“EVALUATION OF PROTECTIVE EFFECT OF HERBAL
CONSTITUENTS ON NEPHROTOXICITY”
Thesis submitted in partial fulfillment
for the award of
Doctor of Philosophy in Pharmaceutical Sciences
by
Ansa Mathew
(Reg. No. PHAR 2009 JA 155)
VINAYAKA MISSIONS UNIVERSITY
SALEM, TAMILNADU, INDIA
JUNE 2015
VINAYAKA MISSIONS UNIVERSITY, SALEM
CERTIFICATE BY THE GUIDE
I, Prof. Dr. B. Jaykar, certify that the thesis entitled “Evaluation of
Protective Effect of Herbal Constituents on Nephrotoxicity” submitted
for the degree of Doctor of Philosophy by Mrs. Ansa Mathew, is the record
of research work carried out by her during the period from January, 2009
to October, 2014 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: Salem Signature of the Supervisor with designation
Date:
Dr. B. Jaykar, M. Pharm, Ph.D.,
Dean, Faculty of Pharmacy
Vinayaka Missions College of Pharmacy
Yercaud Main Road
Kondappanaickenpattty
Salem, Tamil Nadu – 636 008
VINAYAKA MISSIONS UNIVERSITY, SALEM
DECLARATION
I, Ansa Mathew, declare that the thesis entitled “Evaluation of
Protective Effect of Herbal Constituents on Nephrotoxicity” submitted
by me for the award of Degree of Doctor of Philosophy is the record work
carried out by me during the period from January, 2009 to October, 2014
under the guidance of Dr. B. Jaykar 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 Institution of higher learning.
Place: Kannur Signature of the candidate
Date: Ansa Mathew Asst. Professor Crescent College of Pharmaceutical
Sciences, Payangadi R S (P.O),
Kannur, Kerala.
DEDICATED
TO
MY FAMILY, PARENTS AND GUIDE
ACKNOWLEDGEMENT
I extend my sincere and heartfelt gratitude to my esteemed research
guide, who is the backbone of my research Dr. B. Jaykar, Dean, Faculty of
Pharmacy, Vinayaka Missions College of Pharmacy, Salem. His excellent
suggestions, invaluable guidance and constant encouragement with
personal care throughout my research work were unforgettable.
I pay my profound gratefulness and indebtedness to Dr. K.
Rajendran, Dean - Research of Vinayaka Missions University, Salem for
his timely support.
Humble and sincere thanks to Dr. Madhu C Diwakar, Pharmacist,
Oman for early guidance in my research work.
I am very thankful to the Management & Principal Prof. Suja C.
Jayan of Crescent College of Pharmaceutical Sciences, Kannur for
providing necessary facilities required for my research work.
I specially thank Sathya B. of Government Ayurveda Medical
College, Pariyaram for giving more information regarding the research
work.
I would like to thank the Dr. M. Majeed, Managing Director and
Staffs of M/s Sami Labs, Bangalore for helping me to carry out the
analytical studies.
I would like to dedicate this work with my heartfelt gratitude to my
parents and family for their love, and support they showed towards me.
My special thanks to Dr. Suresh Kumar, Dr. Siva Kumar, and Dr.
Manoj K, for helping me to complete the work in time.
I would like to express my deep sense of love and affection to my
colleagues Dr. Sujith S Nair, Dr. Sreena K., Dr. Prasobh G R, Saritha M,
Sreeraj K, Sai Sabari, Sunith D. K., Sreekala P., Yuvaraj S., Radhika G.,
Rajina P., Prasanth V., Ummer P. V. and Radhakrishnan for their kind
help and support during my study period.
I express my sincere thanks to all staff members of Data Printering
Solutions, Kannur, for providing me the technical support.
I thank everyone who made me to complete this work a successful
one. Last but not the least I thank the Almighty for his blessings throughout
the study. His unseen presence gave me the strength and patience to
complete the research work successfully.
Ansa Mathew
List of Tables
Table 1: Documented Plant Parts having Nephroprotective Effect…………………..29 Table 2: Cisplatin Treatment Regimen to assess Cisplatin Toxicity……………..73 Table 3: Gentamicin Treatment Regimen to Assess Gentamicin Toxicity………74 Table 4: Physicochemical Parameters of Crude Drugs……….…………………..79 Table 5: Solvent Extraction of Air Dried Plant Material …….…………………….80 Table 6: Phytochemical Screening of Plant Extracts …….……………………….81 Table 7: Retention Time of Marker Compounds……….………………………….83 Table 8: Percentage of Lupeol, β-Sitosterol and Ellagic Acid in Plant Extracts ……………………………………..……………………………………………………85 Table 9: In vitro Antioxidant Activity of Extracts against Lipid Peroxidase Scavenging Assay……………………………………………………………………..91 Table 10: In vitro Antioxidant Activity of Extracts against Nitric Oxide Scavenging Assay……………………………………………………………………………………91 Table 11: In vitro Antioxidant Activity of Extracts against Superoxide Radical Scavenging Assay……………………………………………………………………..92 Table 12: Comparison of Plant Extracts in Cisplatin-Induced Renal Damage…………………………………………….…………………………………105 Table 13: Effect of Methanolic Extract of the Fruits of Terminalia Bellerica on Histopathological Evidences of Kidney in Cisplatin-Induced Renal Damage ………………………………………………………….……………………………...107 Table 14: Effect of Butanolic Extract of the Leaves of Ficus bengalensis on Histopathological Evidences of Kidney in Cisplatin-Induced Renal Damage….109 Table 15: Effect of Ethyl Acetate Extract of the Leaves of Ixora brachiata on Histopathological Evidences of Kidney in Cisplatin-Induced Renal Damage….112 Table 16: Comparison of Plant Extracts in Gentamicin-Induced Renal Damage……………………………………………………………………………….123 Table 17: Effect of Methanolic Extract of the Fruits of Terminalia bellerica on Histopathological Evidences of Kidney in Cisplatin-Induced Renal Damage…124 Table 18: Effect of Butanolic Extract of the Leaves of Ficus bengalensis On Histopathological Evidences of Kidney in Gentamicin-Induced Renal Damage……………………………………………………………………………….126 Table 19: Effect of Ethyl Acetate Extract of the Leaves of Ixora brachiata on Histopathological Evidences of Kidney In Gentamicin-Induced Renal Damage……………………………………………………………………………….128
List of Figures
Figure 1: Transverse Section of Kidney…………..…………………………………19
Figure 2: Mechanism of Action of Cisplatin Toxicity……………………………….26
Figure 3: Hemidesmus indicus…………………………………………………….…46
Figure 4: Ficus bengalensis……………………………………………………….….48
Figure 5: Sida rhombifolia………………………………………………………….…50
Figure 6: Ixora brachiata……………………………………………………………...52
Figure 7: Terminalia bellerica………………………………………........................54
Figure 8: Camellia sinensis…………………………………………………………...57
Figure 9: HPLC Chromatogram of marker compounds…………………………...84
Figure 10: HPLC Chromatogram of marker compound from crude butanol extract of F. bengalensis and H. indicus……………………………………………………..87 Figure 11: HPLC Chromatogram of marker compound from crude ethyl acetate extract of S. rhombifolia and I. brachiata……………………………………………88 Figure 12: HPLC Chromatogram of marker compound from crude methanolic extract of T. bellerica and C. sinensis……………………………………………….89 Figure 13: Graph showing IC50 values in antioxidant studies for plant extracts...92 Figure 14: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of serum creatinine on cisplatin-induced nephrotoxic models……………………………………………………………………95 Figure 15: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of blood urea nitrogen on cisplatin-induced nephrotoxic model…………………………………………………………………………………………..97 Figure 16: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of SOD on cisplatin-induced nephrotoxic models…………..99 Figure 17: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GSH on cisplatin-induced nephrotoxic models…………..101
