutathione peroxidase (GPx). Superoxide
dismutase has been found to be the first line of defence against superoxide
radical- mediated injury by catalyzing its conversion to H2O2
O2- + O2
- + 2 H
+H2O2 + O2
SOD
20
In mammalian tissues, two types of SOD have been described,
(i) Cytosolic cuprozinc-SOD (Cu Zn SOD) and
(ii) Mitochondrial mangano-SOD (MnSOD).
H2O2 thus produced is detoxified either by catalase or reduced by
glutathione dependent reactions. SOD has an important role in scavenging the
superoxide O2 generated by redox cycling chemicals25,26. Two possible
mechanisms are proposed,
(i) It is likely to act at the level of the cell membrane and remove or
prevent radical formation.
(ii) Removal of oxygen radicals in the growth medium.
Catalase is present virtually in all mammalian cells and is suggested to
play a dual role.
(i) a catalytic role in the decomposition of H2O2.
(ii) a peroxidic role in which the peroxide is utilized to oxidize a range of
hydrogen donors (AH2) such as methanol, ethanol and formate.
It is mostly localized in the peroxisomes (microbodies) of liver and
kidney. The catalase reaction mechanism may be written as follows,
Catalase - Fe (i ii ) + H2O2 Compound I
Compound I + H2O2 Catalase - Fe (i ii ) + 2H2O + O2
AH2 + H2O2 A + 2H2O
1.
2.
At particular conditions, the protective action of superoxide dismutase
and catalase complement each other in a sequential fashion.
21
Glutathione (L-γ-glutamyl-L-Cysteinyl glycine) is important in the
circumvention of cellular oxidative stress, detoxification of electrophiles and
maintenance of intracellular thiol redox status27. Glutathione peroxidase
(GPx), a Selenium (Se) containing enzyme, catalyses the oxidation of GSH to
GSSG at the expense of H2O2.
H2O2 + 2 GSH GSSG + 2 H2O
This enzyme has high activity in liver, moderate activity in heart, lung
and brain and low activity in muscle. Glutathione reductase (GR) catalyses the
regeneration of GSH by the following reaction,
GSSG + NADPH + H+ 2 GSH + NADP+
The oxidative stress in tissues is often reflected as high GSSG level in
the serum. GSH also plays a central role in co-ordinating the synergism of
various crucial antioxidants. Several thiols, dithiolthiones, disulfiram analogous
and selenium compounds are GSH enhancers.
22
1.10 Carbon tetrachloride on liver function
Uncontrolled environmental pollution, poor sanitary conditions,
xenobiotics, alcoholic intoxication and the indiscriminate use of potent drugs
predispose the liver to a vast array of disorders. Direct hepatotoxins are
characterized by a very brief interval between exposure to them and the
development of hepatic injury. In exposed individuals, there are distinctive
hepatic lesions accompanied by lesions in other organs too. Hepatotoxins
exhibit considerable experimental reproducibility and dose-dependence.
These properties and the ability to produce injury in a variety of living things
permit the designation of them as protoplasmic poisons28. This category
includes carbon tetrachloride (CCl4) and other chlorinated hydrocarbons,
inorganic phosphorus and perhaps some heavy metals29.
Liver of most of the higher species of mammals is susceptible to CCl4
damage30,31. Hepatic lipid accumulation was proved as a consistent feature of
CCl4 toxicity as early as 1944. According to lipid peroxidation hypothesis, CCl4
poisoning initiates an intrahepatic process of destructive lipid peroxidation.
Within 30 minutes of CCl4 administration, lipid metabolism is disturbed and at
the end of 24 hrs the blood levels of hepatic enzymes are maximum32. There
are reports that suggest that CCl4 cause liver damage due to liberation of free
radicals33. The aetiology of liver disorder caused by CCl4 ingestion highlights
induced lipid peroxidation. The progression of liver injury after a single i.p
injection of CCl4 (1.0 ml/kg body wt) was observed by Ohta et al34. Ashok
Shenoy et al35 noted a decrease in the activity of hepatic SOD, catalase and
23
glutathione reductase after 24 hrs of CCl4 intoxication; hepatic glutathione
(GSH) and ascorbic acid was reduced and lipid peroxide content was
increased. Dhawan et al36 observed a significant depression of glutathione
concentration following long term treatment of CCl4 to male Albino rats. Wang
et al37 reported a decrease in catalase activity resulting from single i.p.
injection of 20% CCl4 in olive oil/g body weight. Carbon tetrachloride plays a
significant role in inducing liver damage by increasing lipid peroxidation in
membranes whose structural integrity is necessary for lipoprotein release38.
When CCl4 is introduced into the body, CCl4 is reduced to CHCl3 (in
both in vitro and in vivo). The carbon-chlorine bond in CCl4 and CHCl3 is
subjected to homolytic cleavage39 yielding the corresponding free radicals,
which then alkylate the SH groups of enzymes. Also it initiates an intra-hepatic
process of destructive lipid peroxidation which causes enormous production of
MDA (malondialdehyde). Therefore, after treating the animals with CCl4, the
level of hepatic enzymes (SOD, GR, GPx & catalase) and level of reduced
glutathione (GSH) will be decreased. The MDA level will be increased.
1.11 Anti-inflammatory activity
There are increasing suggestions that ROS may play a role in the
pathogenesis of cancer40 and in other diseases including inflammation,
bacterial infection, AIDS, etc41. Phenolic compounds are well known for their
anti-inflammatory activity42,43. So the anti-inflammatory screening confirms the
antioxidant activity of the selected plants.
24
1.12 Plant profile
In the current study, the antioxidant activity of Thespesia populnea (TP)
and Strychnos potatorum (SP) has been carried out.
1.12.1 Plant Material
1.12.1(a) Thespesia populnea
Thespesia populnea or Hibiscus populnea, commonly known as the
Portia Tree is a species of flowering plant in the mallow family; (Malvaceae) is
a typical example of folk remedy. It is a small tree or arborescent shrub that
has a pantropical distribution, found on coasts around the world. However, the
Portia Tree is probably native only to the Old World44, and may have
originated in India45. It is possibly indigenous to the Hawaiian Islands and
elsewhere in the Pacific, but may have been spread by early Polynesians for
its useful wood. The tree reaches a height of 6-10 m (20-33 ft.), tall with a
trunk diameter of 20-30 cm (7.9-12 in)46. It grows at elevations from sea level
to 275 m (902 ft.)47 in areas that receive 500-1600 mm (20-63 in) of annual
rainfall44. The Portia Tree is able to grow in a wide range of soil types that may
be present in coastal environments, including soils derived from quartz (sand),
limestone and basalt; but it favours neutral soils (pH of 6-7.4)46.
The plant is a fairly large, quick growing, evergreen tree up to 18 m in
height with greyish brown fissured bark; leaves simple, alternate, long
petioled, cordate, entire acuminate, prominent nerves 5-7 with peltate scales
on one or both surface; flowers yellow with purple base, slowly changing to
25
purple on withering; fruits globose or oblong brown capsules covered with
minute peltate scales, pubescent, channelled along the back.
Thespesia populnea – Yellow leaves and Flowers
Thespesia populnea plant.
Figure No.1.4: Picture showing different parts of Thespesia populnea
26
The tree has different names like,
English : Portia tree
Hindi : Paraspipal, Parsipu
Kannada : Arasi, Huvarase
Malayalam : Puvarasu, Cilantippatta, Pupparutti
Sanskrit : Haripucchah
Tamil : Puvarasamkallal, Cilanti
Telugu : Gangarvi, Gangarenu, Munigangaravi
Thespesia populnea has been used for its astringent, acrid, cooling,
depurative, anti-inflammatory, haemostatic, vulnerary, alterant, antidiarrhoeal
and antibacterial activity. It is useful in dermatopathy such as scabies,
psoriasis, ring worm and guinea worm, leprosy, urethritis, gonorrhoea,
haemorrhoids, haemorrhages, haemoptysis, inflammations, wounds, ulcers,
diarrhoea, dysentery, cholera, diabetes, ascites, warts, dipsia, cough and
asthma48.
1.12.1(b) Strychnos potatorum
Strychnos potatorum Linn (Fam: Loganiaceae) is a moderate sized tree
found in southern and central parts of India, Sri Lanka and Burma. In
traditional system of medicine the seeds are used for the treatment of various
ailments like jaundice, bronchitis, diabetes, conjunctivitis, chronic diarrhoea,
dysentery etc. They are also used to clear muddy water by its coagulant
action.
27
The plant is a medium sized deciduous tree having height up to 12
meters. Its bark is cracked and scalpy black. Trunk is irregularly fluted. Leaves
are simple, opposite, elliptic, acute, 15 x 6.25 cm, glabrous and shining.
Flowers are white fragrant and axillary cymes. Fruits are ovoid or globose,
glabrous berries and appears black when ripe. Seeds appeared to be one or
two with yellow colour, circular in shape and not much compressed49.
The useful parts of Strychnos potatorum is its seeds, fruits and roots.
According to Ayurveda, seeds are acrid, alexipharmic, lithotriptic and cure
strangury, urinary discharges, head diseases etc. Roots are Leucoderma
whereas fruits are useful in eye diseases, thirst, poisoning and hallucinations.
The fruits are emetic, diaphoretic alexiteric etc. According to Unani system of
medicine, seeds are bitter, astringent to bowels, aphrodisiac, tonic, diuretic
and good for liver, kidney complaints, gonorrhoea, colic etc50.
Seeds are used to purify water. Seeds are rich source of polysaccharide
gum suitable for use in paper and textile industries. In clearing water, one of
the dried nuts is rubbed hard for a short time around the inside of the earthen
water pot; on settling, the water is left pure and tasteless. The seeds contain a
large quantity of an albuminous principle, upon which their virtues probably
depend.
28
Figure No.1.5: Picture showing different parts of Strychnos potatorum
The tree has different names like,
English : Clearing nut tree
Hindi : Nirmali
Kannada : Chilladebeeja, Chilu
Malayalam : Tetranparal, Tetraparel
Sanskrit : Katak, Kataka, Kataka ambuprasada
Tamil : Tetramkotai, Tetta, Tettamaram, Tettran
Telugu : Chillachetu, Indupachettu
29
Chapter 2: REVIEW OF LITERATURE
30
2 REVIEW OF LITERATURE
Siju EN et al51 (2014) conducted the evaluation of antioxidant potential
of ethanolic and aqueous extract of Thespesia populnea fruit (TPF) by in vitro
antioxidant studies like free radical scavenging activity by 1,1-diphenyl,2-
picrylhydrazyl (DPPH) method, Reducing power assay, Superoxide anion
scavenging activity, Hydroxyl radical scavenging activity and Nitric oxide
method. The results suggest that the fruit of Thespesia populnea showed
significant antioxidant property.
Manivachagam Chandrasekaran et al52 (2014) explained the
antibacterial and antifungal activities of different extracts of leaves of
Thespesia populnea. Hexane, chloroform, ethyl acetate and methanol extracts
were screened against Gram positive & Gram negative bacterial strains and
Aspergillus spp & dermatophytic fungal strains. Chloroform extract showed
highest antibacterial activity against Staphylococcus aureus and methanolic
extract showed highest antifungal activity against Aspergillus fumigatus. The
study also explains the presence of phytochemicals such as, flavonoids,
tannins, steroids, glycosides, saponins, phenols, terpenoids and alkaloids in
methanolic extract of leaves of Thespesia populnea than in other extracts.
Mohini A Phanse et al53 (2014) have reported the hypoglycaemic effect
of ethanolic extract of bark of Thespesia populnea in dexamethasone induced
mice. Ethanol extract was administered orally at a dose of 100, 200 and 400
mg/kg in mice which were concomitantly treated with dexamethasone
31
(1mg/kg, orally) for 22 days. There was a significant decrease in plasma-
glucose (p<0.01), serum triglyceride (p<0.01) level and significant increase in
body weight (p<0.01) as compared to dexamethasone control group.
Pratap Chandran R et al54 (2014) have evaluated the antibacterial and
antifungal activities of hot and cold extracts of leaves of Thespesia populnea
Linn. against human pathogens. Hexane, chloroform, dichloromethane, ethyl
acetate, methanol and water extracts were used for the study. Highest
antibacterial activity was shown by the methanol cold extract against
Staphylococcus epidermidis and Bacillus cereus. Both the cold and hot
extracts of all the seven solvents exhibited inhibition zones against Candida
albicans. The study also explained the use of this medicinal plant in different
ailments. The presence of gossypol, tannin, acacetin, quercetin and colouring
matter in bark extracts and presence of lupeol, lupenone, and β-sitosterol in
leaf extracts and kaempferol, kaempferol-7-glucoside and gossypectin in fruit
extracts was also mentioned in the study.
Rajbanshi SL and Pandanaboina CS55 (2014) have done the work to
evaluate chronic alcohol-induced oxidative stress in the cardiac tissue of rat to
explore the effectiveness of Thespesia populnea-induced cardio-protection in
rat heart by utilizing an in vivo model of cardiac injury by alcohol. Activities of
antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx), glutathione reductase (GR), and reduced glutathione content
(GSH) showed a decrease, while glutathione-S-transferase (GST) activity, MDA, and
32
Protein carbonyls (PC) recorded an elevation due to alcohol treatment in the cardiac
tissue compared to the control rats.
Yadav KN et al56 (2014) have written a review which explains the
phytochemical and pharmacological screening of Strychnos potatorum Linn.
This study gives collective information regarding the different medicinal uses
of Strychnos potatorum. The data has provided the basis for its wide use as a
therapeutic agent both in traditional and folk medicines.
In 2014, Packialakshmi N et al57 have reported the phytochemical and
IR spectrum analysis of Strychnos potatorum Linn. The phytochemical
screening revealed the presence of alkaloids, flavonoids, saponins, tannins,
carbohydrates, sterols, glycosides, oils and fats, phenolic compounds, gums
and mucilage in the leaves and bark. The functional groups were identified
through IR spectrum.
Kirankumar Shivasharanappa and Ramesh Londonkar58 (2014)
carried out hepatoprotective activity of methanolic extract of Ficus glomerata
Roxb fruits and the hepatotoxicity was induced by CCl4 in Wistar rats by two
dose of CCl4 (2ml/kg). Methanolic extract at the dose level of 150 mg/kg and
300 mg/kg was administered orally for 7 days. A significant increase in the
levels of reduced liver marker enzymes and antiradical enzymes due to
oxidative stress and histopathological study revealed the regeneration of
damaged hepatic cells in the test animals.
33
Pradeepa Krishnappa et al59 (2014) have explained the antioxidant
potential of ethanolic extract of Delonix elata L. stem bark against CCl4
induced liver damage in Wistar rats. The study also explains the bioassay
guided fractionation of the stem bark. The isolated compounds gallic acid,
rutin, quercetin, ellagic acid and coumaric acid have shown significant
prophylactic effects by restoring the liver function markers (AST, ALT, ALP,
serum bilirubin and total protein) and antioxidant enzymes (SOD, CAT, GSH
and GPx). Silymarin was used as standard.
Kamisan et al60 (2014) have carried out carbon tetrachloride induced
hepatotoxicity study on male Sprague Dawley rats. The experiment explains
the methanolic extract of Dicranopteris linearis leaves possess significant
protection against CCl4 induced hepatotoxicity, which could be due, partly, to
its high total phenolic content value and, antioxidant and anti-inflammatory
properties through scavenging free radicals to ameliorate oxidative stress and
inhibit lipid peroxidation. The phytochemical screening revealed the presence
of various phenolic contents (rutin and quercetin) as the antioxidant moieties.
Srikanth Kagithoju et al61 (2013) conducted a set of pharmacognostic
standardization parameter studies on S. potatorum leaves as per
pharmacopoeia and WHO guidelines. They, in 2012 have published the
physical constituents and phytochemical analytical reports of Strychnos
potatorum seeds and leaves. The petroleum ether extract of seed showed the
presence of linoleic and linolenic acids. Twenty four compounds have been
isolated and identified in the root bark. The ethyl acetate extract and
34
chloroform extract revealed the presence of diaboline, isomotiol, sitosterol,
stigmasterol, ompesterol, norharmane, akuammidine, nor-C-fluroiocuraine,
ochrolifuanine, bis nor dihydrotoxiferine, 11-methoxy-henningsamine, 11-
methoxy-12 hydroxydiaboline and 11-methoxy diaboline and other related
compounds. These compounds showed diuretic activity, antidiarrhoeal
activity, contraceptive efficacy, hepatoprotectivity, antioxidant activity,
antiarthritic activity, antiulcerogenic activity, antinociceptive and antipyretic
effects.
Patil PS et al62 (2012) have evaluated the antioxidant potential of flower
of Thespesia populnea. Antioxidant potential of methanolic extract of flower
was evaluated by in vitro antioxidant studies like free radical scavenging
activity by DPPH method, nitric oxide method, anti-lipid peroxidation study,
and reducing power assay and expressed as % scavenging and IC50. Ascorbic
acid was used as standard. The phytochemical screening of the extract mainly
revealed the presence of terpenoids and flavonoids and the HPTLC profile of
extract confirmed the presence of β- sitosterol in the extract. The extract
showed the significant antioxidant activity as compared to control; but
comparatively less than the ascorbic acid.
Illavarasan R et al63 (2012) have conducted animal studies on the
analgesic and anti-inflammatory activities of aqueous and ethanolic extracts of
Thespesia populnea leaves. Analgesic activity was carried out in chemical-
mechanical and thermally induced pain test models in mice. Anti-inflammatory
activity tested in rats by carrageenan induced paw oedema method. The
35
results show significant analgesic and anti-inflammatory activities of leaves of
Thespesia populnea.
Vijayakumar V et al64 (2012) have done the evaluation of bioactive
compounds and free radical scavenging activity of Strychnos potatorum. The
studies were carried out in hydroalcoholic extracts of leaf and seed to
understand the nature of the phytochemical constituents and free radical
scavenging (antioxidant) properties of Strychnos potatorum Linn. The
preliminary photochemical investigation revealed the presence of alkaloids,
flavonoids, phenols, glycosides, steroids, tannins and saponins and the
absence of resins. The antioxidant activity analyzed by DPPH, LPO, H2O2 and
nitric oxide radical scavenging assays showed that leaf and seed possess
many bioactive compounds exhibiting excellent antioxidant potential.
Muthu et al65 (2012) have conducted in vivo antioxidant studies on
various extracts of Ionidium suffruticosum (Ging.). The methanolic extract
showed significant (p<0.001) results up on an oral dose of 200 mg/kg body
weight. The experiment was carried out in high fat diet Wistar rats. After
administration of the methanolic extract in high fat diet rats were shown
significantly increased levels of antioxidant enzymes such as Superoxide
dismutase (SOD), Catalase (CAT), Glutathione reductase (GR), Glutathione
peroxidase (GPx) and increased levels of non-enzymatic antioxidant
Glutathione (GSH) when compared with high fat diet rats.
36
Venkata Suresh Babu A and Sai Koteswar Sarma D66 (2011) carried
out the pharmacognostic studies and preliminary phytochemical analysis of
different extracts of Thespesia populnea leaves which showed the presence of
alkaloids, flavonoids, carbohydrates, phytosterols, tannins, saponins, proteins
and amino acids, terpenes, phenols, gums and mucilages.
Sai Koteswar Sarma D et al67 (2011) explains the in vitro nitric oxide
antioxidant activity of ethanolic extract of Thespesia populnea leaves. The
antioxidant activity was compared with standard drug (ascorbic acid). In both
cases, as concentration increased, the percentage of free radical scavenging
activity was increased.
Elakkiya S and Ananthi T68 (2011) have conducted anti-inflammatory
activity of ethanolic extract of Thespesia populnea leaves. The study was
carried out by carrageenan induced paw oedema method in rats. 100 mg/kg of
extract was given orally and showed a good anti-inflammatory activity.
Zhang L et al69 (2011) have done the antioxidant, anti-inflammatory and
cytotoxic activities of water and ethanol extracts of 14 Chinese medicinal
plants. The antioxidant activity was evaluated in a biological assay using
Saccharomyces cerevisiae, whereas the radical scavenging activity was
measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Total
phenolics and flavonoid contents were estimated by Folin-Ciocalteu and
aluminium chloride methods, respectively. The anti-inflammatory activities of
the plant extracts were determined by measuring the inhibition of production of
37
nitric oxide (NO) and TNF-α in LPS and IFN-γ activated RAW 264.7
macrophages. Their cytotoxic activities against macrophages were
determined by Alamar Blue assay.
Sewwandi UDS et al70 (2010) have tested anti-inflammatory activity of
aqueous extract of Thespesia populnea barks in conscious rats using
carrageenan induced paw oedema model and three oral doses; 1250, 2500
and 5000mg/kg. Indomethacin was used as the reference drug. The result
showed that the extract significantly and dose-dependently inhibited both early
(1–2h) and late phase (4–5h) of inflammation in the carrageenan model. In
addition, it inhibited the intermediated phase (3h). The anti-inflammatory
activity of the highest dose of the extract was comparable to that of
indomethacin. The extract did not display overt signs of toxicity and was
neither hepatotoxic, renotoxic nor haematotoxic even with chronic
administration.
Mallikharjuna et al71 (2007) have done the phytochemical screening of
therapeutic importance from Strychnos potatorum. The study involves the
preliminary screening, quantitative determination and the qualitative thin layer
chromatographic separation of secondary metabolites from the root, stem,
bark and seeds of Strychnos potatorum. Further, HPLC alkaloid profile of the
seed has been studied. The generated data has provided the basis for its wide
use as the therapeutic agent both in the traditional and folk medicines.
38
Talhouk RS et al72 (2007) have explained the antioxidant and anti-
inflammatory activities of some medicinal plants. The plant extracts contain
natural chemicals such as phenols, carbohydrates, alkaloids and glycosides
and are responsible for showing both antioxidant and anti-inflammatory
activities.
39
Chapter 3: NEED FOR THE STUDY
40
3 NEED FOR THE STUDY
Plants like Thespesia populnea and Strychnos potatorum are widely
used by the tribals as well as common people of Kerala for the treatment of
various ailments, without exactly knowing about their phytoconstituents.
Studies have been carried out by several research workers to explore the
pharmacognostic and phytochemical results of the said plants but not much
pharmacological investigations have been carried out using the said plants.
As many of the diseases are in one way or other related to the excess
production or excess activity of reactive oxygen species, these plants might be
acting via free radical scavenging activity. Hence screening of antioxidant
potential of these drugs will help to evaluate their activities and also a useful
step in the invention of a lead compound or a new formulation without many
side effects.
41
Chapter 4: OBJECTIVE AND HYPOTHESES
42
4 OBJECTIVE AND HYPOTHESES
The objective of the study may be summarized as follows,
1. To conduct the extraction of the desired plant parts and phytochemical
screening of the crude extracts.
2. To carry out the fractionation of the crude extracts.
3. To conduct the in vitro antioxidant pilot study of the different fractions
(DPPH method) to find out the most active fraction.
4. To do the TLC of the active fraction (extract) followed by column
chromatography of the same for isolating the components present in the
extract.
5. To characterize and identify the components using physical characters,
chemical tests and spectral data and compare with those in the spectral
library.
6. To do the in vitro and in vivo antioxidant screening of the active extracts.
7. To do the in vitro antioxidant screening of the isolated compounds.
8. To carry out anti-inflammatory activity of the active extracts in order to
ascertain the antioxidant activity.
It was hypothesized that the selected plants might have antioxidant
activity.
43
Chapter 5: METHODOLOGY
44
Plan of work
Collection and identification
Ash values
Extractive values
Extraction
Phytochemical screening
In vitro studies
DPPH free radical scavenging assay
Nitric oxide radical scavenging assay
Superoxide radical scavenging assay
Hydroxyl radical scavenging assay
Inhibition of lipid peroxide formation
Acute toxicity study
Antioxidant studies – Activity of scavenging enzymes
Assay of superoxide dismutase
Assay of catalase
Assay of Glutathione peroxidase
Assay of Glutathione reductase
Assay of Glutathione content
Estimation of Malondialdehyde
Anti-inflammatory studies – Paw oedema method
P L A N T
S T U D I E S
A N I
M A L
S T U D I E S
45
5 METHODOLOGY
5.1 Collection and identification of plant material
Old and ripened leaves of Thespesia populnea and seeds of Strychnos
potatorum were collected from the suburbs of Thrissur District.
The collected plant materials were identified by the taxonomist, Dr. P.
Sujanapal, Scientist-B, Silviculture Department, Kerala Forest Research
Institute, Peechi, Kerala and a herbarium was made out. The voucher
specimen was kept in the museum of St. James College of Pharmaceutical
Sciences, Chalakudy (No. STJ 010/08 and STJ 011/08).
5.2. Ash values73
The residue remaining after incineration is the ash content of the drug, which
simply represents inorganic salts, naturally occurring in drug or adhering to it
or deliberately added to it, as a form of adulteration. Ash value is a criterion to
judge the identity or purity of crude drugs. Total ash usually consists of
carbonates, phosphates, silicates and silica. Acid insoluble ash, which is a
part of total ash insoluble in dilute HCl, is also recommended for certain drugs.
Adhering dirt and sand may be determined by acid insoluble ash content.
5.2.1 Total ash value74,75
Procedure: 2.5g of the ground drug was accurately weighed and taken in a
tarred silica crucible previously ignited and weighed; scattered the ground
drug in a fine even layer in the bottom of the dish. Incinerated by gradually
increasing the heat until free from carbon. It was then cooled and weighed to
46
constant weight. Calculated the percentage of ash with reference to the air-
dried drug. The experiment was repeated thrice and the average calculated.
5.2.2 Acid insoluble ash value.
Procedure: Boiled the ash obtained after total ash value determination for 5
minutes with 25 ml of dil. Hydrochloric acid. The insoluble matter was
collected in an ash less filter paper, washed with hot water, ignited and
weighed to constant weight. Calculated the percentage of acid insoluble ash
with reference to the air- dried drug. The procedure was repeated thrice and
the average calculated.
5.2.3: Sulphated ash value
Procedure: About 2-3 g of drug was accurately weighed, moistened with
sulphuric acid and ignited gently. Again moistened and re-ignited, cooled and
weighed. Calculated the percentage of sulphated ash with reference to the air
dried drug.
5.2.4 Water-soluble ash value
Procedure: Boiled the total ash with 25 ml of water for 5 minutes; insoluble
matter was collected in an ash less 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 percentage of water-soluble ash
was calculated with reference to the air-dried drug.
47
5.3: Extractive values76
The extracts obtained by exhausting crude drugs are indicative of
approximate measures of their chemical constituents. Taking into
consideration the diversity in chemical nature and properties of contents of
drugs, various solvents are used for determination of extractives. The solvent
used for extraction is in a position to dissolve appreciable quantities of
substances desired.
5.3.1 Alcohol soluble extractive value
Alcohol being an ideal solvent for extraction of various chemicals like
tannins, resins etc. this method is frequently employed to determine the
approximate resin content of drugs. Generally 95% ethyl alcohol is used for
determination of alcohol soluble extractive; dilute alcohol may also be used,
depending upon solubility of the constituents of crude drugs.
Procedure: Macerated 5g of dried coarse powder of the drug with 100ml of
alcohol 95% in a closed flask for 24 hours, shaking frequently during 6 hours
and allowed to stand for 18 hours. It was then filtered immediately taking
precautions against loss of alcohol. 25 ml of the filtrate was evaporated to
dryness in a tarred flat-bottomed shallow dish. Dried at 1050C and weighed.
Calculated the percentage of alcohol soluble extractive with reference to the
shade-dried drug.
48
5.3.2:Water-soluble extractive value
This method is applied to drugs, which contain water-soluble active
constituents, such as tannins, plant sugars, mucilage, glycosides etc.
Procedure: Added 5g of coarse powder of the drug to 50ml of water at 800C
in a stopper flask. Shaken well and allowed to stand for 10 minutes. Cooled to
150C and added 2g of kieselguhr, filtered and transferred 5ml of the filtrate to
a tarred evaporating dish; evaporated the solvent on a water bath and
weighed the residue. Calculated the percentage of water-soluble extractive
with reference to the shade-dried drug. The values were tabulated.
5.4 Extraction and preliminary phytochemical screening77,78,79
The plant may be considered a biosynthetic laboratory, not only for the
chemical compounds like carbohydrates, proteins and lipids that are utilized
as food by man, but also for a multitude of compounds like glycosides,
alkaloids, volatile oils, tannins etc. that exert a physiologic effect. The
compounds that are responsible for therapeutic effect are usually the
secondary metabolites. A systematic study of a crude drug embraces
thorough consideration of both primary and secondary metabolites derived as
a result of plant metabolism. The plant material may be subjected to
preliminary phytochemical screening for the detection of various plant
constituents on the following lines.
49
5.4.1 Extraction
The plant materials were extracted using cold maceration method. For
that 1000 gm (1 kg) each of the plant materials was subjected to cold
maceration using methanol as the menstrum for 5 days. The extracts were
concentrated by rotary evaporator. Each extract was weighed and the
percentage yield was calculated with reference to the air-dried material. The
colour and consistency was also noted.
5.4.2 Qualitative Phytochemical examination of the extracts
The extracts obtained as above were then subjected to qualitative tests
for the identification of various plant constituents.
5.4.2.1 Test for carbohydrates
Molisch’s test: About 300 mg each of the extracts was mixed with 4ml distilled
water and filtered. The filtrate was subjected to Molisch’s test. Observed for
the presence of reddish brown ring.
Fehling’s test: Dissolved a small portion of each extract in water and treated
with Fehling’s solution and noted whether any brown colour is produced.
5.4.2.2 Test for Phenolic compounds
Phosphomolybdic acid test: Each of the extract was spotted on a filter paper.
A drop of phosphomolybdic acid reagent was added to the spot and was
exposed to ammonia vapours. Blue colouration of the spot indicates the
presence of phenolic acids.
50
Ferric chloride test: Small quantities of alcoholic and aqueous extracts of both
the drug were taken in water and treated with dilute ferric chloride (5%).
Observed for the presence of blue colour.
Lead acetate test: Small quantities of alcoholic and aqueous extracts of both
the drugs taken in water were treated with 1% solution of gelatin containing 10
% of sodium chloride and observed for the white precipitate.
5.4.2.3 Test for flavonoids
Shinoda test: To 2 to 3ml of extract, a piece of magnesium ribbon and 1ml of
concentrated HCl was added. A pink or red coloration of the solution indicate
the presence of flavonoids in the drug.
Lead acetate test: To 5ml of extract 1ml of lead acetate solution was added.
Flocculent white precipitate indicated the presence of flavonoids.
Amyl alcohol test: A few millilitre of each extract was shaken with 2 ml of amyl
alcohol and observed for the presence of yellow colour in the amyl alcohol
layer.
5.4.2.4 Test for alkaloids
Dragendorff’s test: A drop of extract was spotted on a small piece of
precoated TLC plate and the plate was sprayed with modified Dragendorff’s
reagent. Orange coloration of the spot indicates the presence of alkaloids.
Hager’s test: The extract was treated with few ml of Hager’s reagent and
observed for the presence of yellow precipitate.
Wagner’s test: The extract was treated with few ml of Wagner’s reagent. The
reddish brown precipitation indicated the presence of alkaloids.
51
Mayer’s test: A small quantity of the extract was treated with Mayer’s reagent
and observed whether any cream coloured precipitate is present.
5.4.2.5 Tests for Glycosides
Legal’s test: Dissolved the extract (0.1g) in pyridine (2ml), added equal
volume of freshly prepared sodium nitroprusside solution (2ml) and made
alkaline with Sodium hydroxide solution. Pink to red colour solution shows the
presence of glycosides.
Borntrager’s test: About 50 mg of powdered extract was boiled with 2 ml of
10% ferric chloride solution and 1ml of concentrated HCl. The extract was
cooled, filtered and the filtrate was shaken with equal volume of chloroform.