Figure 18: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GST on cisplatin-induced nephrotoxic models…………..102 Figure 19: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of MDA on cisplatin-induced nephrotoxic models………….104 Figure 20: Effect of Methanolic Fruit Extract of T. bellerica on Cisplatin-induced Nephrotoxicity on histology of Rat kidneys……………………………………….108 Figure 21: Effect of Butanolic Leaf Extract of F. bengalensis on Cisplatin-induced Nephrotoxicity on histology of Rat kidneys……………………………………….110 Figure 22: Effect of Ethyl Acetate Leaf Extract of I. brachiata on Cisplatin-induced Nephrotoxicity on histology of Rat kidneys……………………………………….113 Figure 23: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of serum creatinine on gentamicin-induced nephrotoxic models…………………………………………………………………..116 Figure 24: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of blood urea nitrogen on gentamicin-induced nephrotoxic model………………………………………………………………………………………….117 Figure 25: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of MDA on gentamicin-induced nephrotoxic models………………………………………………………………………………………..118 Figure 26: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GSH on gentamicin-induced nephrotoxic models………………………………………………………………………………………..119 Figure 27: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GST on gentamicin-induced nephrotoxic models……………………………………………………………………………………….121 Figure 28: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of SOD on gentamicin-induced nephrotoxic models………………………………………………………………………………………..122 Figure 29: Effect of Methanolic Fruit Extract of T. bellerica on gentamicin-induced Nephrotoxicity on histology of Rat kidneys……………………………………….125 Figure 30: Effect of Butanolic Leaf Extract of F. bengalensis on gentamicin-induced Nephrotoxicity on histology of Rat kidneys……………………………..127
Figure 31: Effect of Ethyl Acetate Leaf Extract of I. brachiata on gentamicin -induced Nephrotoxicity on histology of Rat kidneys……………………………..129
Abbrevations Abs Absorbance ANOVA Analysis of variance ARF Acute Renal Failure ATP Adenosine triphosphate BHA Butylated hydroxyl anisole BHT Butylated hydroxyl toluene BUN Blood urea nitrogen b.w Body weight CDD Cis-diaminedichloro platinum Conc Concentration CRF Chronic renal failure dL Decilitre DMSO Dimetyl sulphoxide DTNB 5-5’-dithio-bis-2-nitrobenzoic acid EDTA Ethylene diamine tetra acetic acid g gram GFR glomerular flitration rate GM Gentamicin GSH Glutathione GSSG Glutathione disulphide GST Glutathione-s-transferase HCl Hydrochloric acid HPLC High Performance Liquid Chromatography i.v. intravenous i.p. intraperitoneal IgA Immunoglobulin A iNOS inducible nitric oxide synthetase KCl Potassium chloride M Molar MDA Malondialdehyde µg microgram µMol micromole min minutes mM millimole NADPH Nicotinamide Adenosine Diphosphate NBT Nitro Blue Tetrazolium nM nanometer ODS Octadecyl Saline
Organisation for Economic corporation and Development p.o. per oral RNS Reactive oxygen species
ROM Reactive oxygen metabolite ROS Reactive oxygen species s.c. subcutaneous SC serum creatinine SOD Super oxide dismutase TBA Thiobarbituric acid TCA Trichloro acetic acid WHO World Health Organization
Contents
1.0 Introduction…………………………………………………………………….1 1.1 History of herbal medicine………………………………………...……..1 1.2 Herbal medicine market……………………………………………..…...5 1.3 Biological role of plant compounds………………………………...…..8 1.4 Herbal medicine standardization………………………………….…..10 1.5 Indian Medicinal Plants as a Source of Antioxidants and Free Radical
scavengers………………………………………………………………13 1.5.1 Antioxidants………………………………………………….13 1.5.2 Food as sources of Antioxidants……………………….…15
1.6 Drug Toxicity………………………………………………………….…17 1.6.1 Nephrotoxicity………………………………………………….18 1.6.2 Nephrotoxic Agents………………………………………..…. 21 1.6.3 Mechanisms of renal toxicity………………………………….22
1.7 Cisplatin-induced Nephrotoxicity……………………………………...24 1.7.1 Mechanism of Action of Cisplatin…………………………....24 1.7.2 Mechanism of Action of Cisplatin Nephrotoxicity………....25
1.8 Gentamicin-induced Nephrotoxicity……………………………….….26 1.8.1 Mechanism of action of Gentamicin………………………....26 1.8.2 Mechanism of Gentamicin Nephrotoxicity……………..…..27
1.9 Plants as nephroprotective agents…..………………………………..28 2.0 Aim and Objectives…………………………….……….…………………...36 3.0 Review of Literature………………………………………………………...37
3.1 Hemidesmus indicus………………………………………………..…37 3.2 Ficus bengalensis………………………………………………….…..38 3.3 Sida rhombifolia…………………………………………………….….39 3.4 Ixora brachiata……………………………………………………….....40 3.5 Terminalia bellerica………………………………………………….…40 3.6 Camellia sinensis……………………………………………………..….41
4.0 Plan of work……………………………………………………………….….43 5.0 Materials and Methods……………………………………………………...45
5.1 Plant profile.............................................................………………46 5.2 Extraction of plant material………………………………………….…59 5.3 Physicochemical properties ……………………….……………….…60 5.4 Qualitative phytochemical Analysis…………………………………..63 5.5 HPLC Quantification………………………………………………..….67 5.6 In vitro Antioxidant studies…………………………………...………..68 5.7 Pharmacological studies…………………………….………………....71
6.0 Results and Discussion…………………………………..………………..79 6.1 Physicochemical parameters………………………………………....79
6.2 Percentage yield and phytochemical screening of the plant extracts................................................................................................79
6.3 HPLC determination and quantification of marker compounds in the plant extracts……………………………………………………………83
6.4 In vitro antioxidant studies………………………………….…………90 6.5 Pharmacological studies…………………………………….………...93
7.0 Conclusion…………………………………………………….…………...130 8.0 References…………………………………………………………..……..136
APPENDIX A……………………………………..……………………..…166 APPENDIX B……………………………………………………………….167
1.0 INTRODUCTION
Medicinal plants are plants that provide people with
medicines to prevent disease, maintain health or cure ailments in
one form or another; they benefit virtually everyone on earth.
1.1 History of Herbal Medicine
India has a rich source of medicinal herbs, with high potential for
ayurvedic, unani and siddha systems of medicines. There are more than
2000 species which is spread over a vast area but only a few has been
studied to prove their potential medicinal value.1,2
Medicinal plants constitute an important natural wealth of a country.
They play a significant role in providing primary health care services to
rural people. The use of plants for medicines is by far the biggest use of
plants in term of the number of species targeted. Plants provide the
predominant ingredients of medicines in most traditional system of
medicines. Medicinal plants also constitute a valuable foreign exchange for
most developing countries. Plants have been used for medicinal purposes
from time immemorial. The earliest recordings of use by Chinese and
Egyptians date back to 3000 B.C. Over time the use developed traditional
medicinal systems such as Ayurveda and traditional Chinese Medicine. In
the 19th century, with the development of chemical analysis, the active
ingredients present in plant were segregated to derive modern
pharmaceuticals. WHO estimates that 80% of the people worldwide use
herbal medicine for primary health care. The people of rural areas of most
developed countries still rely on traditional medicine for their health care
needs mainly due to their less side effects and low cost than modern
medicine.3,4
The alternative systems of health such as ayurveda, unani
and homeopathy utilize many herbal drugs that are officially recognized.
Indians generally use herbs as spices, home remedies and over-the-
counter self-medication. There has been a global surge in the usage of
Indian medicinal plants in recent times. But there exists a need to explore
these plants by clinical observations. More than 70 percent of the
population depends on herbal drugs for their primary health requirements.
These herbal drugs provide us with a lot of beneficial compounds like
vitamins, antioxidants, and dietary fiber which acts as functional food.
There is no specific category of herbal drugs or dietary supplements. But
there is vast experimental evidence base for efficacy of many of the natural
drugs. Basic as well as clinical research has to be carried out on the plants
before it is made suitable for human consumption. It is a credit to the
people of India that they are acquainted with a large number of medicinal
plants. The Rig-Veda has mentioned the use of medicinal plants for
various treatments almost about 5000 years ago.
Herbal products are medicinal agents obtained from plants. Many of
the isolated compounds like atropine, colchicine, taxol, vincristine, etc. are
being extensively used.5 As medicinal agents, herbal products should be
considered separate from other medicinal forms of therapies. The herbal
products that are not regulated as medicine cannot be used in alternative
therapies. So the herbs used as medicines should have some potential
effects that are patented as drugs. A recent review on the trials evaluating
herbal medicine showed that only 15% of the study provided information of
safety or side effects.
The World Health Organization has established medicinal plant
monographs that divide the use of botanicals into three major categories –
use supported by clinical data, use described in pharmacopoeias and
traditional systems of medicines and use described in folk medicine which
are not supported by experimental or clinical data. The consumers and
clinicians should have a concern for the safety, efficacy, contents,
bioavailability and dose regimen of a variety of products available in the
market. Bulk herbs are plant raw materials used to prepare dosage forms.
They are used for the preparation of various dosage forms. The chemical
stability of herbal extracts makes it difficult to determine the date of expiry
or shelf life. Botanicals packed in dosage forms for medicinal purposes
must obey pharmacokinetic principles. The pharmacological effect of the
raw plant material varies from those of isolated constituents.6 The
pharmacologically active moiety may not be known even if the chemical
composition of the extract is known. Environmental factors, climate and
growth conditions have got an influence in the chemical composition of
plants.
To manufacture a product with safety and efficacy, manufacturing
standards are required. Quality control and assurance methods should be
defined for each product in the market. Herbal medicines like all other
medicinal agents have unexpected effects including toxicity. The
unexpected effects are influenced by age, gender, genetics, nutrition
status, concurrent disease state and statements. The unexpected effects
of herbal medicines may be either intrinsic or extrinsic to the compound.
The intrinsic effects are due to the presence of phytochemicals that are
present in them. Extrinsic effects are due to misidentification of plants, lack
of standardization, impurities through contamination, substitution or
adulteration. Adverse effects may also affect the organ systems. Easily
affected organ systems are skin, liver, and gastrointestinal tract.
The phytochemicals can be classified as primary and secondary
metabolites. The primary metabolites are widely distributed in nature and
are needed for physiological development in cell metabolism. The
secondary metabolites on the other hand are biosynthetically derived from
primary metabolites and play an important role in combating diseases
directly or indirectly.7 The secondary metabolites are accumulated in lesser
amount than primary metabolites in plants. They can be obtained from
plant materials by steam distillation or by extraction with organic or
aqueous solvents. Some of the biologically active plant compounds have
found their use as drug entities or as models for drug synthesis. About
25% of the prescribed drugs are derived from plants.8,9
The general research methods include proper selection of plant
materials, preparation of plant extracts, biological screening, a detailed
chemo pharmacological investigations, toxicological and clinical studies,
standardization and use of active moiety as lead molecules for drug
design.7
1.2 Herbal medicine market
Herbal medicines have easily earned reputation as the people’s
medicines because of their easy accessibility, safety and the ease with
which they are prepared. Use of herbal medicines in the treatment of
diseases has a long tradition. In some Asian and African countries 80% of
population depends on traditional herbal medicine for health care. In many
developed countries 70 - 80% of population has some complementary or
alternative medicine (CAM) composed primarily of herbal medicines. Many
drugs commonly used today in the developing countries are of herbal
origin and most modern prescription drugs contain at least one active
ingredient derived from plant extracts. According to WHO, approximately
25% of modern drugs used in the world have been derived from plants.
More than 120 active constituents have been isolated from higher plants
are widely used in allopathic medicine today and 80% of them show a
positive correlation between therapeutic use and the traditional use of
plants from which they are derived. At least 7000 medicinal compounds
derived from plants the ingredients of herbal medicines are included in the
modern pharmacopoeia of drugs. Annual revenue from herbal medicines
and herbal products in Western Europe reached US $5 billion in 2003 and
sales of herbal medicine revenue in Brazil was US $160 million in 2007.
Herbs and plants have been used for medicinal or therapeutic purposes
long before recorded history. In earlier days, herbal medicines were
dispensed in the form of simple preparations such as tinctures, tea,
poultice, powders and other herbal preparations which formed the basic
therapeutic items of all forms of traditional system of medicines.
According to a study by Associated Chambers of Commerce
(ASSOCHAMS) the herbal industry will grow rapidly in the coming years
and by 2015, the size of the market will rise to Rs.15,000 crore. The Indian
domestic market can be broadly categorized into two. The first one which
covers raw materials required by the industrial units and the second one
covers ready to use finished medicines, health supplements, etc.
As per the data on herbal medicines market in 1991, the total trade
is about $6 billion in European countries, $3 billion in Germany and $1.6
billion in France. The herbal medicine market in India is about $1 billion.
And the export of herbal crude extract is about $80 million. About 60 % of
export is psyllium seeds and husk, castor oil and opium extract. In Arab
countries, the most prevalent alternative system of medicine is Unani and
they use drugs worth US $120 million annually. In Japan, herbal medicine
preparations are more in demand than the pharmaceutical preparations.
National Medicinal Plant Board has been constituted to facilitate the
conservation, propagation and marketing of important medicinal plants.
The Government of India adopted many measures to give a boost to the
export of medicinal plants. India exports crude drugs worth US $31 million.
The global herbal supplement and remedies market is expected to reach
$93 billion by 2015 (Drugs and Pharmaceuticals, 1998).
There has been resurgence in the interest of herbal medicines
particularly in Europe and North America. About 75–80% of the world
population depends on herbal medicines for their primary health care
because of better acceptability better compatibility and lesser side effects.
In the last few decades a drastic change has happened to botanical
medicine. Instead of being killed by medicinal science and pharmaceutical
chemistry it has made to come back. Herbal medicines has benefited from
the objective analysis of medicinal science while the cures claimed by
herbals and plant medicines have been acknowledged. Phytochemicals
which is evolved from natural product chemistry mainly involves the study
of products obtained by plants and in recent years has developed into
plant organic chemistry and plant biochemistry. The study mainly involves
the chemical structure of the plant constituents, its biogenesis natural
distribution and biological functions.10
1.3 Biological role of Plant Compounds
Phenolic compounds
Polyphenolics constitute a distinct group among phytochemicals.
Natural polyphenols are simple molecules like phenolic acids to highly
polymerized compounds such as condensed tannins.11 They are widely
distributed among secondary metabolites. They can interact with primary
materials like polysaccharides and proteins. They possess various
pharmacological actions on the human body. They have cardiovascular
benefits by altering concentration of lipid components. A high intake of
polyphenols can reduce the risk of cardiovascular diseases.12 They can
also scavenge the harmful effects of free radicals in the biological
system.13
Phenols and phenolic acids
They are useful for hydrogen donating and radical scavenging
reactions. Phenolic compounds act as good antioxidants because they
have the ability to donate electrons and forms stable radical intermediates
that prevents oxidation at cellular and physiological levels.14
Catechins
Catechins are components responsible for the flavor of red wines.
They are produced in stems and seeds. They can polymerise themselves
or with other flavonoids and non-flavanoids to form tannins. Flavanols like
quercetin absorb UV rays of the sun and can act as protective agents of
skin. Quercetin also play important role in human health.15
Tannins
They are a large group of polyphenolic compounds that is capable to
cure a variety of diseases.16 They are divided as hydrolysable tannins and
proanthocyanidins (condensed tannins). Hydrolysable tannins are gallic
acid and ellagic acid esters. Proanthocyanidins are polymers of flavan-3-
ols and flavan-3,4 diols linked by a interflavin bond which cannot be
hydrolyzed.17 Tannins form complex with proteins by hydrogen bonding,
hydrophilic effects and also by covalent binding.18 Tannic acid is known to
exhibit various types of health benefits.19,20,21
Terpenes
They are a large group of compounds responsible for fragrance of
plants and are called essential oil fractions. They are synthesized from
isoprene units. Terpenes may be classified as monoterpenes, diterpenes
tetraterpenes, hemiterpenes and sesquiterpenes. They may be called
terpenoids when oxygen is added.22 Terpenes and terpenoids possess
antimicrobial activity.23,24,25
1.4 Herbal medicine standardisation
The World Health Assembly emphasized the need to ensure quality
control of medicinal plants with appropriate techniques and suitable
standards as it is estimated that 80% of the people living in developing
countries mainly depend upon herbal drugs for their primary health care
needs. So, to deliver a good, quality and safe medication, standardization
of herbs and formulations has become important. WHO gives different
quality parameters to standardize the raw materials as well as finished
products.