The chloroform extract was further extracted with strong ammonia. Pink or red
colouration in the ammoniacal layer indicates the presence of anthraquinone
glycosides (C-glycosides).
5.4.2.6 Test for Saponins
Foam test: 1ml of extract was diluted with 20ml of distilled water and shaken
in a graduated cylinder for a few minutes. A 1cm layer of foam formation
indicates the presence of Saponins.
Haemolysis test: One drop each of methanolic and aqueous extracts in little
warm water were added to blood samples on glass slides and mixed well.
Observed for the presence of clear haemolytic zones.
52
5.4.2.7 Test for Amino acids
Ninhydrin test: Dissolved a small quantity of the extract in few ml of water and
added 1ml of ninhydrin reagent. Blue colour indicates the presence of amino
acids.
5.4.2.8 Test for steroid / terpenoid
Liebermann-Burchard’s test: To 1ml of extract, 1ml of chloroform, 2 to 3 ml of
acetic anhydride and 1 to 2 drops of concentrated Sulphuric acid were added.
Dark green coloration of the solution indicated the presence of steroids and
dark pink or red coloration of the solution indicated the presence of
terpenoids.
5.5 Fractionation of the crude extracts
The crude extracts were defatted with petroleum ether. Then it was
fractioned using the solvents chloroform, ethyl acetate, acetone and water.
Each extract was concentrated by distilling off the solvent and then
evaporated to dryness on a water bath.
5.6 In vitro antioxidant pilot study of the fractions
Each fraction was subjected to DPPH radical scavenging assay. Of
these ethyl acetate of Thespesia populnea and aqueous fraction of Strychnos
potatorum showed maximum positive results.
53
5.7 Phytochemical studies
5.7.1 TLC of the most effective fractions
TLC of the most effective fractions was done to find out the number of
compounds present in them and the possible type of compounds by spraying
with appropriate reagents.
5.7.2 Detection of compounds from the bio-active fractions using LCMS
Ethyl acetate fraction of Thespesia populnea and aqueous fraction of
Strychnos potatorum were then used for LCMS to get a probable idea about
the compounds present. Then column chromatography of effective fractions
was carried out to isolate the compounds.
5.7.3(a): Isolation of compounds from ethyl acetate fraction of Thespesia
populnea (TP)
Ethyl acetate extract of TP was subjected to column chromatography on
silica gel using solvents of increasing polarities starting from chloroform, ethyl
acetate, and methanol in different ratios to yield the fractions.
Fractions 2-10 (96%chloroform in methanol)were pooled due to their
similar TLC pattern and were coded as EaTP-1. All the above fractions with
same Rf were pooled, concentrated, crystallized and recrystallized using
methanol to get 110 mg of EaTP-1.
EaTP-2 was isolated from fractions 11-20 (50% chloroform in
methanol). They were pooled together and TLC of this pooled fraction with a
solvent system n-butanol : acetic acid : water in the ratio 5 : 3 : 5 showed a
54
single spot with an Rf value 0.8. The eluted EaTP-2 when concentrated and
crystallized gave 133 mg. of the compound.
EaTP-3 was isolated from 21-32 fractions (5% chloroform in methanol).
TLC of these pooled fractions was done with a solvent system of petroleum
ether : ethyl acetate : acetic acid in the ratio 10 :91 : 4 and Rf value was found
to be 0.51. The eluted EaTP-3 gave 182 mg of the sample.
Fractions 33-42 were pooled and TLC was performed with a solvent
system n-butanol : acetic acid : water in the ratio 3 : 1 : 1. Rf value was
determined as 0.47. The 20% of methanol in ethyl acetate (43rd - 54th fraction)
gave a single spot in TLC. This was collected, dried and recrystallized with
methanol. EaTP-4 was about 240 mg.
5.7.3 (b): Isolation of compounds from aqueous fraction of Strychnos
potatorum (SP)
Aqueous fractions of SP were subjected to column chromatography on
silica gel using solvents as in the case of TP, to get the compounds. Each
fraction was about 20 ml in volume.
Fractions 1-10 (50 % chloroform in methanol) were mixed due to their
similar TLC pattern. This pooled fraction was coded as ASP-1. The solvent
system chloroform : methanol in the ratio 4 : 1 showed an Rf value of 0.52 for
the spot. All the fractions with same Rf were pooled, concentrated, dried and
recrystallized using methanol to get 280 mg of ASP-1.
55
ASP-2 isolated from fractions 11-22 (20% chloroform in methanol) and
subjected to TLC using the solvent system toluene : ethyl acetate : formic acid
in the ratio 1.1 : 2.2 : 1.1. The TLC showed a sharp single spot in UV and the
Rf value was found to be 0.59. These fractions were collected, pooled,
concentrated, dried and recrystallized and named as ASP-2 (230 mg).
5.7.4 Characterization of isolated compounds from TP and SP
The isolated compounds were subjected to their characterization
studies. Initially, their physical characters such as colour and appearance,
melting point, Rf value and solubility were observed. The chemical tests were
done to confirm the chemical moiety. Then, the spectra (UV, IR, H-NMR, C-
NMR and Mass) of each compound were taken and analyzed to explore their
complete chemical characterization. Co-TLC studies helped to confirm their
structure.
5.8 Antioxidant studies (IN VITRO)
As pilot studies with fractionated extracts did not show significant results
except for ethyl acetate fraction of Thespesia populnea and aqueous fraction
of Strychnos potatorum, further programmed studies were concentrated on
these fractions only.
In vitro anti-oxidant screening was carried out using spectrophotometer.
The absorbance obtained for test and control are noted and the percentage
inhibition of radical scavenging activity was calculated using the equation,
56
Where, A Control - Absorbance of control
A Test - Absorbance in the presence of the samples of
extracts.
5.8.1 DPPH free radical scavenging assay80
A stock solution of DPPH (1.3 mg/ml in methanol) was prepared such
that 75 μl of it in 3 ml methanol gave an initial absorbance of 0.9. The
decrease in the absorbance in the presence of sample extract and standard at
different concentrations (10-100 μg/ml) were noted at 517 nm after 30
minutes, using methanol as blank in UV-Visible Spectrophotometer. IC50
(Inhibitory concentration to scavenge 50% free radicals) value which denotes
the concentration of sample required to scavenge 50% of the DPPH free
radicals was also determined.
5.8.2 Nitric Oxide Radical Scavenging Assay81
The reaction mixture (3ml), containing 2.0ml of sodium nitroprusside,
0.5ml of phosphate buffered saline and 0.5ml of different concentrations
(10μg-100μg/ml) of various extracts was incubated at 25°C for 5 hours.
Control experiments without the test compounds, but with equivalent amounts
of buffer were conducted in an identical manner. After 5 hours, 0.5ml of Griess
reagent was added. The absorbance of the chromophore formed during
diazotization coupling with naphthylethylenediamine was measured at 546nm.
5.8.3 Superoxide Radical Scavenging Assay
57
Superoxide radical was generated from the photo reduction of riboflavin
and was deducted by nitroblue tetrazolium dye (NBT) reduction method82. The
scavenging activity towards the superoxide radical was measured in terms of
generation of O2. The reaction mixture consisted of phosphate buffer (50mM,
pH 7.6), riboflavin (2 μm), EDTA (6 μm), nitroblue tetrazolium (NBT) (50 μm)
and sodium cyanide (3 μg). Test compounds at various concentration of 10μg
to 100μg / ml were added to make a final volume of 3ml. The absorbance was
read at 530nm before and after illumination under UV lamp for 15 minutes
against a control instead of sample. Ascorbic acid was used as the reference
compound. All the tests were performed in triplicate and the results averaged.
The percentage inhibition was calculated by comparing the results of control
and test samples.
5.8.4 Hydroxyl Radical Scavenging Activity
The scavenging capacity for hydroxyl radical was measured according
to the modified method Rajeshwar Y et al83. The assay is based on
quantification of degradation product of 2-deoxy ribose by condensation with
TBA. Hydroxyl radical was generated by the Fe3+- Ascorbate-EDTA-H2O2
system (Fenton reaction). The reaction mixture contained 0.1ml of
deoxyribose, 0.1ml of EDTA, 0.1ml of H2O2, 0.1ml of ascorbic acid, 0.1ml of
KH2PO4-KOH buffer and various concentrations of different extracts in a final
volume of 1.0ml. The reaction mixture was incubated for an hour at 370C. At
the end of the incubation period, colour developed was measured at 535nm in
a spectrophotometer. Deoxyribose degradation was measured as
58
thiobarbituric acid reactive substance (TBARS) and the percentage inhibition
was calculated. The percentage inhibition was calculated from the control
where no test extracts were added.
5.8.5 Lipid peroxide inhibition84
Different concentrations (10-100 μg/ml) of the plant extracts were
incubated at 370C with 0.1ml of rat liver homogenate (25%) containing 30mM
KCl, Tris-HCl buffer (0.04M; pH 7), ascorbic acid (0.06mM) and ferrous iron
(0.16mM) in a total volume of 0.5ml for 1 hour. At the end of incubation time,
TBARS produced was measured at 532 nm using UV-Visible
spectrophotometer. The percentage of inhibition was calculated from the
control where no test extract was added.
5.9 In vitro antioxidant study of isolated compounds
In vitro anti-oxidant screening for the isolated compounds were also
carried out using the same methods. The different concentrations of isolated
compounds (0-10 μg) were screened using the same standard drugs which
were used for the ‘active fractions’ in the above studies.
5.10 Experimental studies (Animal)
5.10.1 Animal maintenance and care
Wistar strains of rats (Rattus norvigicus) were the animals used for the
study. They were bred in colony and brought up in our own animal house.
Animals were housed in well-ventilated polypropylene cages and fed with a
standard pellet diet (Gold Mohur laboratory animal feed) and water ad libitum.
They were kept in standard environmental conditions. (Temperature 25-280C
59
and 12 hours light/dark cycle) Rats of both sexes weighing 150-250g were
used for animal experiments. (All ethical formalities were cleared for the
conduct of animal experiments using albino rats.)
5.10.2 Acute toxicity studies
Albino rats of either sex weighing 100 – 200 g were used for carrying
out acute toxicity studies. Before the actual study, a pilot study was carried out
in small group of animals (2 each) giving them widely spaced doses to select
the dose ranges for the actual acute toxicity studies in all animals. Finally the
assay was carried out as per OECD guide lines No. 425. All animals were
fasted overnight before the acute toxicity studies. After oral administration of
the drug, the animals were observed continuously for 2 hours and then
intermittently for another 4 hours. After 24 hours, the deaths if any were noted
to calculate the LD50.
5.10.3 Dose response assay of the most effective extract
For the methanol extract, a dose response assay was carried out using
1/20th, 1/10th and 1/5th of the maximum dose given to the animals to determine
the dose required to produce maximum biological activity.
5.10.4. Impairment of liver function by CCl4
Hepatic injury was induced using Carbon tetrachloride (CCl4). CCl4 was
mixed with olive oil in the ratio 1:1 and given intraperitoneally at a dose of
0.5ml/kg body wt. for 5 days. After 24 hours of the last dose, the animals were
sacrificed, the liver taken and used for the study of scavenging enzymes.
60
5.10.4.1 Grouping of animals and different treatment
Animals were grouped and given different treatments as follows,
Male Wistar Albino rats weighing 100-120 g were used for
hepatoprotective studies. The animals were divided into four groups of six
each.
Group I – Control (without any drug or toxicant)
Group II – CCl4 treated (0.5 ml / kg body wt.)
Group III – CCl4 + Thespesia populnea / Strychnos potatorum extract
(200 mg / kg body wt.)
Group IV – CCl4 + Thespesia populnea / Strychnos potatorum extract
(400 mg / kg body wt.).
This procedure was repeated for 5 consecutive days and on the 6th day
animals were sacrificed. The liver homogenate was prepared and used for
anti-oxidant enzyme studies.
5.11 Activity of Scavenging Enzymes
5.11.1 Assay of Superoxide dismutase (SOD) [EC 1.15.1.1]
SOD activity was determined by the method of Kakkar et al85. The tissue
was homogenized in 0.25M sucrose and differentially centrifuged at 10,000
rpm under cold conditions to get the cytosol fraction. The initial purification
was done by precipitating the protein from the supernatant with 90%
ammonium sulphate and after dialysis against 0.0025M Tris-HCl buffer, pH
7.4. The supernatant was used as the enzyme source.
61
The assay mixture contained 1.2ml Sod.pyrophosphate buffer (0.052M,
pH8.3), 0.1ml 186μM phenazine methosulphate (PMS), 0.3ml 300μm nitroblue
tetrazolium (NBT), 0.2ml 780μM NADH, appropriately diluted enzyme
preparation and water in a total volume of 3ml. Reaction was started by the
addition of NADH. After incubation at 300C for 90 seconds reaction was
stopped by the addition of 1ml glacial acetic acid. The reaction mixture was
stirred vigorously and shaken with 4ml of n-butanol. Mixture was then allowed
to stand for ten minutes. Centrifuged and butanol layer was taken out. Colour
intensity of the chromogen in the butanol fraction was measured at 560nm
against butanol. A system devoid of enzyme served as control.
One unit of enzyme activity is defined as the enzyme concentration
required inhibiting chromogen production (OD of 560 nm) 50% in one minute
under the assay conditions. The specific activity is expressed in units/ mg
protein. Unit is defined as the velocity constant per second.
5.11.2 Assay of Catalase [EC 1.11.1.6]
(H2O2: Hydrogen peroxide oxido reductase)
The catalase activity was assayed by the method of Maehly and
Chance86. The tissue was homogenized with 0.91M-phosphate buffer (pH 7.0)
at 1- 40C and centrifuged at 5000 rpm. The estimation was done
spectrophotometrically following the decrease in absorbance at 240nm. The
reaction mixture contained 0.91 M phosphate buffer pH 7.0, 2mM H2O2
(diluted 0.1ml H2O2 to 100ml using buffer) and 50μl enzyme extract. Specific
62
activity is expressed in terms of units / mg protein. Unit is defined as the
velocity constant per second.
5.11.3 Assay of Glutathione peroxidase (EC.1.11.1.9)
(Glutathione: hydrogen peroxide, oxido reductase)
The activity of glutathione peroxidase was determined by the method of
Lawrence and Burk87 as modified by Agerguard and Jense88. Tissue
homogenate (10%) was prepared in 0.25M sucrose, centrifuged at 10,000 rpm
for 30 minutes and the supernatant fraction was used for the assay. Activity
was determined in phosphate buffer (50mM pH7.0) containing EDTA (1.5mM),
sodium azide (1.0mM), reduced glutathione (1.0mM), NADPH (0.1mM) and
glutathione reductase (1.0μM/ml). Absorbance was measured at 340nm at 20
seconds interval. Enzyme activity is defined as μM of NADPH oxidized/min
/mg protein using 0.25mM H2O2 as substrate.
5.11.4 Assay of Glutathione reductase (EC.1.6.4.2)
(Reduced NAD (P): oxidized glutathione oxido reductase)
Glutathione reductase activity was determined by the method described
by Bergmeyer89. Tissue homogenate (10%) was prepared in 0.25 M sucrose,
centrifuged at 10,000 rpm for 30 minutes and supernatant fraction was used
for the enzyme assay. The assay system contained 1ml phosphate buffer
(0.12M, PH 7.2) 0.1ml EDTA, 0.1ml sodium azide (10mM/l), 0.1ml oxidized
glutathione (6.3mM) and 0.1 ml enzyme source. It was kept for 3 minutes.
Then 0.1 ml NADPH (9.6 mM/l) was added. The absorbance at 340 nm was
63
measured at an interval of 15 seconds for 2 minutes. The activity is expressed
as μM NADPH oxidized per minute / mg protein.
5.11.5 Estimation of Glutathione content
Glutathione content was estimated by the method of Benke et al90. 20%
tissue homogenate prepared in 5% TCA containing 0.001M EDTA, was
centrifuged at 2000 rpm for 5 minutes; 0.2ml aliquot of each supernatant
fraction was transferred to another tube containing 4.75ml of 0.1M sodium
phosphate buffer (pH 8) and to it 0.05ml of 0.01m 5,5 dithiobis-2-nitrobenzoic
acid (DTNB) was added. The absorbance was read at 412 nm within 4
minutes.