Phytomedicine are standardized herbal preparations that consist of
complex mixtures of one or more plants used for the treatment of various
diseases. Quality can be defined as the status of a drug that is determined
by identity, purity, content and other physical, chemical or biological
properties or by manufacturing process. For the production of quality
herbal products strict guidelines has to be followed. The traditional system
of medicine dispenses drugs as water decoction or ethanolic extracts.
Thus the plant parts used should be free from harmful materials like
pesticides, heavy metals, microbial or radioactive contamination. The
extraction of medicinal plants may use single solvent or water or as
described in ancient texts. The extracts are then checked for their
biological activity in experimental animal models. The bioactive extract
should also be standardized for the basic active principles or major
compound along with fingerprints. The next important step is stabilization
of the bioactive extract with a minimum shelf life over a year. The stabilized
bioactive extract should undergo regulatory or limited safety studies. The
WHO in 1991, has developed guidelines for the assessment of herbal
medicine. The main features of the guidelines are:
Quality control of crude drug material, plant parts and finished products
Stability
Safety assessment
Assessment of efficacy
Quality control of botanicals including phytomedicine and dietary
supplements is a basic requirement to ensure their safety and
effectiveness. Qualitative and quantitative analysis of the marker
component is an important need for the study as they represent the quality
of the product. Nowadays marker profiling is done to standardize herbal
medicine so as to establish the lead molecules of therapeutically important
plants. The presence of a marker compound in plant materials may be
useful for quantifying the marker in the total extract. This study will be a
useful tool in the quality control of isolated molecules as well as extracts. If
a marker itself is a biologically active principle of the plant, qualitative and
quantitative analysis of the biomarkers not only help us to control the
quality of herbal materials used but can also be used to estimate the
quantity of the biologically active chemical entities required to produce the
desired pharmacological effects. The quality and the phytoconstituents
contents of the plant materials may vary in season, growing condition and
harvesting and storage conditions. Quality control through qualitative
chromoprofiling may be a fast and useful tool for monitoring the quality of
plant materials under these variations. Quality assurance of herbal
products may be ensured by proper control of the herbal ingredients and
by means of good manufacturing practices. Some herbal products have
many herbal ingredients with only small amounts of individual herbs being
present. Chemical and chromatographic tests are useful for developing
finished product specifications.26
High Performance Liquid Chromatography (HPLC) is used as a
suitable analytical method for the determination of phytoconstituents in
plant material due to following reasons:
a) Simplicity, high speed of separation and sensitivity to low concentrations
b) Specificity in detection and compatibility with wide range of organic
solvents miscible with water
c) High reproducibility and repeatability of data that leads to reliable results
d) HPLC offers analysis of samples in low quantities
Suitable animal models help to understand the mechanism of action
as well as the pharmacodynamics of the medicines. To bring more
objectivity and also to confirm traditional claims, clinical trials are
necessary. In Ayurvedic medicine research, clinical experiences,
observations or available data becomes a starting point. Thus, the drug
discovery based on ayurveda follows a reverse pharmacology path. All the
pharmacopeial tests must be in accordance with good manufacturing
procedures for herbal products. There have been concerns about quality
standards and safety issues of herbal medicines.
1.5 Indian Medicinal Plants as a Source of Antioxidants and Free
Radical scavengers
1.5.1 Antioxidants
Antioxidants are a type of complex compounds found in our diet that
act as a protective shield for our body against certain disastrous diseases
such as arterial and cardiac diseases, arthritis, cataracts and also
premature ageing along with several chronic diseases.
Oxygen is used by the cells of our body to breakdown
carbohydrates, proteins and fats and produce free radicals. Free radicals
are atoms or a group of atoms possessing an unpaired electron that
makes them highly reactive. The human body has an elaborate antioxidant
defense system. Antioxidants exert their action by giving up their own
electrons to free radicals. When a free radical gains an electron from an
antioxidant it no longer attacks the cell and the chain reaction is broken.
After donating an electron an electron becomes a free radical and is not
harmful like the reactive oxygen species. Antioxidants are manufactured
within the body and can also be extracted from food that humans eat such
as fruits, vegetables, seeds, nuts, meat and oils. Free radicals including
reactive oxygen species is formed in the human body through aerobic
metabolism, detoxification of toxic compounds during the denaturing of
foreign proteins like antigens as a part of phagocytosis other
environmental factors like UV radiation, inadequate exercise, pollution,
cigarette smoking etc. In normal conditions, body maintains a balance
between free radicals and antioxidants but deficiency of antioxidants leads
to increase in free radicals which in turn leads to changes in the genetic
material damaging the immune system and leads to various types of
diseases. Antioxidants give electron to the free radicals and break the
chain reaction. Free radicals damage the molecules in cell membranes,
mitochondria DNA and are very unstable. Oxidative reactions produce free
radicals which can damage the cells. Antioxidants act by terminating these
chain reactions by removing free radical intermediates and inhibit other
oxidation reaction by being oxidized themselves. So, antioxidants are
mostly reducing agents like thiols or polyphenols.
Reactive oxygen species (ROS) is a collective term that includes
not only oxygen radicals but also some nonradical derivatives of oxygen.
These include hydrogen peroxide, hypochlorous acid and ozone. Various
sources of ROS have been identified in the living organisms. The
superoxide anion radical appears to play the central role and other reactive
intermediates are formed from it. Even though there is defence
mechanisms against ROS it has been observed that the level of the
cellular antioxidant system goes down or when the level of ROS reaches
substantially high oxidative damage to cells occurs leading to pathological
conditions.
Antioxidant defence comprise agents that catalytically remove free
radicals and other reactive species like SOD, CAT, peroxidases, thiol
specific antioxidants, low molecular mass agents that scavenge ROS and
RNS.
1.5.2 Food as sources of Antioxidants
Antioxidants are abundant in fruits and vegetables as well as other
foods including nuts, some meat, poultry and fish. Beta carotene is found
in many foods that are orange in colour, sweet potatoes, carrots, apricots,
pumpkin and mangoes. Some green leafy vegetables like spinach, kale
are rich source of beta carotene. Lutein another antioxidant is needed for
healthy eyes and is abundant in green leafy vegetables. Another potent
source of antioxidant is lycopene found in apricots, watermelon, tomatoes,
papaya, oranges and other foods. Foods that contain Vitamin A are also a
rich source of antioxidants. Foods rich in Vitamin A include liver, carrots,
sweet potatoes and milk. Vitamin C also called ascorbic acid is another
rich source of antioxidants. It is found in many fruits, vegetables, cereals,
beef, poultry and fish. Vitamin E is another antioxidant source. It is also
called alpha tocopherol is found in many oils like wheat germ, safflower,
corn and soya bean oils and also in mangoes, nuts, broccoli and other
foods.
Many antioxidant substances are present in plants (fruits,
vegetables, medicinal herbs, etc.) and the free radicals present in them are
in the form of phenolic compounds like tannins, lignans, coumarin, and
endogenous metabolites.27,28 Intakes of foods rich in antioxidants lower the
risk of chronic health problems.29,30,31 Synthetic antioxidants like butylated
hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) are unsafe
due to their carcinogenic effects.32,33 So naturally occurring antioxidants
can be used for treating free radical related disorders.34,35
Report has shown that proper intake of antioxidant will help quench
all these inevitable free radicals in the body, thus, improving the health by
lowering the risk of various diseases such as cancer. Antioxidants are also
important in body lotions and creams so as to protect the skin from sun
exposure and to decrease skin roughness, wrinkle depth, ultraviolet
induced skin cancer and skin swelling from sunlight. To cap it up, there is a
need for proper orientation on the necessity of proper intake of balanced
diet which will definitely supply the much needed antioxidants. The RDA
has been previewed so that people will have lower health risks and tend to
live longer and have fewer disabilities.