5.11.6 Estimation of Malondialdehyde
Determination of Malondialdehyde was based up on Nichans and
Samuelson method91. To 1ml of liver homogenate in a test tube, 2ml of freshly
prepared TBA-TCA-HCl mixture was added and thoroughly mixed. Kept in
boiling water bath for 15 minutes. The mixture was cooled and centrifuged at
1000 rpm for 10 minutes to remove the flocculent precipitate. The supernatant
was recovered and absorbance was recorded at 535 nm in UV-VIS
spectrophotometer against reagent blank. A Standard calibration curve was
obtained by plotting absorbance values against various concentrations
(0.5nm/ml, 1.0 nm/ml, 2 nm/ml, 2.5 nm/ml, 3 nm/ml and 3.5 nm/ml). The effect
on lipid peroxidation was expressed in nM of malondialdehyde/gram of tissue.
64
5.12 Anti-inflammatory screening of the extracts by carrageenan induced
paw oedema in Albino rats.
Principle
The redness and swelling of the paw produced by congestion of the
capillaries in the skin after the inflammatory process occurred within few
minutes by carrageenan application. Thus, the oedema response of the skin
was expressed as an increase in paw volume (ml) due to the acute
inflammatory process. It has been established that inflammation activates
PLA-2, which releases arachidonic acid from the cell membrane is
metabolized to prostaglandins and leukotrienes (Paula, 2003)92. The release
of inflammatory mediator’s response for the clinical signs of inflammation.
Vasodilatation and its resulting increased blood flow causing the
redness and increased heat. Increased permeability of the blood vessels
results in an exudation of plasma proteins and fluid into the tissue, which
manifests itself as swelling or oedema.
5.12(i) Test animals
Anti-inflammatory activity against carrageenan induced oedema and
acute toxicity study was investigated in female rats weighing 100-150 g/kg
body wt.
The animals were housed in standard cages under 12 hour light and
dark cycle and fed with a standard pellet diet. Groups of 6 test animals were
used. (The study protocol was approved by the institutional ethical
committee). The animals were kept for 2 weeks to get acclimatized with
65
laboratory conditions. Then they were fasted overnight before the experiment,
but with water ad libitum.
5.12(ii) Acute toxicity studies:
All the extracts were subjected for acute toxicity studies by following the
OECD guidelines No. 425 of CPCSEA and 1/10th
of the LD50
dose was
selected for the pharmacological activity.
5.12(iii) Anti-inflammatory study using carrageenan induced paw
oedema method
Paw oedema was produced by subcutaneous injection of 0.05ml of (3%
w/v) carrageenan in saline solution into the sub plantar region of the left hand
paw in Wistar rats93,94. The volume (ml) of induced oedema was measured
with the aid of a plethysmometer.
Table No.5.1 Protocol of the Anti-inflammatory study using carrageenan
induced paw oedema method
Groups Treatment
Group-I
(Control) Carrageenan only (0.05ml)
Group-II
(Standard) Carrageenan + Indomethacin, 10 mg/kg.
Group-III
(TP-50) Carrageenan + EtOAc fraction (TP), 50 mg/kg.
66
Group-IV
(TP-100) Carrageenan + EtOAc fraction (TP), 100 mg/kg.
Group-V
(SP-50) Carrageenan + Aq. fraction of SP, 50 mg/kg.
Group-VI
(SP-100) Carrageenan + Aq. fraction of SP, 100 mg/kg.
Aq. – Aqueous; EtOAc – Ethyl acetate; TP – Thespesia populnea; SP – Strychnos potatorum
The standard (indomethacin, 10 mg/kg.), TP-50 (Ethyl acetate fraction
of Thespesia populnea, 50 mg/kg.), TP-100 (Ethyl acetate fraction of
Thespesia populnea, 100 mg/kg.), SP-50 (Aqueous fraction of Strychnos
potatorum, 50 mg/kg.) and SP-100 (Aqueous fraction of Strychnos potatorum,
100 mg/kg.) were given orally 1 hour before carrageenan administration. Paw
volume was measured by water plethysmography before injection of the
phlogistic agent and at 0, 3, 5 and 7 hour afterwards. The percentage
inhibition was calculated using the formula,
Where, Vc - mean increase of paw volume of control animals
Vt - mean increase of paw volume of treated animals.
5.13 Statistical analysis
The experimental results are expressed as the mean + SEM. Data were
assessed by the method of analysis of ANOVA followed by student’s t- test.
P< 0.05 was considered as statistically significant.
67
Chapter 6: RESULTS AND DISCUSSION
68
6 RESULTS AND DISCUSSION
6.1 Collection and identification of plant materials
The plant materials were collected from the suburbs of Thrissur District
and got identified by a taxonomist as described earlier.
6.2 Ash values
The total ash value as well as sulphated ash value and water- soluble
and acid insoluble ash values were determined according to the procedure
given in the Indian Pharmacopoeia (I.P). The results are tabulated in Table
No.6.2. The percentage yield of sulphated ash was more than total ash
content for both plants. Also the water-soluble ash was more than acid
insoluble ash.
Table No.6.1: Data showing ash values of TP and SP
Plant Extracts
Total ash (%w/w)
Acid insoluble ash
(% w/w)
Sulphated ash (%w/w)
Water soluble ash (%w/w)
TP 6.33 0.73 9.53 5.60
SP 8.42 1.06 10.68 7.36 TP - Thespesia populnea ; SP - Strychnos potatorum
6.3 Extractive values
The alcohol soluble extractive and water- soluble extractive values were
determined as specified in the I. P. (The results are tabulated in Table No.6.3)
Alcohol soluble extractives were more in both plants when compared to water
soluble extractives.
69
Table No.6.2: Data showing extractive values of methanolic extract of TP and SP
Plant Extracts
Alcohol extractive value (% w/w)
Water extractive value (%w/w)
TP 22.98 15.12
SP 12.78 9.68 TP - Thespesia populnea ; SP - Strychnos potatorum
6.4 Extraction and Preliminary phytochemical screening
After 5 days cold maceration process, the colour and consistency and
the percentage yield of the methanolic extracts of the leaves of Thespesia
populnea and seeds of Strychnos potatorum were noted and are shown in
Table No.6.3. Extracts of both plants were brown gummy in nature and
Thespesia populnea gave good percentage yield than Strychnos potatorum.
Table No.6.3: Results of methanolic extract of Thespesia populnea
(leaves) and Strychnos potatorum (seeds)
No. Methanolic extracts Colour and consistency
Percentage of extractive (w/w)
1 TP Brown gummy 20.46
2 SP Brown gummy 11.29 TP – Thespesia populnea ; SP – Strychnos potatorum
The preliminary phytochemical screening of the methanolic extract of
leaves of Thespesia populnea and seeds of Strychnos potatorum was done
using different chemical tests. Extract of Thespesia populnea have shown the
presence of carbohydrates, phenolic compounds, flavonoids, alkaloids,
terpenoids and steroids. Extract of Strychnos potatorum seeds have also
70
shown the presence of same components along with saponins. (The results
are shown in Table No.6.4).
Table No.6.4: Results of qualitative phytochemical screening of methanolic extract of Thespesia populnea and Strychnos potatorum
Classes of Compounds Chemical Tests Performed Observations TP SP
Carbohydrates Molisch’s test Fehling’s test
+ +
+ +
Phenolic compounds Phosphomolybdic acid test
Ferric chloride test Lead acetate test
+++ + +
++ ++ ++
Flavonoids Shinoda test
Lead acetate test Amyl alcohol test
+++ +++ +++
++ ++ ++
Alkaloids
Dragendorff’s test Hager’s test Wagner’s Mayer’s
+ + + +
+++ +++ +++ +++
Glycosides Legal’s test Borntrager’s test
- -
++ ++
Saponins Foam test Haemolysis test
- -
+ +
Aminoacids Ninhydrin test - -
Terpenoids and Steroids Liebermann-Burchard’s test ++ +
TP - Thespesia populnea ; SP - Strychnos potatorum
NB:
- indicate not present
+ in traces
++ present in moderate amount
+++ more amount is present
71
6.5 Fractionation of the crude extracts
After defatting the crude methanolic extract with petroleum ether, the
extracts were fractionated with chloroform, ethyl acetate, acetone and water.
Each fraction was concentrated by distilling off the solvents and the dry
product was collected.
6.6 In vitro antioxidant pilot study of the fractions
Each fraction was subjected to DPPH free radical scavenging assay. Of
these, ethyl acetate fraction of Thespesia populnea and aqueous fraction of
Strychnos potatorum showed maximum percentage inhibition. (Results are
shown in Table No.6.5).
Table No.6.5: Results of antioxidant activity of different fractions of TP
and SP by DPPH free radical scavenging assay
Crude extract Fraction Percentage inhibition
Ethanol extract of TP
Chloroform 17.78 Ethyl acetate 85.72
Acetone 36.25 Water 24.86
Ethanol extract of SP
Chloroform 19.51 Ethyl acetate 41.73
Acetone 20.62 Water 71.39
TP - Thespesia populnea ; SP - Strychnos potatorum
72
6.7 Phytochemical studies
6.7.1 TLC of most effective fractions
After doing TLC of most effective fractions of TP and SP, it was found to
contain four spots in the ethyl acetate fraction of Thespesia populnea and two
spots in the aqueous fraction of Strychnos potatorum (Figure No.6.1 and 6.2).
Moreover, these fractions showed maximum percentage inhibition by
preliminary DPPH free radical scavenging assay (Table No.6.5) and they were
called as bio-active fractions and these fractions were used for the
programmed study.
73
Figure No.6.1: TLC of ethyl acetate fraction of TP
Figure No.6.2: TLC of aqueous fraction of SP
4 spots
2 spots
74
6.7.2 Detection of compounds from bio-active fractions using LCMS
LCMS of ethyl acetate fraction of TP (Figure No.6.3) and aqueous
fraction of SP (Figure No6.4) were taken. This gave a probable idea about the
compounds present in each fraction.
Figure No.6.3: LCMS of ethyl acetate fraction of Thespesia populnea
Figure No.6.4: LCMS of aqueous fraction of Strychnos potatorum
75
Then column chromatography of these fractions was carried out to isolate the
compounds.
6.7.3(a) Isolation of compounds from ethyl acetate fraction of TP
After carrying out the column chromatography and following elution, four
compounds were isolated from TP and were named as EaTP-1, EaTP-2,
EaTP-3 and EaTP-4 respectively. Table No.6.6(a) explains the isolated
compounds and their Rf values.
Table No.6.6(a): Results showing isolated compounds and their Rf
values from ethyl acetate fraction of TP
Compounds separated
Rf Value Solvent system
Ethyl
acetate
fraction of
TP
EaTP-1 0.65 chloroform : methanol
4 : 1
EaTP-2 0.8 n-butanol : acetic acid : water
5 : 3 : 5
EaTP-3 0.51 pet.ether : ethyl acetate : acetic acid
10 : 91 : 4
EaTP-4 0.47 n-butanol : acetic acid : water
3 : 1 : 1
6.7.3(b) Isolation of compounds from aqueous fraction of SP
In the same way, two compounds were isolated from SP and were
named as ASP-1 and ASP-2. Table No.6.6(b) explains the isolated
compounds and their Rf values.
76
Table No.6.6(b): Results showing isolated compounds and their Rf
values from aqueous fraction of SP.
Compounds separated
Rf Value Solvent system
Aqueous
fraction of
SP
ASP-1 0.52 chloroform : methanol
4 : 1
ASP-2 0.59 toluene : ethyl acetate : formic acid
1.1 : 2.2 : 1.1
6.7.4 Characterization of isolated compounds from ethyl acetate fraction
of Thespesia populnea (TP)
6.7.4.1 Identification of EaTP-1 (110 mg)
Physical characters
Colour and appearance : White crystalline solid
Melting point : 178 – 1800C
Rf value : 0.65 [Chloroform (4) : water (1)]
Solubility : Soluble in water.
77
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) The alkaline solution of the sample in pyridine and sodium nitroprusside
solution gave red colour.
ii) Sample solution after hydrolysis gave a positive test for fehling’s solution.
Spectral Analysis
UV Spectrum
λmax (Methanol) : 258, 360 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3570 (O-H), 2927 (C-H vibrations), 1752 (C=O), 1540, 1498 (C=C),
1308 (C-O), 1260 (C-H bending), 796, 768, 722, 686 (aromatic C-H).
Figure No.6.5: IR spectrum of EaTP-1
78
1H NMR (Chloroform-d, 500 MHz)
8.08, 8.07, 8.07, 8.06, 8.06, 8.05, 8.05, 8.05, 7.58, 7.58, 7.57, 7.57,
7.56, 7.56, 7.56, 7.56, 7.55, 7.55, 7.54, 7.45, 7.44, 7.44, 7.43, 7.43, 7.42,
7.41, 7.41 CH-31, 41), 7.22, 7.21, 7.20, 7.20, 7.18, 6.99, 6.99, 6.98, 6.97, 6.86,
6.85, 6.84, 6.84, 6.83, 6.82 (aromatic C-H), 5.34, 5.32 (H of CH linked to
phenoxy group), 4.72, 4.72, 4.70, 4.70, 4.70, 4.68, 4.68, 4.64, 4.63, 4.61
(methanolic C-H), 4.41, 4.40, 4.39, 4.39, 4.38, 4.37, 4.36 (C-H in glucose
CH2OH), 4.08, 4.07, 4.06, 4.04, 4.01, 4.00, 3.99, 3.98 (C-H in pyranose ring
connected to CH2OH), 3.51, 3.49, 3.48, 3.44, 3.43, 3.41 (glucose C-H,
attached to OH groups), 2.11, 2.07, 1.96 (glucose O-H), 1.48, 1.46 (O-H in
CH2OH).
Figure No.6.6: 1H NMR spectrum of EaTP-1
79
13C NMR (C6D5N, 125 MHz)
167.23 (C-9), 157.31 (C-11), 133.35, 132.03, 130.96, 129.89, 129.77,
128.91, 123.39, 116.45 (aromatic carbon), 103.47 (C-6), 76.90 (C-4), 75.10
(C-5), 74.31 (C-2), 70.48 (C-3), 65.55 (C-7), 60.23 (C-71).
Figure No.6.7: 13C NMR spectrum of EaTP-1
LC-ESI-MS
m/z: 390 (90 %, M+), 286 (72 %, C13H18O7+), 180 (67 %, C6H12O6+),
162 (59 %, C6H9O5+), 124 (40 %, C7H8O2+).
80
Figure No.6.8: Mass spectrum of EaTP-1
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of populin, the compound isolated
is confirmed as the same.
OO
O
O
OH
OH
OH
OH
12
34
5
67
89
10
1112
13
1415
11
21
31
41
51
61
71
3,4,5-trihydroxy-6-[2-(hydroxymethyl)phenoxy]oxan-2-yl]methyl benzoate
(Populin)
81
6.7.4.2: Identification of EaTP-2 (133 mg)
Physical characters
Colour and appearance : Crystalline yellow powder
Melting point : 315 - 317 ºC
Rf value : 0.8 [n-butanol (5): acetic acid (3): water (5)]
Solubility : Soluble in acetone and alcohol.
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) To one ml of the extract, a few drops of dilute sodium hydroxide are
added. An intense yellow colour was produced in the plant extract,
which became colourless on addition of few drops of dilute acid.
ii) With amyl alcohol, yellow colour was produced.
iii) A little of the salt solution was treated with dilute HCl and a piece of
magnesium ribbon was added to it. A red colour was obtained.
82
Spectral Analysis
UV Spectrum
λmax (Methanol) : 255, 372 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3375 (O-H), 1619 (C=O), 1539 (C=C), 1451, 1338 (C-O), 1250 (C-H
bending), 1025 (symmetric C-O-C), 866, 766, 731, 701 (aromatic C-H).