1.6 Drug Toxicity
Toxicity testing in animals is carried out on new drugs to identify
potential hazards before it is administered to humans. It involves the use of
a wide range of tests in different species with long term administration of
the drug, regular monitoring for physiological or biochemical abnormalities
and a detailed postmortem examination at the end of the trial to detect any
gross or histological changes. Toxicity testing is performed with doses
above the therapeutic range and establishes which tissues or organs are
likely targets of toxic effects of the drugs. Recovery studies are performed
to assess whether toxic effects are reversible and particular attention is
paid to irreversible changes. The basic premises are similar in humans and
other animals. This is due to similarities between higher organisms at the
cellular and molecular levels. Toxic effects can range from negligible to
severe as to preclude further development of the compound. Intermediate
levels of toxicity are more acceptable in drugs intended for severe illness
(e.g.: AIDS or cancer) and decisions on whether or not to continue
development are often difficult. Toxic effects of drugs can be related to
pharmacological action (e.g. bleeding with anticoagulants) or unrelated to
the principal pharmacological action (liver damage with Paracetamol).
Toxic concentration of drug or drug metabolites can cause necrosis.
Chemically reactive drug metabolites can form covalent bonds with target
molecules or alter the target molecule by non-covalent interactions. Drugs
and their polar metabolites are concentrated in renal tubular fluids as water
is reabsorbed, so renal tubules to expose to higher concentrations than
other tissues. Renal vascular mechanism is critical to the maintenance of
glomerular filtration and is vulnerable to drugs that interfere with control of
afferent and efferent arteriolar contractility.
1.6.1 Nephrotoxicity
Paired kidneys are situated retro peritoneal on either side of the
vertebral column. Each kidney is made of a large number of nephrons
groups which unite to form collecting ducts or tubules and these in turn
combine to form ducts of Bellini, around the papilla tip. The papilla opens
into a calyx which then narrows to the ureter. Each kidney is supplied by a
renal artery.32
Fig 1: Transverse section of kidney
The kidneys are organs that are essential in the urinary system and
also serve homeostatic functions such as regulation of electrolytes,
maintenance of acid base balance and regulation of blood pressure. They
serve the body as a natural filter of the blood and remove waste that is
diverted to the urinary bladder. Diseases of the kidney are diverse but
individual with kidney disease with frequently display characteristics clinical
features.32,33,34 Common clinical conditions of the kidney include nephritic
and nephritic and nephritic syndrome, renal cysts, kidney injury, chronic
kidney disease, urinary tract diseases and nephrolithiasis. Renal failure is
mainly determined by a decrease in glomerular filtration rate, the rate at
which blood is filtered in the glomeruli of the kidney. This can be detected
by a decrease or absence of urine production or determination of waste
products (creatinine or urea) in the blood. The kidneys are affected by an
array of chemicals. Man is exposed to medicines, industrial and
environmental chemicals and a variety of naturally occurring substances.34
Exposure may be for a long period of time or limited or single event and
toxicity may be due to a single substance or multiple chemicals.
Nephrotoxicity is an adverse effect of certain antibiotics, anticancer agents
and other synthetic agents. Some chemicals cause an acute injury and
others may produce chronic renal changes that may lead to end stage
renal failure and renal malignancies.
Renal failures are mainly of two types - acute renal failure and
chronic renal failure. Acute renal failure (ARF) is characterized by a
reversible loss of kidney function and azotemia that progress rapidly by
several hours to days. Acute renal failure is often symptomatic. It can be
detected by measuring the levels of creatinine and blood urea nitrogen in
the blood. Chronic renal failure (CRF) is characterized by progressive
irreversible deterioration of the kidneys due to slow destruction of renal
parenchyma. Toxic agents like amphotericin and polyene antibiotics
directly affect the permeability of membrane. Phospholipids present in the
membrane degrade to form lysophospholipids and free fatty acids which
act as detergents.37 Even in the absence of major changes in membrane
permeability, the failure of plasma membrane pump cause changes in
cation homeostasis of the cell. Toxins also cause remodeling of the
surface of renal tubular cell, thereby changing the area available for
transportation. An early change detected by toxins on the kidney is the
accumulation of intracellular calcium. This increase in calcium is found in
plasma membrane, mitochondria, endoplasmic reticulum and also in the
cytoplasm. Due to an increase in calcium the permeability of the internal
membrane of the mitochondria is affected that changes the
electrochemical gradient across it that decreases the oxidative
phosphorylation capacity of the mitochondria. Disordered permeability
leads to the loss of enzymes and nucleotides.36,37
In vivo and in vitro studies have demonstrated the effect of free
radicals like superoxide hydroxyl ions and hydrogen peroxide that are
important mediators of tissue injury. Free radical injury and oxidative stress
have been implicated in many renal diseases like acute renal failure, IgA
nephropathy, anemia of chronic renal failure and ischemic kidney. Most
risk assessment decisions are based on information concerning
aminoglycosides, halogenated anesthetic, and several heavy metals where
an excellent concordance between animals and findings in humans
exposed to these agents.
1.6.2 Nephrotoxic Agents35
There are a number of drugs, diagnostic agents and chemicals that
cause nephrotoxicity. Some of the important nephrotoxic agents are
a) Heavy metals - Mercury, Arsenic, Lead, Bismuth.
b) Antineoplastic agents
Alkylating agents - Cisplatin, Cyclophosphamide
Nitrosoureas – Streptozotocin, Carmustine, Lomustine, Semustine.
Antimetabolites – Methotrexate, Cytosine arabinose, high dose of 6-
Thioguanine, 5-Flurouracil.
Antitumour antibiotics – Mitomycin, Mithramycin, Doxorubicin.
c) Biological agents – Recombinant leukocyte and Interferon.
d) Antimicrobial agents – Tetracycline, Acyclovir, Pentamidine,
Sulphadiazine, Trimethoprim, Rifampicin
e) Aminoglycosides – Gentamicin, Amikacin, Kanamycin, Streptomycin.
1.6.3 Mechanisms of renal toxicity
There are several mechanisms for toxicity caused by toxins like
impaired lysosomal function, membrane changes and oxidative stress.
Calcium homeostasis in the cell and calcium mediated cell functions are
the targets for the various pathophysiological process and also cell death
caused by toxicants.38,39 A number of pharmaceuticals and other
chemicals impair the calcium messenger system. The disturbances in the
intracellular calcium cause cell death by disruption of the plasma
membrane, cytoskeleton, endoplasmic reticulum and mitochondria.
Chemicals produce toxicity by causing changes in the DNA or by
apoptosis. The cellular accumulation of calcium causes generation of
oxygen free radicals and damage cellular components especially
mitochondrial membrane. Chemicals produce nephrotoxicity by lipid
peroxidation and cause membrane damage and cell death.40,41,42 Free
radicals formed directly by metabolism of chemicals or from reduction of
oxygen initiate lipid peroxidation by hydrogen abstractions from PUFA.
This forms lipid per oxy radicals and lipid hydroxyl peroxides propagating
chain reactions. Such chain reactions destroy cellular membranes which
results in increased plasma membrane permeability or altered fluidity and
cell death. Lipid peroxidation also causes cell death by forming potent toxic
lipid metabolites like hydroxyl alkenes.42
Proximal convoluted tubules are highly vulnerable to toxic action of
chemicals owing to their high energy demand. Oxidative stress or
reduction of oxidized glutathione (GSSG) to GSH by NADPH dependent
GSSG reductase is lower than the rate of GSH oxidation. This leads to
depletion of glutathione and cause oxidation of cellular enzymes, depletion
of cellular ATP and loss of mitochondrial function.43
Super oxidase dismutase enzymes catalyze the dismutation of super
oxide into oxygen and hydrogen peroxide. It has been shown in animal
experiments that the enzyme super oxidase dismutase and catalase can
be used to prevent renal lesion.44
1.7 Cisplatin-induced Nephrotoxicity
Cisplatin [cis-diamine, dichloro, platinum (II)] CDDP is a divalent
platinum compound used for the treatment of tumour of bladder, testis and
ovary. The toxic effects caused by the use of cisplatin include
nephrotoxicity, ototoxicity, neurotoxicity and bone-marrow suppression and
renal toxicity.45,46 Inspite of the availability of some newer and less toxic
drugs, Cisplatin remains a major antineoplastic drug for the treatment of
solid tumors. Cisplatin nephrotoxicity may occur as acute kidney injury (20-
30%), hypomagnesaemia (40-100%), chronic renal failure, thrombotic
microangiopathy etc. Cisplatin-induced nephrotoxicity was first reported in
1971 in animal models.47 In 14 to 100% of the patients treated with
cisplatin, a dose related induced acute nephrotoxicity was observed. Renal
toxicity caused by cisplatin occurs several days after its treatment and is
evident by a rise in serum creatinine and BUN levels. The main risk factors
for nephrotoxicity induced by cisplatin are old age, gender, smoking, and
hypoalbuminemia.47,48
1.7.1 Mechanism of Action of Cisplatin
Cisplatin enters the cell by diffusion. A positively charged molecule is
formed by replacement of chloride ion with water. This active form
(electrophile) of the drug reacts with nucleic acids and proteins. Interaction
with DNA is the primary mode of cisplatin activity. Intrastrand cross links
are produced and cause changes in DNA confirmation that affect
replication. Nephrotoxicity of cisplatin occurs at the S3 segment of proximal
tubule which causes a decrease in the glomerular filtration rate.36
1.7.2 Mechanism of Action of Cisplatin Nephrotoxicity
Animal studies show that cisplatin undergoes metabolic activation in
the kidney to a more potent toxin. This begins with the formation of
glutathione which conjugates in the presence of glutathione-s-transferase.
The gamma glut amyl transpeptidase seen on the proximal tubule cells
cleaves the glutathione conjugate to cystinyl lysine conjugates as they
pass through the kidney. Further amniopeptides present in the proximal
tubules metabolize the cystinyl lysine conjugate to cysteine conjugate.
They are then transported into the proximal tubule cells where they are
further metabolized by cysteine-s-conjugate β-lyase to highly reactive
thiol.48,49
Mitochondria produce reactive oxygen metabolites like super oxide.
It scavenges the ROM with the help of antioxidants, enzymes like
superoxide dismutase, glutathione peroxidase, catalase and GSH.
Cisplatin accumulates in the mitochondria. Cisplatin induces ROS in renal
epithelium cells by decreasing the activity of antioxidant enzymes and by
depleting intracellular concentration of GSH. Antioxidants like SOD,
dimethyl thiourea and GSH have shown beneficial effects in the degree of
cisplatin-induced nephrotoxicity.48
Cisplatin Cisplatin uptake by renal tubular cells ROS P53 P21
MAPK TNF∞ vascular changes
Renal tubular cell death
Renal tissue damage Ischemia Decrease in GFR Acute renal failure
Fig 2: Cisplatin enters renal cells by passive and facilitated mechanism. The exposure of tubular cells activates the signaling pathways that lead to renal cellular cell death factors. Cisplatin induces the production of TNF in the tubular cells that trigger the inflammatory response, further leads to tubular injury and death. The tubular cell death causes a decrease in GFR which finally causes acute renal failure.
1.8 Gentamicin-induced Nephrotoxicity
1.8.1 Mechanism of action of Gentamicin
Gentamicin is an aminoglycoside antibiotic used to treat many types
of bacterial infections particularly gram negative organism. The use of
gentamicin is limited due its ototoxic and nephrotoxic side effects.
Gentamicin is not absorbed in the small intestine so is not active when
given orally. It is administered by IV, IM or topical route. Gentamicin is a
bacterial antibiotic that binds irreversibly to 30s subunits of ribosome, thus
interrupting protein synthesis.50
1.8.2 Mechanism of Gentamicin Nephrotoxicity
There are many hypotheses for the mechanism of nephrotoxicity
induced by gentamicin, but a precise mechanism remains unclear.
In animal models it has been shown that gentamicin enters tubular
cells by endocytosis which is mediated by meglin-cubutin complex that
requires electrostatic binding to the negative charges of membrane
phospholipids. Gentamicin then passes by pinocytosis to endosomal
compartment. The accumulation of the drug is mostly in the lysosomes but
they travel through a secondary pathway and enter the Golgi apparatus
and endothelial reticulum which alters the vesicular movements. In the
lysosomes gentamicin produces membrane destabilization, lysosomal
aggregation, alteration of lipid metabolism and phospholipidosis which is
associated with cell death. It has also shown in invitro models that
increased concentration of aminoglycosides cause mistranslation or block
incorporation of aminoacids by ribosomes.51
1.9 Plants as nephroprotective agents
Nephroprotective agents are compounds that possess protective
activity against nephrotoxicity. Medicinal plants that contain such active
ingredients are often used for their curative property in kidney disease.
Ancient literature prescribed the use of various herbs for the cure of kidney
disease.52 When such herbs are co-administered along with nephrotoxic
drugs they reduce their toxic effects. Certain plants which are documented
in literature for their nephroprotective effect are cited below.
Table 1: Documented Plant having Nephroprotective Effect
Sl.No:
Name of the plant
Family Part used
Active constituents
Extract Animal model
Nephro toxic agent used
Remarks
1. Aerva lanata53
Amaranthaceae Entire plant
Lupeol, alkaloids, β-sitosterols, tannic acids, sterols
Ethanolic Albino rats
Cisplatin and gentamicin
In gentamycin group, animals in preventive regimen showed nephron- protective activity at 300 mg/kg with elevated levels of s. urea and creatinine & normalized histopathological cha, extract at dose 75, 150, 300 mg/kg showed dose dependent reduction in the elevated levels of renal parameters.
2. Harungana madagascariensis54
Clusiaceae/ hyperiaceae/ gultiferae
Root Anthrones-harunganol (harunganol B)
Aqueous Albino rats
Acetaminophen
Oral pretreatment with graded 100-500 mg/kg/day single oral doses of the root extract of Harungana madagascariensis attenuated the elevated serum concentration of blood parameters in dose related pattern. The biochemical results were also confirmed by histopathological findings.
3. Carica papaya55
Caricaceae Seed Benzylisothiocyanate, proteins, lipids, crude fibres.
Aqueous Wister rats
Carbon tetrachloride
Maximum nephroprotection was offered by the extract at 400mg/kg/day CPE which lasted up to 3hrs post carbon tetrachloride exposure and the biochemical evidences were correlated by improvements in renal histological lesions induced by carbon tetrachloride intoxication.
4. Ficus racemosa56
Moraceae leaf Triterpenoids,steroids
Ethanolic and aqueous
Mice cisplatin The extracts if Ficus racemosa had a significant effect on the serum parameters.
5. Crataeva nurvala57
Caparidaceae Stem bark
Flavonoids, glusinol, sterols, lupeol, saponin
Petroleum ether
Wistar rats
Cisplatin The plant extract is effective significantly altering the indices of cisplatin-induced dysfunction of renal proximal tubule cells under study by decreasing concentration of BUN, creatinine and lipid peroxide.
6. Withania somnifera 58
Solanaceae Roots Alkaloids-withanine somniferine, somnine, tropine. Steroidal lactones.
Ethanolic Albino rats
gentamicin Withania somnifera (500mg/kg) significantly reversed the changes of nephrotoxicity evidenced microscopically when compared to other two doses of Withania somnifera (250mg/kg and 750mg/kg).
7. Cassia auriculata 59
Leguminosae fruits Lupeol,betasitosterol,tannins
Petroleum ether, chloroform, methanolic extract
Wistar rats
gentamicin Oral administration of extract in Wistar rats showed nephroprotective effect in gentamycin induced renal failure was evident by a decrease in the renal toxic parameteres
8. Grape seed60
Labiateae Diterpenoids, acylhydroquinones, phenyl anthroquinones
50% alcohol Mice Ethylene glycol
100mg/kg grape seed extract produced a significant reduction inurinary LDH blood urea and creatinine.