Figure No.6.9: IR spectrum of EaTP-2
83
1H NMR (d6-acetone, 300 MHz)
8.30 (CH-7), 7.04, 7.03, 7.02, 7.02 (CH-21, 61), 6.77, 6.77, 6.76, 6.75
(CH-51), 6.56, 6.55 (CH-8), 6.34, 6.34 (CH-6), 6.16 (OH-3), 5.94 (OH-31), 5.24
(OH-41).
Figure No.6.10: 1H NMR spectrum of EaTP-2
84
13C NMR (d6-acetone, 300 MHz)
176.24 (C-4), 165.02 (C-7), 160.24 (C-5), 157.72 (C-9), 148.78 (C-41),
146.88 (C-2), 145.57 (C-31), 136.08 (C-3), 122.71 (C-11), 120.94 (C-61),
116.06 (C-21), 115.71 (C-51), 103.38 (C-10), 99.25 (C-6), 94.34 (C-8).
Figure No.6.11: 13C NMR spectrum of EaTP-2
85
LC-ESI-MS
m/z: 302 (97 %, M+), 301 (70 %, M-1), 286 (78 %, M-O), 273 (58 %, M-
CHO), 153 (56 %, M-C8H5O3), 69 (67 %, CH3-CH=CH-C=O).
Figure No.6.12: Mass spectrum of EaTP-2
λmax from UV spectrum indicated the presence of conjugation and two
chromophores which is a specific character of flavonoids. FT-IR spectra
resulted in the presence of functional groups hydroxyl (-OH) stretch, C-H
stretch of alkenes, C=O stretch for lactone and aromatic benzenoid ring. 1H
NMR and 13C NMR showed the aromatic proton and hydroxyl proton and
presence of 15 carbons in structure. The molecular weight of compound (m/z
302) was confirmed by mass spectrum. The data correlates the structure of
the isolated compound to phenyl propanoid flavanol.
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of quercetin, the compound
isolated is confirmed as the same.
86
O
OH
OH
OH
OH
OOH
12
34
56
78
9
10
112
1 31
41
51
61
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one
(Quercetin)
6.7.4.3 Identification of EaTP-3 (182 mg)
Physical characters
Colour and appearance : Yellow pigment
Melting point : 177 - 179 ºC
Rf value : 0.51 [petroleum ether (10): ethyl acetate (91) :
acetic acid (4)]
Solubility : Soluble in methanol, ethanol and insoluble in
water.
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) One drop of sample solution was spotted on a filter paper. A drop of
phosphomolybdic acid reagent was added to the spot and was exposed
to ammonia vapours. Blue colouration of the spot occurred.
ii) With ferric chloride solution gives blue colour.
87
Spectral Analysis
UV Spectrum
λmax (Methanol) : 385 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3421 (phenolic O-H), 2959, 2929 (C-H vibrations), 2608 (aldehyde C-
H), 1710, 1611, 1577 (aromatic C=C), 1440, 1379 (methyl bending vibrations).
Figure No.6.13: IR spectrum of EaTP-3
1H NMR (Chloroform-d, 500 MHz)
7.29 (CH-4, 7.18 (CH-41), 5.43 (OH-6, 61), 5.36 (OH-1, 11), 3.53, 3.51,
3.50, 3.49, 3.47 (CH-12 iso propyl), 3.33, 3.32, 3.30, 3.29, 3.27 (CH-121 iso
propyl), 2.55 (CH-15), 2.48 (CH-151), 1.51, 1.51, 1.50, 1.50, 1.45, 1.44 (CH-
13, 14, 131, 141).
88
Figure No.6.14: 1H NMR spectrum of EaTP-3
13C NMR (C6D5N, 125 MHz)
198.20 (C-11, 111), 154.80 (C-7, 71), 149.90 (C-1, 11), 141.90 (C-6,
61), 135.20 (C-5, 51), 133.10 (C-3, 31), 129.20 (C-10, 101), 123.36 (C-2, 21),
117.60 (C-4, 41), 114.10 (C-9, 91), 111.20 (C-8, 81), 27.55 (C-12, 121), 22.73
(C-13, 14), 21.93 (C-131, 141).
89
Figure No.6.15: 13C NMR spectrum of EaTP-3
LC-ESI-MS
m/z: 518 (83 %, M+), 500 (64 %, C30H30O-), 234 (97 %, C13H14O4+), 191
(68 %, C10H7O4+).
Figure No.6.16: Mass spectrum of EaTP-3
90
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of gossypol, the compound
isolated is confirmed as the same.
CH3 CH3
OH
OH
OOH
CH3
CH3
CH3 CH3
OH
OH
OOH
12
3
45
6
78
910
11
12
13 14
15
11
21
31 41 51
61
71
8191
101
111
121131
141
151
2,2′-bis-(Formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene)
(Gossypol)
6.7.4.4: Identification of EaTP-4 (240 mg)
Physical characters
Colour and appearance : Crystalline yellow powder
Melting point : 276 - 278 ºC
Rf value : 0.47 [n-butanol (3): acetic acid (1): water (1)]
Solubility : Soluble in hot ethanol and diethyl ether.
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) To one ml of the extract, a few drops of dilute sodium hydroxide are
added. An intense yellow colour was produced in the plant extract,
which became colourless on addition of few drops of dilute acid.
91
ii) With amyl alcohol, yellow colour was produced.
iii) A little of the salt solution was treated with dilute HCl and a piece of
magnesium ribbon was added to it. A red colour was obtained.
Spectral Analysis
UV Spectrum
λmax (Methanol) : 357, 255 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3401 (O-H stretching), 1635 (C=O stretch), 1510 (C=C), 1482, 1408 (C-
O), 1208 (C-H bending), 827, 756, 712 (aromatic C-H).
Figure No.6.17: IR spectrum of EaTP-4
1H NMR (Chloroform-d, 500 MHz)
9.95 (OH-41), 8.30 (OH-7), 7.47, 7.47, 7.46, 7.45, 7.45, 7.45 (CH-21,
61), 6.96, 6.95, 6.95, 6.94, 6.94, 6.93 (CH-31, 51), 6.59, 6.59 (CH-8), 6.31, 6.31
(CH-6), 6.22 (OH-3).
92
Figure No.6.18: 1H NMR spectrum of EaTP-4
13C NMR (Chloroform-d, 125 MHz)
176.78 (C-4), 165.02 (C-7), 160.24, 160.15 (C-5, 41), 157.89 (C-9),
147.04 (C-2), 136.30 (C-3), 129.96 (C-21, 61), 122.11 (C-11), 115.79 (C-31, 51),
103.38 (C-10), 99.25 (C-6), 94.34 (C-8).
93
Figure No.6.19: 13C NMR spectrum of EaTP-4
LC-ESI-MS
m/z: 287 (88 %, M+, C15H10O6+), 153 (49 %, M-C8H5O3), 93 (45 %,
C6H5O+), 69 (54 %, CH3-CH=CH-C=O).
94
Figure No.6.20: Mass spectrum of EaTP-4
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of kaempferol, the compound
isolated is confirmed as the same.
O
O
OH
OH
OH
OH
12
34
56
78
9
10
11
21
31
41
51
61
2-(4-hydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one
(Kaempferol)
95
6.7.5. Characterization of isolated compounds from aqueous fraction of
Strychnos potatorum (SP)
6.7.5.1 Identification of ASP-1 (280 mg)
Physical characters
Colour and appearance : Crystalline colourless powder
Melting point : 235 - 237 ºC
Rf value : 0.52 [Chloroform (4): methanol (1)]
Solubility : Soluble in methanol and distilled water.
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) The solution (methanol) of compound when treated with ferric chloride
solution gave a blue colour.
ii) Lead acetate solution when added to the salt solution gave a white bulky
precipitate.
iii) With 2% gelatine solution containing 10% NaCl it gave a white
precipitate.
Spectral Analysis
UV Spectrum
λmax (Methanol) : 272.8 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3410 (O-H stretch), 1665 (C=O), 1611 (Carboxylic O-H), 1561 (C=C),
1262 (C-O), 1013 (C-C).
96
Figure No.6.21: IR spectrum of ASP-1
1H NMR (Chloroform-d, 500 MHz)
7.08 (CH-2, 6, s), 5.93 (OH-4, s), 5.52 (OH-3, 5, s).
Figure No.6.22: 1H NMR spectrum of ASP-1
97
13C NMR (C6D5N, 125 MHz)
169.43 (C-7), 145.99 (C-3, 5), 139.46 (C-4), 121.65 (C-1), 110.00 (C-
2, 6).
Figure No.6.23: 13C NMR spectrum of ASP-1
98
LC-ESI-MS
m/z: 170 (98 %, M+, C6H6O5+), 168 (32 %, M+-1), 153 (84 %, M-OH),
136 (49 %, C7H5O3+), 125 (58 %, M+-COOH), 107 (38 %, C7H7O+).
Figure No.6.24: Mass spectrum of ASP-1
λmax from UV spectrum indicated the presence of conjugation and
chromophore. FT-IR spectra resulted in presence of functional groups
hydroxyl (-OH) stretch, C-H stretch of alkenes, C=O stretch for acid and
aromatic benzenoid ring. 1H NMR and 13C NMR showed the aromatic proton,
acidic proton and hydroxyl proton and presence of 7 carbons in structure. The
molecular weight of compound (m/e 170) was confirmed by mass spectrum.
The data correlates the structure of the isolated compound to phenolic acid.
99
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of gallic acid, the compound
isolated is confirmed as the same.
OH
OHOH
O OH
12
34
5
6
7
3,4,5-trihydroxybenzoic acid
(Gallic aid)
6.7.5.2 Identification of ASP-2 (230 mg)
Physical characters
Colour and appearance : Crystalline pale yellow powder
Melting point : 339 - 341 ºC
Rf value : 0.59 [toluene (1.1): ethyl acetate (2.2): formic
acid (1.1)]
Solubility : Slightly soluble in methanol, water and
soluble in pyridine.
Chemical tests
Chemical tests gave negative results for nitrogen and sulphur.
The positive results obtained were,
i) With lead acetate solution, the sample solution gives a flocculent white
precipitate.
ii) Sample solution with dilute ferric chloride solution gives a blue colour.
100
Spectral Analysis
UV Spectrum
λmax (Methanol) : 365, 254 nm.
FT-IR Spectrum (KBr, vmax, cm-1)
3557, 3472, 3095 (O-H stretching), 1699 (C=O), 1618 (O-H bending),
1581, 1510, 1446, 1396, 1331 (C=C), 1258, 1193 (C-O), 1109, 1052 (C-C),
920, 879, 830, 811, 776, 685, 638 (aromatic C-H).
Figure No.6.25: IR spectrum of ASP-2
101
1H NMR (Chloroform-d, 500 MHz)
7.61 (CH-3, 31), 6.02 (OH-4, 41), 5.93 (OH-5, 51).
Figure No.6.26: 1H NMR spectrum of ASP-2
102
13C NMR (C6D5N, 125 MHz)
159.72 (C-7, 71), 148.70 (C-4, 41), 140.82 (C-5, 51), 134.32 (C-6, 61),
112.35 (C-3, 31), 110.83 (C-2, 21), 108.64 (C-1, 11).
Figure No.6.27: 13C NMR spectrum of ASP-2
103
LC-ESI-MS
m/z: 301 (97 %, M(-), 257 (72 %, M+-H2O+O2), 229 (46 %, M+-CH3O4).
Figure No.6.28: Mass spectrum of ASP-2
The spectral data were compared with the standard data present in the
spectral library. As it was matching with that of ellagic acid, the compound
isolated is confirmed as the same.
O
O
O
O
OH
OH
OH
OH
2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione
(Ellagic acid)
Mass spectra have been co-related with the known compounds of NBS
inbuilt library of mass spectra at Regional research Laboratory,
104
Thiruvananthapuram and was identified. Moreover, the Co-TLC performed for
quercetin, gossypol, kaempferol and gallic acid confirmed the identified
compounds.
Figure No.6.28(a): Co-TLC for quercetin, gossypol, kaempferol and
gallic acid
EaTP-2 (Quercetin) Rf Value = 0.8
[n-butanol (5) : acetic acid (3): water (5)]
EaTP-3 (Gossypol) Rf Value = 0.51
[pet ether (10) : EtAc (91): acetic acid (4)]
EaTP-4 (Kaempferol) Rf Value = 0.47
[n-butanol (3) : acetic acid (1): water (1)]
ASP-1 (Gallic acid) Rf Value = 0.52
[chloroform (4) : methanol (1)]
Sample
Reference
Sample
Reference
105
6.8 Antioxidant Studies (in vitro)
6.8.1: Antioxidant studies of Ethyl Acetate fraction of TP
Using different in vitro methods, free radical scavenging activity was
analyzed and the percentage inhibition was calculated. Results are tabulated
in Table No.6.7 to Table No.6.11 and Figure No.6.29 to Figure No.6.33. The
methods used were DPPH free radical scavenging assay, nitric oxide radical
scavenging assay, super oxide radical scavenging assay, hydroxyl radical
scavenging assay and inhibition of peroxide formation method.
106
Table No.6.7: Results showing DPPH radical scavenging activity of ethyl
acetate extract of Thespesia populnea and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Thespesia populnea
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 26.87±0.54 20 25.46±0.65
40 41.98±1.07 40 42.80±1.19
60 51.42±0.77 60 57.92±1.51
80 55.38±0.42 80 68.87±0.99
100 61.56±0.60 100 75.77±0.88 IC50 for Thespesia populnea = 56 μg/ml
IC50 for Ascorbic acid = 48 μg/ml Values are Mean±SD (n=3)
Figure No.6.29: DPPH radical scavenging activity of ethyl acetate extract
of Thespesia populnea and Ascorbic acid
26.87
41.98
51.42 55.38
61.56
25.46
42.8
57.92
68.87 75.77
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
DPPH radical scavenging assay of ethyl acetate extract of Thespesia populnea and Ascorbic acid
Percentage Inhibition by Thespesia populnea
Percentage Inhibition by Ascorbic acid
107
Table No.6.8: Nitric oxide radical scavenging activity of ethyl acetate
extract of Thespesia populnea and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Thespesia populnea
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 19.84±1.47 20 24.78±1.35
40 39.5±0.75 40 39.95±1.41
60 54.37±0.71 60 57.52±0.13
80 65.84±1.10 80 71.18±0.26
100 71.06±0.35 100 77.69±1.29 IC50 for Thespesia populnea = 53 μg/ml
IC50 for Ascorbic acid = 51 μg/ml Values are Mean±SD (n=3)
Figure No.6.30: Nitric oxide radical scavenging activity of ethyl acetate
extract of Thespesia populnea and Ascorbic acid
19.84
39.5
54.37
65.84
71.06
24.78
39.95
57.52
71.18
77.69
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Nitric oxide radical scavenging assay of ethyl acetate extract of Thespesia populnea and Ascorbic acid
Percentage Inhibition by Thespesia populnea
Percentage Inhibition by Ascorbic acid
108
Table No.6.9: Results showing Super oxide radical scavenging activity of
ethyl acetate extract of Thespesia populnea and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Thespesia populnea
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 29.71±1.36 20 18.79±1.33
40 42.24±0.65 40 35.93±1.03
60 51.00±1.15 60 45.93±0.93
80 55.62±0.83 80 51.74±0.90
100 61.32±0.82 100 56.13±0.80
IC50 for Thespesia populnea = 58 μg/ml IC50 for Ascorbic acid = 73 μg/ml
Values are Mean±SD (n=3)
Figure No.6.31: Super oxide radical scavenging activity of ethyl acetate
extract of Thespesia populnea and Ascorbic acid
29.71
42.24
51 55.62
61.32
18.79
35.93
45.93 51.74
56.13
0
10
20
30
40
50
60
70
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Super oxide radical scavenging assay of ethyl acetate extract of Thespesia populnea and Ascorbic acid
Percentage Inhibition by Thespesia populnea
Percentage Inhibition by Ascorbic acid
109
Table No.6.10: Results showing Hydroxyl radical scavenging activity of
ethyl acetate extract of Thespesia populnea and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Thespesia populnea
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 10.44±0.67 20 24.23±0.68
40 27.68±0.96 40 44.35±1.31
60 39.66±0.75 60 58.05±0.88
80 50.96±1.00 80 71.27±0.70
100 59.2±1.33 100 78.55±1.17 IC50 for Thespesia populnea = 76 μg/ml
IC50 for Ascorbic acid = 44 μg/ml Values are Mean±SD (n=3)
Figure No.6.32: Results showing Hydroxyl radical scavenging activity of
ethyl acetate extract of Thespesia populnea and Ascorbic acid
10.44
27.68
39.66
50.96 59.2
24.23
44.35
58.05
71.27 78.55
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Hydroxyl radical scavenging assay of ethyl acetate extract of Thespesia populnea and Ascorbic acid
Percentage Inhibition by Thespesia populnea
Percentage Inhibition by Ascorbic acid
110
Table No.6.11: Results showing Inhibition of lipid peroxide formation of
ethyl acetate extract of Thespesia populnea and α-Tocopherol
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Thespesia populnea
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by α-
Tocopherol (Standard)
Control 0 Control 0
20 10.93±1.23 20 17.3±0.53
40 24.79±1.53 40 27.49±0.81
60 37.31±1.64 60 34.36±1.07
80 46.14±1.61 80 42.7±0.98
100 53.75±1.06 100 51.28±0.87 IC50 for Thespesia populnea = 90 μg/ml
IC50 for α-Tocopherol = 88 μg/ml Values are Mean±SD (n=3)
Figure No.6.33: Inhibition of lipid peroxide formation of ethyl acetate
extract of Thespesia populnea and α-Tocopherol
10.93
24.79
37.31
46.14
53.75
17.3
27.49 34.36
42.7
51.28
0
10
20
30
40
50
60
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Inhibition of lipid peroxide formation by ethyl acetate extract of Thespesia populnea and α-tocopherol
Percentage Inhibition by Thespesia populnea
Percentage Inhibition by α-tocopherol
111
6.8.2 Anti-oxidant studies of aqueous fraction of SP
In vitro anti-oxidant study for SP had been carried out using the same
methods as that of TP. Results are tabulated in Table No.6.12 to Table
No.6.16 and Figure No.6.34 to Figure No.6.38.