9. Bauhinia variegate61
Caesalpiniacae Whole stem
Flavonone,(2,5), 5,7-dimethoxy 3’,4’ methylene dio flavonone dihydrodibenzoxepin
Methanolic Albino rats
Cisplatin/ Gentamicin
Ethanolic extract exhibited significant and comparable nephroprotective effect to that of standard poly herbal drug cystone in rats Cisplatin 400 mg/kg Gentamicin 200, 400, 500 mg/kg/bw.
10. Prosthechea michuacana 62
Orchidaceae Bulb Flavonoids Chloroform, hexane, methanolic
Albino rats
Cisplatin Extracts studied for cisplatin-induced renal injury model in rats and was found to be nephroprotective
11. Acorus calamus 63
Acoraceae leaves Lignans, epieudesmin, sakuranin, flavanoid-retusin, 5 rachi-galgravin
Ethanolic Male albino rats
Acetaminophen
Extract in male albino rats showed histopathological changes in APAP induced narcotic damage of renal tissues, thereby decreased nephrotoxicity and oxidative stress
12. Authoxanthum odoratum 64
Poaceae Leaves Alkaloids Ethanolic rats Acetaminophen
In rats with acetaminophen induced toxicity the ethanolic extract of the drug prevent renal damage likely through its antioxidant activity which is evident with biochemical findings 250 & 500 mg/kg
13. Embelia ribes 65
Myrsinaceae Fruits Vilangin, leaves-embelin
Alcoholic Wistar rats
Cisplatin Combination of fruit and vitamin E showed a better nephroprotective effect than groups treated with fruit alone
14. Ficus religiosa 66
Moraceae Latex Quercetin flavonoids
Methanolic Wistar rats
Cisplatin Methanolic extract in Wistar rats showed nephroprotective and curative activity in cisplatin-induced
nephrotoxicity
15. Kalanchoe pianata67
Crassulaceae Whole plant leaf
Arachidonic acid,β-stitosterolbenzenoids, bufadienolides, steroids, quercetin
Aqueous extract
Rat Gentamicin Aqueous extract protected rat kidneys from gentamycin induced histopathological changes
16. Momordica dioica
Root Alpha-spinasterol octadecanoate.alpha-spinasterol-3-0-beta-D –glucopyranoside,3-0-beta-D-glucuranopyranosylgypsogenin,3-0-beta-D-glucopyranosylgypsogenin
Ethanolic Albino rats
cisplatin Nephroprotective activity was studied in 6 cisplatin-induced nephrotoxicity. It was shown that the extract at the dose of 250 mg/kg attenuated the effects of nephrotoxicity caused by cisplatin
17. Macrothelypteris oligophlebia69
Thelypteridaceae Rhizome Flavonoids, tannins
Ethanolic Albino rats
Gentamicin 250 & 500 mg/kg significantly decreased levels of BUN, creatinine, MDA and NO and also restored activities of renal
antioxidant enzymes.
18. Zingiber officinalis 70
Zingiberaceae seeds Volatile oils, gingerol, shogaol, resin, starch
Aqueous extract
Rat Doxorubicin Nephroprotection mediated by preventing Doxorubicin induced decline of renal antioxidants.
19. Curcuma longa71,72
Zingiberaceae Rhizome Curcumin ,terpenoids
Ethanolic Rat Cisplatin The ethanolic extract of Curcuma longa exhibits effective protection against cisplatin-induced renal toxicity.
20. Cassia auriculata73
Fabaceae Flower Tannins, alkaloids
Ethanolic Albino rats
Cisplatin and Gentamicin
Nephroprotective effect due to antioxidant and free radical scavenging properties.
21. Aegle marmelos74
Rutaceae Leaf, fruit and bark
Rutin, β-sitosterol, lupeol, tannins, flavonoids
Aqueous Albino & Wistar rats
Gentamicin Aqueous extract of Aegle marmelosa decreased serum creatinine, urea and BUN showing nephroprotective activity.
2.0 Aim and Objectives of the Study
1. To compile the pharmacognostic profile of selected medicinal plants
in order to confirm their authenticity as per pharmacopeial standards.
2. To quantify the biomarkers in the crude drug extract using high
performance liquid chromatography -
1. Lupeol content in Ficus bengalensis and Hemidesmus
indicus
2. β-sitosterol content in Ixora brachiata and Sida retusa
3. Ellagic acid content in Terminalia bellerica and Camellia
sinensis
4. To conduct in vitro antioxidant studies on the crude extract and
phytoconstituents.
5. To evaluate the nephroprotective activity of total extract and
phytoconstituents per se of the plants containing higher
concentration of the phytoconstituents.
3.0 Review of Literature
3.1 Hemidesmus indicus
1. Rajan S. et al worked on the pharmacognostical and phytochemical
properties of Hemidesmus indicus roots. Roots of Hemidesmus
indicus can be used for a number of disorders. In this study
pharmacognostic and phytochemical analysis of market samples
were conducted and results were compared with the authentic
samples.75
2. Gurudutt KN et al studied the chemical composition of the volatiles in
the roots of Hemidesmus indicus. From this study it was concluded
that nerolidol (1.2%), borneol (0.3%), salicylaldehyde (0.1%) are the
active principles responsible for the aromaticity of the roots.76
3. Moideen MM et al performed the wound healing activity of ethanolic
extract of leaves of Hemidesmus indicus in rats. It was shown that
the rats treated with the ethanolic extract showed an increase in the
rate and percentage of wound contraction when compared to groups
of animals treated with nitrofurantoin.77
4. Kaur A et al studied the effect of ethanolic extract of the roots of
Hemidesmus indicus in cisplatin-induced nephrotoxicity in rats. It
was shown that at the dose levels of 250 and 500 mg/kg showed a
dose dependent decrease in the elevated serum urea and
creatinine. There was also an increase in the levels of GSH and
GST in the curative group of animals.78
3.2 Ficus bengalensis
1. Shukla S et al showed that on treatment with 50 mg/kg/day bark
extract of F. bengalensis decreased serum cholesterol levels by 59%
and triglycerides by 54% and also a decrease in lipid peroxidation.
There was also an increase in the levels of antioxidant enzymes like
SOD, catalase, glutathione peroxidase and glutathione reductase.79
2. Aswar A et al studied anthelminthic activity of methanolic, aqueous,
chloroform, petroleum ether extract of roots of F.bengalensis. The
aqueous and methanolic extracts at dose of 20 mg/ml showed
potent anthelminthic activity when compared to standard drug
albendazole.80
3. Thakare VN et al evaluated the anti-inflammatory and analgesic
activity of methanolic stem bark of F. bengalensis on experimental
animal models. The methanolic stem barks extract had significant
acute and sub-acute anti-inflammatory activity compared to aqueous
extract of stem.81
4. Sawarkar HA et al82 compared the anthelminthic activity of aqueous
fruit extract of different species of Ficus (Ficus bengalensis, Ficus
carica and Ficus religiosa). From the study it was concluded that the
aqueous fruit extract of Ficus bengalensis at dose of 37.5 mg/kg was
more effective as it killed all the test worms within an hour of
exposure.
3.3 Sida rhombifolia
1. Dhalwal K et al studied the antioxidant activity of the ethanolic
extract of roots and stems of Sida rhombifolia by superoxide radical
scavenging, nitric oxide scavenging and lipid peroxidase assay. It
was proved that the plant parts possessed antioxidant property83.
2. Dhalwal K et al studied the hepatoprotective activity of the aqueous
extract of roots of Sida rhombifolia against thioacetamide and allyl
alcohol induced toxicity in rats. The elevated levels of serum
enzymes alanine transaminase and aspartate transaminase were
lowered in the rats treated with the extracts.84
3. Gupta SR et al carried out the anti-arthritic activity of the aerial parts
of Sida rhombifolia. The study concluded that the polar constituents
of the plant were useful in the treatment of arthritis.85
4. Sarangi RR et al studied the antimicrobial activity of the petroleum
ether fruit extracts of Sida rhombifolia.86
5. Islam ME87 et al carried out the cytotoxicity and antimicrobial activity
of Sida rhombifolia grown in Bangladesh. It was shown that the ethyl
acetate extract of S.rhombifolia had a potent cytotoxic activity with
LC50 value of 5.41 ppm compared to gallic acid. The extract also had
mild antibacterial activity against both gram positive and gram
negative bacteria.