112
Table No.6.12: Results showing DPPH radical scavenging activity of
aqueous extract of Strychnos potatorum and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Strychnos potatorum
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 6.19±1.12 20 25.46±0.65
40 21.71±0.63 40 42.80±1.19
60 48.48±0.84 60 57.92±1.51
80 64.31±1.71 80 68.87±0.99
100 75.26±1.29 100 75.77±0.88 IC50 for Strychnos potatorum = 61 μg/ml
IC50 for Ascorbic acid = 48 μg/ml Values are Mean±SD (n=3)
Figure No.6.34: DPPH radical scavenging activity of aqueous extract of
Strychnos potatorum and Ascorbic acid
6.19
21.71
48.48
64.31 75.26
25.46
42.8
57.92
68.87 75.77
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
DPPH radical scavenging assay of aqueous extract of Strychnos potatorum and Ascorbic acid
Percentage Inhibition by Strychnos potatorum
Percentage Inhibition by Ascorbic acid
113
Table No.6.13: Results showing Nitric oxide radical scavenging activity
of aqueous extract of Strychnos potatorum and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Strychnos potatorum
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 22.18±1.46 20 24.78±1.35
40 30.77±1.54 40 39.95±1.41
60 47.76±0.85 60 57.52±0.13
80 61.21±1.64 80 71.18±0.26
100 71.29±0.51 100 77.69±1.29 IC50 for Strychnos potatorum = 63 μg/ml
IC50 for Ascorbic acid = 51 μg/ml Values are Mean±SD (n=3)
Figure No.6.35: Nitric oxide radical scavenging activity of aqueous
extract of Strychnos potatorum and Ascorbic acid
22.18 30.769
47.76
61.21
71.29
24.78
39.95
57.52
71.18 77.69
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Nitric oxide radical scavenging assay of aqueous extract of Strychnos potatorum and Ascorbic acid
Percentage Inhibition by Strychnos potatorum
Percentage Inhibition by Ascorbic acid
114
Table No.6.14: Results showing Super oxide radical scavenging activity
of aqueous extract of Strychnos potatorum and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Strychnos potatorum
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 17.46±1.28 20 18.79±1.33
40 35.22±1.54 40 35.93±1.03
60 54.91±1.08 60 45.93±0.93
80 63.89±1.55 80 51.74±0.90
100 73.18±1.54 100 56.13±0.80 IC50 for Strychnos potatorum = 54 μg/ml
IC50 for Ascorbic acid = 73 μg/ml Values are Mean±SD (n=3)
Figure No.6.36: Super oxide radical scavenging activity of aqueous
extract of Strychnos potatorum and Ascorbic acid
17.46
35.22
54.91
63.89
73.18
18.79
35.93
45.93 51.74
56.13
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Super oxide radical scavenging assay of aqueous extract of Strychnos potatorum and Ascorbic acid
Percentage Inhibition by Strychnos potatorum
Percentage Inhibition by Ascorbic acid
115
Table No.6.15: Results showing Hydroxyl radical scavenging activity of
aqueous extract of Strychnos potatorum and Ascorbic acid
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Strychnos potatorum
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by Ascorbic
acid (Standard)
Control 0 Control 0
20 10.25±0.83 20 24.23±0.68
40 23.18±1.39 40 44.35±1.31
60 39.85±0.86 60 58.05±0.88
80 64.94±1.63 80 71.27±0.70
100 68.87±1.78 100 78.55±1.17 IC50 for Strychnos potatorum = 68 μg/ml
IC50 for Ascorbic acid = 44 μg/ml Values are Mean±SD (n=3)
Figure No.6.37: Hydroxyl radical scavenging activity of aqueous extract
of Strychnos potatorum and Ascorbic acid
10.25
23.18
39.85
64.94 68.87
24.23
44.35
58.05
71.27
78.55
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Hydroxyl radical scavenging assay of aqueous extract of Strychnos potatorum and Ascorbic acid
Percentage Inhibition by Strychnos potatorum
Percentage Inhibition by Ascorbic acid
116
Table No.6.16: Results showing Inhibition of lipid peroxide formation of
aqueous extract of Strychnos potatorum and α-Tocopherol
Concentration of the extract
(μg/ml)
Percentage Inhibition
by Strychnos potatorum
extract
Concentration of the
standard (μg/ml)
Percentage Inhibition
by α-
Tocopherol (Standard)
Control 0 Control 0
20 21.35±0.51 20 17.3±0.53
40 32.89±0.51 40 27.49±0.81
60 50.32±1.75 60 34.36±1.07
80 55.22±1.37 80 42.7±0.98
100 63.2±1.13 100 51.28±0.87 IC50 for Strychnos potatorum = 60 μg/ml
IC50 for α-Tocopherol = 88 μg/ml Values are Mean±SD (n=3)
Figure No.6.38: Results showing Inhibition of lipid peroxide formation of
aqueous extract of Strychnos potatorum and α-Tocopherol
21.35
32.89
50.32 55.22
63.2
17.3
27.49 34.36
42.7
51.28
0
10
20
30
40
50
60
70
0 20 40 60 80 100 120
Perc
enta
ge In
hibi
tion
Concentration (μg/ml)
Inhibition of lipid peroxide formation by aqueous extract of Strychnos potatorum and α-tocopherol
Percentage Inhibition by Strychnos potatorum
Percentage Inhibition by α-tocopherol
117
6.9 In vitro antioxidant screening of isolated compounds of TP and SP
In vitro anti-oxidant screening for the isolated compounds had also been
carried out to find out their anti-oxidant potential. The same methods used for
‘bio-active fractions’ anti-oxidant screening was adopted here. The standard
used was curcumin which is a well-known natural anti-oxidant. The IC50 values
for each isolated compound have also been determined. The isolated
compounds from bio-active fraction of TP were populin, quercetin, gossypol
and kaempferol. The results are shown in Table No.6.17(i) to 6.21 (ii) and
Figure No.6.39 to 6.43. All were found to possess a significant
antioxidant potential.
Similarly the compounds isolated from SP were identified as gallic aid and
ellagic acid. These compounds also showed significant antioxidant potential.
Results are shown in Table No.6.22(i) to 6.26(ii) and Figure No.6.44 to 6.48.
118
Table No.6.17(i): Results showing DPPH free radical scavenging activity of isolated compounds from bio-active
fraction of TP
Populin Quercetin Gossypol Kaempferol
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 16.68±0.93 2 14.62±0.93 2 32.61±0.26 2 21.96±0.19 4 33.38±0.79 4 30.62±1.08 4 47.24±1.42 4 36.45±0.67 6 41.52±0.49 6 39.45±0.49 6 56.14±1.81 6 46.57±0.31 8 47.85±0.26 8 45.73±0.29 8 58.17±0.52 8 54.07±0.60 10 53.83±0.94 10 53.14±0.90 10 62.43±1.48 10 58.55±0.77
Values are Mean±SD (n=3)
Table No.6.17(ii): Results showing DPPH free radical scavenging activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 36.15±0.25 4 50.77±1.43 6 58.49±0.76 8 64.06±0.48 10 68.91±0.52 Values are Mean±SD (n=3)
119
Figure No.6.39: DPPH free radical scavenging activity of isolated compounds from TP
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Thespesia populnea by DPPH free radical scavenging method
Percentage inhibition by Populin
Percentage inhibition by Quercetin
Percentage inhibition by Gossypol
Percentage inhibition by Kaempferol
Percentage inhibition by Curcumin (Standard)
120
Table No.6.18(i): Results showing nitric oxide radical scavenging activity of isolated compounds from bio-active
fraction of TP
Populin Quercetin Gossypol Kaempferol
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 13.26±1.40 2 11.11±1.41 2 32.22±0.50 2 22.32±0.48 4 30.64±1.17 4 27.78±1.20 4 46.72±2.15 4 36.62±0.64 6 39.12±0.25 6 36.97±0.24 6 52.87±0.10 6 46.71±0.33 8 45.71±0.15 8 43.50±0.05 8 56.87±0.15 8 54.19±0.62 10 52.64±0.56 10 51.21±0.70 10 62.44±0.72 10 58.66±0.75
Values are Mean±SD (n=3)
Table No.6.18(ii): Results showing nitric oxide radical scavenging activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 25.64±0.77 4 40.92±0.66 6 49.57±0.92 8 56.34±0.62 10 62.24±0.62 Values are Mean±SD (n=3)
121
Figure No.6.40: Nitric oxide radical scavenging activity of isolated compounds from TP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Thespesia populnea by Nitric oxide radical scavenging method
Percentage inhibition by Populin
Percentage inhibition by Quercetin
Percentage inhibition by Gossypol
Percentage inhibition by Kaempferol
Percentage inhibition by Curcumin (Standard)
122
Table No.6.19 (i): Results showing super oxide radical scavenging activity of isolated compounds from bio-
active fraction of TP
Populin Quercetin Gossypol Kaempferol
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 9.23±1.40 2 13.73±0.72 2 34.57±0.46 2 18.72±0.60 4 27.41±1.16 4 24.42±1.38 4 46.48±0.48 4 33.68±0.75 6 36.29±0.27 6 34.04±0.26 6 50.69±0.19 6 44.30±0.37 8 43.19±0.04 8 45.38±0.12 8 54.87±0.23 8 52.06±0.75 10 50.44±0.57 10 51.20±0.82 10 60.70±0.86 10 56.74±0.67
Values are Mean±SD (n=3)
Table No.6.19 (ii): Results showing super oxide radical scavenging activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 23.03±0.22 4 38.17±0.76 6 47.23±0.82 8 54.53±0.72 10 60.48±0.73 Values are Mean±SD (n=3)
123
Figure No.6.41: Results showing super oxide radical scavenging activity of isolated compounds from TP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Thespesia populnea by Super oxide radical scavenging method
Percentage inhibition by Populin
Percentage inhibition by Quercetin
Percentage inhibition by Gossypol
Percentage inhibition by Kaempferol
Percentage inhibition by Curcumin (Standard)
124
Table No.6.20 (i): Results showing hydroxyl radical scavenging activity of isolated compounds from bio-active
fraction of TP
Populin Quercetin Gossypol Kaempferol
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 9.47±1.28 2 8.43±0.51 2 26.93±0.48 2 14.70±0.53 4 23.84±1.07 4 20.69±1.35 4 36.77±0.54 4 30.40±0.90 6 35.50±0.41 6 36.28±1.14 6 44.36±1.56 6 41.55±0.41 8 44.85±0.48 8 47.39±0.21 8 52.64±0.21 8 49.70±0.73 10 51.96±0.99 10 51.14±0.93 10 58.76±0.91 10 54.60±0.73
Values are Mean±SD (n=3)
Table No.6.20 (ii): Results showing hydroxyl radical scavenging activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 24.34±0.12 4 35.12±0.90 6 44.63±0.85 8 52.05±0.72 10 58.53±0.81 Values are Mean±SD (n=3)
125
Figure No.6.42: Results showing hydroxyl radical scavenging activity of isolated compounds from TP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Thespesia populnea by Hydroxyl radical scavenging method
Percentage inhibition by Populin
Percentage inhibition by Quercetin
Percentage inhibition by Gossypol
Percentage inhibition by Kaempferol
Percentage inhibition by Curcumin (Standard)
126
Table No.6.21 (i): Results showing inhibition of lipid peroxide formation by isolated compounds from bio-active
fraction of TP
Populin Quercetin Gossypol Kaempferol
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 25.07±1.05 2 28.00±0.55 2 35.02±0.99 2 32.42±0.65 4 39.57±0.70 4 39.40±0.77 4 48.86±0.90 4 45.75±0.56 6 47.33±0.41 6 48.52±0.26 6 59.25±0.14 6 53.97±0.11 8 52.84±0.61 8 54.86±0.27 8 62.71±0.10 8 60.38±0.37 10 54.73±0.83 10 59.54±0.42 10 67.52±0.51 10 64.25±0.81
Values are Mean±SD (n=3)
Table No.6.21 (ii): Results showing inhibition of lipid peroxide formation by curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 36.39±0.73 4 48.88±1.02 6 57.26±0.88 8 62.02±0.72 10 66.85±0.41 Values are Mean±SD (n=3)
127
Figure No.6.43: Results showing inhibition of lipid peroxide formation by isolated compounds from TP
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Thespesia populnea by Lipid peroxide formation inhibition method
Percentage inhibition by Populin
Percentage inhibition by Quercetin
Percentage inhibition by Gossypol
Percentage inhibition by Kaempferol
Percentage inhibition by Curcumin (Standard)
128
Table No.6.22(i): Results showing DPPH free radical scavenging activity
of isolated compounds from bio-active fraction of SP
Gallic acid Ellagic acid
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 22.23±0.30 2 19.07±1.16 4 40.04±1.74 4 35.01±1.14 6 49.44±0.94 6 44.41±0.31 8 56.22±0.59 8 51.91±0.60 10 62.13±0.62 10 57.83±0.61
Values are Mean±SD (n=3)
Table No.6.22(ii): Results showing DPPH free radical scavenging activity
of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 36.15±0.25 4 50.77±1.43 6 58.49±0.76 8 64.06±0.48 10 68.91±0.52 Values are Mean±SD (n=3)
129
Figure No.6.44: Results showing DPPH free radical scavenging activity
of isolated compounds from SP
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Strychnos potatorum by DPPH free radical scavenging method
Percentage inhibition by Gallic acid
Percentage inhibition by Ellagic acid
Percentage inhibition by Curcumin (Standard)
130
Table No. 6.23(i) Results showing nitric oxide radical scavenging activity
of isolated compounds from bio-active fraction of SP
Gallic acid Ellagic acid
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 22.44±0.36 2 18.14±0.38 4 40.20±1.69 4 35.19±1.10 6 48.86±0.32 6 44.92±0.81 8 56.31±0.57 8 52.04±0.64 10 62.17±0.62 10 60.81±0.75
Values are Mean±SD (n=3)
Table No.6.23(ii): Results showing nitric oxide radical scavenging
activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 25.64±0.77 4 40.92±0.66 6 49.57±0.92 8 56.34±0.62 10 62.24±0.62 Values are Mean±SD (n=3)
131
Figure No.6.45: Results showing nitric oxide radical scavenging activity
of isolated compounds from SP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Strychnos potatorum by Nitric oxide radical scavenging method
Percentage inhibition by Gallic acid
Percentage inhibition by Ellagic acid
Percentage inhibition by Curcumin (Standard)
132
Table No.6.24(i): Results showing super oxide radical scavenging
activity of isolated compounds from bio-active fraction of SP
Gallic acid Ellagic acid
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 18.84±0.54 2 14.34±0.57 4 37.42±1.72 4 32.18±0.99 6 46.48±0.48 6 41.99±0.50 8 54.31±0.73 8 49.81±0.76 10 60.11±0.36 10 58.99±0.69
Values are Mean±SD (n=3)
Table No.6.24(ii): Results showing superoxide radical scavenging
activity of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 23.03±0.22 4 38.17±0.76 6 47.23±0.82 8 54.53±0.72 10 60.48±0.73 Values are Mean±SD (n=3)
133
Figure No.6.46: Super oxide radical scavenging activity of isolated
compounds from SP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Strychnos potatorum by Super oxide radical scavenging method
Percentage inhibition by Gallic acid
Percentage inhibition by Ellagic acid
Percentage inhibition by Curcumin (Standard)
134
Table No.6.25(i): Results showing hydroxyl radical scavenging activity of
isolated compounds from bio-active fraction of SP
Gallic acid Ellagic acid
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 14.83±0.52 2 1011±0.54 4 34.34±1.93 4 29.68±0.55 6 43.84±0.52 6 39.12±0.54 8 52.05±0.71 8 47.34±0.74 10 58.46±0.82 10 56.96±0.75
Values are Mean±SD (n=3)
Table No.6.25(ii): Results showing hydroxyl radical scavenging activity
of curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 24.34±0.12 4 35.12±0.90 6 44.63±0.85 8 52.05±0.72 10 58.53±0.81 Values are Mean±SD (n=3)
135
Figure No.6.47: Hydroxyl radical scavenging activity of isolated compounds from SP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Strychnos potatorum by Hydroxyl radical scavenging method
Percentage inhibition by Gallic acid
Percentage inhibition by Ellagic acid
Percentage inhibition by Curcumin (Standard)
136
Table No.6.26(i): Results showing inhibition of lipid peroxide formation
by isolated compounds from bio-active fraction of SP
Gallic acid Ellagic acid
Concentration (μg/ml)
Percentage Inhibition
Concentration (μg/ml)
Percentage Inhibition
2 32.96±0.12 2 29.03±0.31 4 48.28±1.65 4 43.33±0.64 6 55.77±0.16 6 52.06±0.14 8 62.24±0.37 8 58.53±0.37 10 67.29±0.52 10 63.57±0.55
Values are Mean±SD (n=3)
Table No.6.26(ii): Results showing inhibition of lipid peroxide formation
by curcumin (standard)
Concentration (μg/ml) Percentage Inhibition 2 36.39±0.73 4 48.88±1.02 6 57.26±0.88 8 62.02±0.72 10 66.85±0.41 Values are Mean±SD (n=3)
137
Figure No.6.48: Inhibition of lipid peroxide formation by isolated compounds from TP
The minimum concentration required to scavenge 50% of free radicals
(IC50) was determined from the graphs and is tabulated in the Table No.6.27
Table No.6.27: Results showing IC50 values of isolated compounds from
TP and SP
Isolated compound
DPPH free radical
scavenging assay
Nitric oxide radical
scavenging assay
Superoxide radical
scavenging assay
Hydroxyl radical
scavenging assay
Inhibition of lipid
peroxide formation
Populin 8.6 9.2 9.9 7.4 6.9
Quercetin 9.2 9.6 9.6 8.7 7.0
Gossypol 4.6 5.2 5.8 6.6 4.6
Kaempferol 6.8 7.0 7.5 8.1 5.1
Gallic acid 6.2 6.5 6.9 7.5 4.7
Ellagic acid 7.4 7.2 8.1 8.6 5.5 Curcumin (Standard) 4.1 6.1 6.4 5.5 4.3
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12
Perc
enta
ge in
hibi
tion
Concentration (µg/ml)
Percentage inhibition by isolated compounds from Strychnos potatorum by Lipid peroxide formation inhibition method
Percentage inhibition by Gallic acid
Percentage inhibition by Ellagic acid
Percentage inhibition by Curcumin (Standard)
138
The IC50 values of isolated compounds were compared with that of the
standard. All the four compounds (populin, quercetin, gossypol and
kaempferol) isolated from Thespesia populnea and the two compounds (gallic
acid and ellagic acid) isolated from Strychnos potatorum showed significant
anti-oxidant activity.
In the case of Thespesia populnea, the isolated gossypol showed an
excellent anti-oxidant activity. The IC50 values for isolated gossypol and
standard curcumin stood nearby in all in vitro methods. Even, in nitric oxide
radical scavenging assay and in super oxide radical scavenging assay the
IC50 values found less than that of the standard drug (curcumin) used, which
explores the high anti-oxidant potential of the isolated gossypol.
Gallic acid isolated from Strychnos potatorum also showed IC50 values
close to the standard drug (curcumin) in all in vitro methods.
6.10 Experimental studies (Animal)
6.10.1 Animal maintenance and care
Animals were given proper care, food and water ad libitum as specified
by CPCSEA guidelines in our own animal hose.
6.10.2 Acute toxicity studies
Acute toxicity studies were carried out as per OECD guide lines
No.425.The maximum dose given was 2000 mg/Kg and no death was
reported
139
6.10.3 Dose response assay of most effective extract
In order to find out the dose required for imparting maximum
pharmacological effect, a dose response assay was carried out using 1/5th,
1/10th and 1/20th of the maximum dose given in the previous studies. From
these 1/10th and 1/20th dose were selected for the programmed studies.
6.10.4 Impairment of Liver function using CCl4.for antioxidant studies
Wistar rats of either sex were treated with CCl4 (0.5 ml/kg,
intraperitoneally), which impairs liver function. There was a variation in the
levels of MDA, GSH, SOD, GR, GPx and catalase. When the liver was
poisoned with CCl4, the MDA level was increased than in the normal liver. The
bioactive fractions of both plant materials were found to decrease the elevated
MDA level. GSH level was reduced in CCl4 treated animals. After treating the
animals with bioactive fractions of both plant materials, there was a
considerable increase in the GSH level. The decreased levels of anti-oxidant
enzymes in CCl4 treated animals also were found to be increased by the
bioactive fractions. The results are tabulated in Table No.6.28 to Table
No.6.31a.
A comparative graphical study was carried out to analyse the variation
in the levels of MDA, GSH, SOD, GR, GPx and catalase present in normal
liver, CCl4 treated liver and the liver treated with CCl4 & bioactive fractions in
different concentrations (200 mg/kg and 400 mg/kg, orally). (Figure No.6.49 to
Figure No.6.60)
140
Table No.6.28: Effect of ethyl acetate extract of TP on the activity of
Malondialdehyde, Glutathione and Superoxide dismutase in CCl4 treated
Wistar rats
Groups Malondialdehyde
(MDA) (nmols / g wet
tissue)
Glutathione (GSH)
(mg / g)
Superoxide dismutase(SOD)
(Units / mg)
I 0.75±0.16a 3.79±1.14a 7.39±0.93d II 3.11±0.62c 2.97±0.41a 2.46±0.73a III 1.28±0.46b *** 3.18±0.91a 4.41±0.93b * IV 1.17±0.16ab *** 3.75±0.93a 6.27±0.57c ***
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl
acetate extract (400 mg / kg.). Means in the same column scored by the same alphabet are not significantly different at 1%
level. MDA- ***p <0.001 compared to group II ;
GSH- Comparison between treatments are non-significant (p > 0.05). SOD- *p<0.05 compared to group II ; ***p<0.001 compared to group II
Values are Mean±SD (n=6)
Table No.6.28(a): Statistical analysis (One-Way ANOVA) of data in Table
No.6.28
Groups compared
F value (2, 15) MDA GSH SOD
I, III & IV 5.52 0.71 19.84 II, III & IV 34.26 1.57 38.19
141
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.49: Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of
Malondialdehyde in CCl4 treated Wistar rats
0
0.5
1
1.5
2
2.5
3
3.5
0.75
1.28
0.75 1.17
nmol
s / g
Group I & III Group I & IV
Group comparitive evaluation of the amount of malondialdehyde by ethyl acetate extract of Thespesia
populnea in liver homogenate
I
III
IV
0
0.5
1
1.5
2
2.5
3
3.5 3.11
1.28
3.11
1.17
nmol
/ g
Group II & III Group II & IV
Group comparitive evaluation of the amount of malondialdehyde by ethyl acetate extract of Thespesia
populnea in liver homogenate
II
III
IV
142
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.50: Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of
Glutathione in CCl4 treated Wistar rats
0
1
2
3
4
3.79
3.18
3.79 3.75
mg
/ g
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione by ethyl acetate extract of Thespesia populnea in liver
homogenate
I
III
IV
0
1
2
3
4
2.97 3.18
2.97
3.75
mg
/ g
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione by ethyl acetate extract of Thespesia populnea in liver
homogenate
II
III
IV
143
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.51: Results showing effect of ethyl acetate extract of TP on the activity of Superoxide dismutase in
CCl4 treated Wistar rats
0
1
2
3
4
5
6
7
8 7.39
4.41
7.39
6.27
units
/ m
g
Group I & III Group I & IV
Group comparitive evaluation of the amount of Super oxide dismutase by ethyl acetate extract of Thespesia populnea
in liver homogenate
I
III
IV
0
1
2
3
4
5
6
7
8
2.46
4.41
2.46
6.27
units
/ m
g
Group II & III Group II & IV
Group comparitive evaluation of the amount of Super oxide dismutase by ethyl acetate extract of Thespesia populnea
in liver homogenate
II
III
IV
144
Table No.6.29: Effect of ethyl acetate extract of TP on the activity of
Glutathione reductase, Glutathione peroxidase and Catalase in CCl4
treated Wistar rats
Groups
Glutathione reductase(GR)
(nmol of NADPH oxidized / min /
mg protein)
Glutathione peroxidase(GPx) (nmol of NADPH oxidized / min /
mg protein)
Catalase (moles of H2O2 decomposed /
min / mg protein)
I 59.20±1.45d 187.35±1.18d 69.22±1.09d
II 43.84±1.86a 96.76±0.98a 38.32±1.08a
III 51.11±1.80b *** 118.30±1.75b *** 60.63±0.99b ***
IV 54.98±1.11c *** 125.95±1.42c *** 63.36±1.70c ***
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl
acetate extract (400 mg / kg.). Means in the same column scored by the same alphabet are not significantly different at 1%
level. ***p<0.001 compared to group II in GR, GPx and Catalase.
Values are Mean±SD (n=6) Table No.6.29(a): Statistical analysis (One-Way ANOVA) of data in Table
No.6.29
Groups compared
F value (2, 15)
GR GPx Catalase
I, III & IV 44.78 3986.75 68.07
II, III & IV 72.80 683.75 672.12
145
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200
mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.52: Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of
Glutathione reductase in CCl4 treated Wistar rats
0
10
20
30
40
50
60
59.2
51.11
59.2 54.98
nmol
sof N
ADPH
oxi
dize
d / m
in /
mg
prot
ein
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione reductase by ethyl acetate extract of Thespesia populnea in liver homogenate
I
III
IV
0
10
20
30
40
50
60
43.84
51.11
43.84
54.98
nmol
sof N
ADPH
oxi
dize
d / m
in /
mg
prot
ein
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione reductase by ethyl acetate extract of Thespesia populnea in liver homogenate
II
III
IV
146
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200 mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.53: Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of
Glutathione peroxidase in CCl4 treated Wistar rats
0
50
100
150
200 187.35
118.3
187.35
125.95
nmol
sof N
AD
PH o
xidi
zed
/ min
/ m
g pr
otei
n
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione peroxidase by ethyl acetate extract of Thespesia populnea
in liver homogenate
I
III
IV
0
50
100
150
200
96.76
118.3
96.76
125.95
nmol
sof N
ADPH
oxi
dize
d / m
in /
mg
prot
ein
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione peroxidase by ethyl acetate extract of Thespesia populnea
in liver homogenate
II
III
IV
147
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Thespesia populnea ethyl acetate extract (200
mg / kg.); Group IV – CCl4 + Thespesia populnea ethyl acetate extract (400 mg / kg.).
Figure No.6.54: Results showing effect of ethyl acetate extract of Thespesia populnea on the activity of Catalase
in CCl4 treated Wistar rats
0
10
20
30
40
50
60
70
69.22
60.63
69.22 63.36
mol
es o
f H2O
2 dec
ompo
sed
/ min
/ m
g pr
otei
n
Group I & III Group I & IV
Group comparitive evaluation of the amount of Catalase by ethyl acetate extract of Thespesia populnea in liver homogenate
I
III
IV
0
10
20
30
40
50
60
70
38.32
60.63
38.32
63.36
mol
es o
f H2O
2 dec
ompo
sed
/ min
/ m
g pr
otei
n
Group II & III Group II & IV
Group comparitive evaluation of the amount of Catalase by ethyl acetate extract of Thespesia populnea in liver homogenate
II
III
IV
148
Table No.6.30: Effect of aqueous extract of Strychnos potatorum on the
activity of Malondialdehyde, Glutathione and Superoxide dismutase in
liver homogenate of CCl4 treated Wistar rats
Groups Malondialdehyde
(MDA) (nmols / g wet
tissue)
Glutathione (GSH)
(mg / g)
Superoxide dismutase(SOD)
(Units / mg)
I 0.75±0.16a 3.79±1.14a 7.39±0.93c
II 3.11±0.62b 2.97±0.41a 2.46±0.73a
III 1.27±0.28a *** 3.38±0.72a 4.55±0.92b *
IV 1.07±0.48a *** 3.84±0.66a 5.60±1.19b ***
Group I – Control; Group II – CCl4 treated (0.5 ml / kg body wt.); Group III – CCl4 +
Strychnos potatorum extract (200 mg / kg body wt.); Group IV – CCl4 + Strychnos potatorum extract (400 mg / kg body wt.).
Means in the same column scored by the same alphabet are not significantly different at 1%
level. MDA- ***p<0.001 compared to group II ;
GSH- Comparison between treatments are non-significant (p > 0.05). SOD- *p<0.05 compared to group II ; ***p<0.001 compared to group II
Values are Mean±SD (n=6)
Table No.6.30a: Statistical analysis (One-Way ANOVA) of data in Table
No.6.30
Groups compared
F value (2, 15)
MDA GSH SOD
I, III & IV 3.88 0.51 11.80
II, III & IV 33.22 3.05 16.57
149
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract (400 mg / kg.).
Figure No.6.55: Results showing effect of aqueous extract of Strychnos potatorum on the activity of
Malondialdehyde in CCl4 treated Wistar rats
0
1
2
3
4
0.75 1.27
0.75 1.07
nmol
s / g
Group I & III Group I & IV
Group comparitive evaluation of the amount of Malondialdehyde by aqueous extract of Strychnos
potatorum in liver homogenate
I
III
IV
0
1
2
3
4 3.11
1.27
3.11
1.07
nmol
s / g
Group II & III Group II & IV
Group comparitive evaluation of the amount of Malondialdehyde by aqueous extract of Strychnos
potatorum in liver homogenate
II
III
IV
150
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract(400 mg / kg.).
Figure No.6.56: Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione
in CCl4 treated Wistar rats
0
1
2
3
4
3.79 3.38
3.79 3.84
mg
/ g
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione by aqueous extract of Strychnos potatorum in liver
homogenate
I
III
IV
0
1
2
3
4 2.97
3.38 2.97
3.84
mg
/ g
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione by aqueous extract of Strychnos potatorum in liver
homogenate
II
III
IV
151
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract(400 mg / kg.).
Figure No.6.57: Results showing effect of aqueous extract of Strychnos potatorum on the activity of Superoxide
dismutase in CCl4 treated Wistar rats
0
2
4
6
8 7.39
4.55
7.39
5.6
units
/ m
g
Group I & III Group I & IV
Group comparitive evaluation of the amount of Super oxide dismutase by aqueous extract of Strychnos potatorum in
liver homogenate
I
III
IV
0
1
2
3
4
5
6
7
8
2.46
4.55
2.46
5.6
units
/ m
g
Group II & III Group II & IV
Group comparitive evaluation of the amount of Super oxide dismutase by aqueous extract of Strychnos potatorum in
liver homogenate
II
III
IV
152
Table No.6.31 Effect of aqueous extract of Strychnos potatorum on the
activity of Glutathione reductase, Glutathione peroxidase and Catalase
in CCl4 treated Wistar rats
Groups
Glutathione reductase(GR)
(nmol of NADPH oxidized / min /
mg protein)
Glutathione peroxidase(GPx) (nmol of NADPH oxidized / min /
mg protein)
Catalase (moles of H2O2 decomposed /
min / mg protein)
I 59.20±1.45d 187.35±1.18d 69.22±1.09d
II 43.84±1.86a 96.76±0.98a 38.32±1.08a
III 51.76±1.70b *** 121.32±1.18b *** 59.93±1.17b ***
IV 56.29±1.57c *** 147.58±1.08c *** 62.47±1.83c ***
Group I – Control; Group II – CCl4 treated (0.5 ml / kg body wt.); Group III – CCl4 +
Strychnos potatorum extract (200 mg / kg body wt.); Group IV – CCl4 + Strychnos potatorum extract (400 mg / kg body wt.).
Means in the same column scored by the same alphabet are not significantly different at 1%
level. ***p<0.001 compared to group II in GR, GPx and Catalase.
Values are Mean±SD (n=6)
Table No.6.31a: Statistical analysis (One-Way ANOVA) of data in Table No.6.31
Groups compared
F value (2, 15)
GR GPx Catalase
I, III & IV 33.88 5013.36 70.15
II, III & IV 81.11 3292.56 538.74
153
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract(400 mg / kg.).
Figure No.6.58: Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione
reductase in CCl4 treated Wistar rats
0
10
20
30
40
50
60
59.2 51.76
59.2 56.29
nmol
sof N
AD
PH
oxi
dize
d / m
in /
mg
prot
ein
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione reductase by aqueous extract of Strychnos potatorum in
liver homogenate
I
III
IV
0
10
20
30
40
50
60 43.84
51.76 43.84
56.29
nmol
sof N
AD
PH
oxi
dize
d / m
in /
mg
prot
ein
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione reductase by aqueous extract of Strychnos potatorum in
liver homogenate
II
III
IV
154
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract(400 mg / kg.).
Figure No.6.59: Results showing effect of aqueous extract of Strychnos potatorum on the activity of Glutathione
peroxidase in CCl4 treated Wistar rats
0
20
40
60
80
100
120
140
160
180
200 187.35
121.32
187.35
147.58
nmol
sof N
ADPH
oxi
dize
d / m
in /
mg
prot
ein
Group I & III Group I & IV
Group comparitive evaluation of the amount of Glutathione peroxidase by aqueous extract of Strychnos potatorum in
liver homogenate
I
III
IV
0
50
100
150
200
96.76
121.32
96.76
147.58
nmol
sof N
ADPH
oxi
dize
d / m
in /
mg
prot
ein
Group II & III Group II & IV
Group comparitive evaluation of the amount of Glutathione peroxidase by aqueous extract of Strychnos potatorum in
liver homogenate
II
III
IV
155
Group I– Control; Group II – CCl4 treated (0.5 ml / kg.); Group III – CCl4 + Strychnos potatorum aqueous extract (200 mg
/ kg.); Group IV – CCl4 + Strychnos potatorum aqueous extract(400 mg / kg.).
Figure No.6.60: Results showing effect of aqueous extract of Strychnos potatorum on the activity of Catalase in
CCl4 treated Wistar rats
0
10
20
30
40
50
60
70
69.22
59.93
69.22 62.47
mol
es o
f H2O
2 dec
ompo
sed
/ min
/ m
g pr
otei
n
Group I & III Group I & IV
Group comparitive evaluation of the amount of Catalase by aqueous extract of Strychnos potatorum in liver homogenate
I
III
IV
0
10
20
30
40
50
60
70
38.32
59.93
38.32
62.47
mol
es o
f H2O
2 dec
ompo
sed
/ min
/ m
g pr
otei
n
Group II & III Group II & IV
Group comparitive evaluation of the amount of Catalase by aqueous extract of Strychnos potatorum in liver homogenate
II
III
IV
156
6.11 Anti-inflammatory screening of the extracts by carrageenan induced
paw oedema in Albino rats.
Ethyl acetate fraction of Thespesia populnea and aqueous fraction of
Strychnos potatorum was screened for acute anti-inflammatory activity by
carrageenan induced paw oedema in Albino rats. Both fractions showed good
anti-inflammatory activity. The percentage inhibition of oedema produced by
carrageenan by both fractions at two different concentrations (50 mg/kg and
100 mg/kg) was compared with the standard drug, indomethacin (10 mg/kg).
After 3 hr. itself, both extracts have shown anti-inflammatory effect. At 5th hr.
anti-inflammatory effect by them was maximum and at 7th hr. onwards the
effect was found to be declining.
The anti-inflammatory effect by Thespesia populnea extract was found
to be more than the Strychnos potatorum extract. Both extracts at 100 mg/kg
dose was found to possess more activity than the standard drug. The results
are shown in Table No.6.32 and in Figure No.6.61.
157
Table No.6.32: Effect of ethyl acetate extract of Thespesia populnea and
aqueous extract of Strychnos potatorum on plantar oedema in Albino
rats.
Treatment/Dose (mg/kg)
Percentage inhibition of oedema produced 3 h 5 h 7 h
Group I (Control) 0 0 0 Group II (Standard) 44.59 67.81 65.34 Group III (TP-50) 47.62** 65.22** 50** Group IV (TP-100) 71.43** 82.61** 72.22** Group V (SP-50) 42.86** 65.21** 61.11** Group VI (SP-100) 61.9** 69.57** 66.67**
Group I – Control (Carrageenan, 0.05 ml only); Group II – Standard (Carrageenan, 0.05 ml + Indomethacin, 10 mg/kg); Group III – TP-50 (Carrageenan, 0.05 ml + ethyl acetate fraction
of Thespesia populnea, 50 mg/kg); Group IV – TP-100 (Carrageenan, 0.05 ml + ethyl acetate fraction of Thespesia populnea, 100 mg/kg); Group V – SP-50 (Carrageenan, 0.05
ml + aqueous fraction of Strychnos potatorum, 50 mg/kg); Group VI – SP-100 (Carrageenan, 0.05 ml + aqueous fraction of Strychnos potatorum, 100 mg/kg)
**p<0.01 compared to group II (standard). Values are Mean±SD (n=6)
Figure No.6.61: Effect of ethyl acetate extract of Thespesia populnea and
aqueous extract of Strychnos potatorum on plantar oedema in Albino
rats
0 20 40 60 80
100
Per
cent
age
Inhi
bitio
n
Treatment
Percentage inhibition by ethyl acetate fraction of Thespesia populnea and aqueous fraction of Strychnos potatorum up on Carrageenan induced paw oedema in
Albino rats
Percentage Inhibition after 3 hr
Percentage Inhibition after 5 hr
Percentage Inhibition after 7 hr
158
Chapter 7: CONCLUSIONS
159
7 CONCLUSIONS
Indigenous drugs are used widely for the treatment of many diseases.
Many of the herbal drugs are said to be rich sources of antioxidants. It has
been proved that without continuous supply of antioxidants that can scavenge
oxygen radicals, survival of aerobic living beings would be impossible. Clinical
and epidemiological studies have conclusively indicated that nutrients with
antioxidant activity are effective in the prevention of diseases. Molecular and
cellular approaches have demonstrated that ROS and antioxidants can
directly affect the cellular signalling apparatus and consequently the control of
gene expression. This provides a link between ROS and antioxidants in the
mechanism of disease processes and prevention.
A wide range of antioxidants, both natural and synthetic has been
proposed for use in the prevention and treatment of human diseases where
the role of oxygen free radicals has been implicated. Number of useful
antioxidants is rather limited and discovery of new antioxidants will be highly
valued in this context. The present study thus was an attempt to identify the
bioactive fractions of the plants Thespesia populnea (leaves) and Strychnos
potatorum (seeds) with a view of understanding its anti-oxidant activity, both in
vitro and in vivo. The isolation of the chemical constituents from the bioactive
fractions of both plant parts and their in vitro anti-oxidant screening has also
been carried out. The in vivo anti-oxidant study of the bioactive fractions from
the selected plant materials was done in albino rats.
160
The quantitative analysis of the plant materials were carried out
according to the procedure in Indian Pharmacopoeia (IP). The total ash value,
sulphated ash value, water soluble ash value and acid insoluble ash value for
both plant materials were determined (Table 6.1). The alcohol extractive value
and water extractive values were also determined as specified in IP (Table
6.2).
The methanolic extracts of both plant materials were prepared by cold
maceration procedure. Both plant extracts were brown gummy in nature. The
methanolic extract of leaves of Thespesia populnea yield 20.46 %w/w and that
of seeds of Strychnos potatorum yield 11.29 %w/w (Table 6.3).
The preliminary phytochemical screening of the methanolic extract of
leaves of Thespesia populnea and seeds of Strychnos potatorum showed the
presence of carbohydrates, phenolic compounds, flavonoids, alkaloids,
terpenoids and steroids. Seeds of Strychnos potatorum have also shown the
presence of glycosides and saponins which was found to be absent in
Thespesia populnea leaves (Table 6.4).
The methanolic extracts were defatted with petroleum ether and then
partitioned with solvents like chloroform, ethyl acetate, acetone and water.
The TLC study and preliminary in vitro anti-oxidant study by DPPH free radical
scavenging assay of these fractions were carried out to identify the bioactive
fractions. Ethyl acetate fraction of Thespesia populnea and aqueous fraction
of Strychnos potatorum were selected as the bioactive fractions. Further
studies were concentrated on these fractions only.
161
As a next step to isolate the chemical constituents present in the ethyl
acetate fraction of TP and aqueous fraction of SP, column chromatography
was carried out. After doing the column chromatography, four compounds
were isolated from TP and two compounds from SP.
The compounds isolated from ethyl acetate fraction of TP were,
1. EaTP-1 - Populin
2. EaTP-2 - Quercetin
3. EaTP-3 - Gossypol and
4. EaTP-4 - Kaempferol
The compounds isolated from aqueous fraction of SP include,
1. ASP-1 - Gallic acid
2. ASP-2 - Ellagic acid
The data which was obtained in the LCMS of the bio-active fractions
were compared with the LCMS data of the isolated compounds from the same
fractions. The molecular mass of the isolated compounds were found to match
with the mass peak of the bio-active fraction.
In vitro anti-oxidant study was conducted for the bioactive fractions as
well as for the isolated compounds. The methods used were DPPH free
radical scavenging assay, nitric oxide radical scavenging assay, super oxide
radical scavenging assay, hydroxyl radical scavenging assay and inhibition of
lipid peroxide formation. The ethyl acetate fraction of TP and aqueous fraction
of SP have shown a good in vitro anti-oxidant activity. The activity was
162
compared with a standard drug. Ascorbic acid was used as the standard drug
for all in vitro methods except for inhibition of lipid peroxide formation method
(α- tocopherol). From the IC50 values it was understood that the ethyl acetate
fraction of TP was having higher anti-oxidant property than aqueous fraction of
SP.
In vitro antioxidant screening for the isolated compounds from ethyl
acetate fraction of TP and aqueous fraction of SP was also carried out. The
IC50 values were calculated. In the case of TP, the isolated gossypol showed
an excellent anti-oxidant activity (IC50 values for gossypol - 4.6 μg/ml and that
of the standard, curcumin – 4.1 μg/ml). Gallic acid isolated from SP was found
to be more active than the isolated ellagic acid. (Table 6.27)
In vivo anti-oxidant studies of the bioactive fractions from both plant
materials were mainly based up on the anti-oxidant enzymes study (the
activity of anti-oxidant enzymes such as super oxide dismutase, glutathione
reductase, glutathione peroxidase and catalase were estimated). The Wistar
rats were used for the in vivo studies. After conducting the acute toxicity study,
the animals were treated with CCl4, which causes impaired hepatic function.
Under this condition, the superoxide dismutase (SOD), glutathione reductase
(GR), glutathione peroxidase (GPx), catalase and reduced glutathione (GSH)
contents will be reduced in the liver. The plant extracts (bioactive fractions)
were found to increase the level of the same. According to the lipid
peroxidation hypothesis, CCl4 poisoning initiates an intrahepatic process of
destructive lipid peroxidation. Therefore, malondialdehyde (MDA), one of the
163
products of lipid peroxidation was found to have increased level after CCl4
poisoning of liver of rats. After treating with plant extracts, the MDA generation
was decreased. All these reveals the antioxidant potential of the ethyl acetate
fraction of TP and aqueous fraction of SP.
The in vitro and in vivo studies of the selected plant materials reveal the
anti-oxidant potential of ethyl acetate fraction of TP and aqueous fraction of
SP, which is due to the presence of active constituents like polyphenols,
flavonoids etc. present in them. Since the above said plant constituents
causes anti-inflammatory activity also, an acute anti-inflammatory study was
also conducted to confirm the activity of the isolated compounds and thereby
their anti-oxidant potential95,96,97. Bioactive fractions from both TP and SP
showed a good anti-inflammatory activity towards plantar paw oedema
method in rats.
The observed effects of TP and SP lead to the conclusion that ethyl
acetate fraction of Thespesia populnea (leaves) and aqueous fraction of
Strychnos potatorum (seeds) possess anti-oxidant potential and anti-
inflammatory activity due to the presence of the phytoconstituents, either
alone or in a synergistic manner.
164
Chapter 8: REFERENCES
165
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179
Chapter 9: ANNEXURE
180
9 ANNEXURE
9.1 Annexure 1: List of Materials
Sl. No. List of Materials Source
1 Nitroblue Tetrazolium (NBT) Sisco Research Lab Pvt. Ltd. Mumbai
2 Reduced glutathione (GSH) Sisco Research Lab Pvt. Ltd. Mumbai
3 Glutathione reductase (GR) Sisco Research Lab Pvt. Ltd. Mumbai
4 Thiobarbituric acid E-merck
5 Carbon tetrachloride (CCl4) E-merck
6 Indomethacin Sigma Aldrich
7 1,1-diphenyl-2-picrylhydrazyl (DPPH) Sigma Aldrich
8 Sodium nitroprusside Sigma Aldrich
9 Griess reagent Sigma Aldrich
10 Naphthylethylenediamine Sigma Fluka
11 Riboflavin Sigma Fluka
12 Tris-HCl buffer Sigma Fluka
13 Phenazine methosulphate (PMS) CDH Chemicals, New Delhi
14 NADH CDH Chemicals, New Delhi
15 5,5 dithiobis-2-nitrobenzoic acid (DTNB) CDH Chemicals, New Delhi
All the chemicals used were of Analytical grade. Other chemicals
were purchased from Nice Chemicals, Kochi.
181
9.2 Annexure 2: List of Equipment
Sl. No. List of Equipment Manufacturer
1 UV-Visible spectrophotometer Shimadzu Analytical (India) Pvt. Ltd
2 FTIR spectrometer Shimadzu Analytical (India) Pvt. Ltd
3 NMR spectrometer Bruker, USA
4 Liquid chromatography-Mass spectrometer Finnigan Matt, Germany.
5 Macerator Perfit, India
6 Pulverisor Khalsa Foundary Works Ltd., India
7 Soxhlet Apparatus Perfit, India
182
9.3 Annexure 3(i): Published Journal copy
183
9.3 Annexure 3(ii): Published Journal copy