3.4 Ixora brachiata
1. Sadeghi-Nejad B et al carried out Invitro antifungal activity of Ethanolic
leaf and root extract of I.brachiata by Dilution Agar method. It was
shown that the extracts prevented the growth of tested dermophytic
species with MIC values between 5.0 to 10.0 and 2.5 to 10 mg/ml
respectively. Phytochemical screening of the extracts showed the
presence of starch, saponins, reducing sugars, anthraquinones,
phenols and proteins.88
3.5 Terminalia bellerica
1. Khan A-U & Gilani AH screened the effect of Terminalia bellerica in
hypertension. After administration of Terminalia bellerica observed
the fall in the arterial blood pressure of rats under anesthesia in
isolated guinea pig atria, inhibition of force and rate of atrial
contractions noted.89
2. Madani A et al studied the in vitro cellular toxicity and antisalmonella
activity of petroleum ether, chloroform, acetone, alcohol and
aqueous extract of the fruits of Terminalia bellerica. From the study it
was shown that alcoholic and aqueous extract had significant activity
against Salmonella and also good Invitro cellular toxicity study.90
3. Elizabeth KM et al conducted the antimicrobial activity of the
aqueous and methanolic fruit extract of Terminalia bellerica. The
study showed that the dry aqueous dry fruit extract at 4 mg
concentration showed highest zone of inhibition against S. aureus.
Methanolic extract of Terminalia bellerica also showed activity
against E.coli and P.aeruginosa.91
4. Saha S and Verma RJ studied the effect of aqueous extract of
Terminalia bellerica against DPPH activity. The fruits showed potent
antioxidant and hydroxyl radical scavenging activity.92
5. Saha et al has shown that herbal paste made from the fruits of
Terminalia bellerica and Terminalia chebula at 500 mg/kg showed
potent wound healing activity.93
3.6 Camellia sinensis
1. Ibrahim DA and Albadani RN studied the potential nephroprotective
and antimicrobial effect of Camellia sinensis and concluded that
Green tea-treated groups had nephroprotective effects as they
reduced the elevation in nonenzymatic kidney markers.94
2. S. Ramya and G. Prasanna studied the protective effect of Camellia
sinensis L. leaf extract on lead acetate-induced nephrotoxicity in
albino wistar rats by analyzing various renal parameters like urea,
uric acid creatinine and serum electrolytes sodium, potassium,
chloride, calcium, phosphorous and TBARS and decrease the levels
of haemoglobin and protein. The oral administration of aqueous
extract of Camellia sinensis along with lead acetate reversed these
altered parameters to normal level which indicated the
nephroprotective efficacy of the leaf extract against lead acetate-
induced kidney injury. From this, they concluded that phytochemical
constituents such as flavonoids which are present in the plant are
responsible for the nephroprotective activity of Camellia sinensis. 95
3. Akinyemi AJ et al studied the in vitro effect of tannic acid and
gallic acid and showed that cisplatin–induced thiobarbituric acid
reactive substances (TBARS) production was inhibited in rat kidney
in a dose-dependent manner. This inhibitory effect could be due to
their antioxidant properties which were proved by their DPPH radical
scavenging, Fe2+ chelating and reducing abilities. Furthermore, the
study provided further insight into the mechanism of action of their
nephroprotective properties from previous reported experimental
studies and confirm their antioxidant potential. However, tannic acid
possesses better antioxidant properties than gallic acid which could
be due to the number of functional groups present indicating that
hydrolysis affects its potency.96
4.0 Plan of Work
1. Collection and authentication of plant species.
2. Organoleptical and physicochemical studies of the dried plant
materials.
3. Extraction of dried plant material in soxhlet apparatus using various
solvents.
4. Preliminary chemical test of extracts to identify phytoconstituents.
5. Total Phenolic content
6. HPLC Quantification of extract.
7. Determination of In vitro antioxidant activity
Nitric oxide scavenging (Sreejayan, 1997)
Superoxide dismutase scavenging activity (Alkaline DMSO
method)
Lipid peroxidase activity (Rajkumar, 1994)
8. Acute toxicity studies of fruit extract of Terminalia bellerica, leaf
extract of Ficus bengalensis and Ixora brachiata
9. Effect of plant extract on Gentamicin and Cisplatin-induced
nephropathy in rats using following tests.
Determination of biochemical parameters:
Serum creatinine (Varley, 1980)
Blood urea nitrogen (Varley, 1980)
In vivo Antioxidant studies:
Superoxide dismutase (Winterbourn et al, 1975)
Glutathione-s-transferase [GST](Habig et al, 1974)
Glutathione [GSH] (Mannervik, 1985)
Lipid peroxidase activity (Uchiyama, 1978)
10. Histopathological studies
5.0 MATERIALS AND METHODS
Plants Selected for the Study
Medicinal plants for the study were selected on the basis of the
traditional use of these plants by the Paniyas, Karimbalas and Kurichia
tribes of Iritty hills in Kannur district. The selected plants are Ficus
bengalensis, Hemidesmus indicus, Sida rhombifolia, Ixora brachiata,
Camellia sinensis and Terminalia bellerica. The plants selected were
authentified by Dr. Sathya M., Professor, at Government Ayurveda
College, Pariyaram, Kannur. A voucher specimen (CCOPS-124) was kept
at the department of Pharmacognosy at Crescent College of
Pharmaceutical Sciences for future reference.
5.1 Plant Profile
5.1.1 Hemidesmus indicus
Botanical classification
Kingdom: Plantae
Family: Apocynaceae
Genus: Hemidesmus
Species: indicus
Fig 3: Hemidesmus indicus
Synonym: Indian sarsaparilla
Vernacular names
English: Country sarsaparilla
Hindi: Anantamul
Malayalam: Nannari, Naruninte
Tamil: Saribam, Nannari
Telugu: Sungadi pala
Biological origin: Hemidesmus indicus belonging to family Apocynaceae
is a perennial, fast growing thin creeper vine with woody root-stock and
numerous slender stems having thickened nodes. The leaves are simple,
opposite, elliptic oblong to linear lanceolate, variegated with white above,
silvery white and pubescent beneath. Flowers are greenish purple crowded
in sub sessile cymes in the opposite leaf axils. Fruits are slender follicles,
cylindrical, 10 cm (l) tapering to a point at the apex. It is found throughout
India.
Plant parts used: leaves, roots, whole plant.
Chemical constituents: Triterpenoid, lupeol, saponin, stigma sterol,
tannin, sarsaponin, β-sitosterol, smilagenin, hemidesminine, hemidescine.
Uses: Plant extract regulates the release of IGG by lymphocytes and
adenosine deaminase (ADA) activity, reduces excessive glucose levels
and increases the number of insulin receptors in humans by stimulating
glucose dependent insulin secretion from pancreatic beta cells. It lowers
the amount of total cholesterol, LDL, VLDL and triglycerides significantly.
Shows inhibitory effect on DNA synthesis and causes cytotoxicity against
tumor cells.97
5.1.2 Ficus bengalensis
Botanical classification
Kingdom: Plantae
Family: Moraceae
Genus: Ficus
Species: Bengalensis
Fig. 4: Ficus bengalensis
Vernacular names
English: Banyan
Hindi: Bal, Borgad
Malayalam: Peral
Tamil: Alomaram
Telugu: Peddamarri
Biological origin and parts used: Ficus bengalensis belonging to the
family Moraceae is a very large evergreen tree, extending laterally by
sending down aerial roots. It grows up to a height of 30 m and have wide
spreading branches with many aerial roots that function as prop roots.
Leaves are ovate to elliptic with sub cordate or rounded base. The parts
used are root bark, tender root, fruits, buds, latex, aerial roots and leaves.
Leaves are simple, alternate, stipulate 10-20 cm (l) and 5-12.5 cm (w).
Chemical constituents: The plant contains leucoanthocyanins, rutin,
beta-sitosterol, lupeol, β-amyrin, tannins.
Uses: The majority of traditional remedies of medicinal tonics are made
from plant roots. It is a good diuretic and increases flow of urine and are
useful in nephritic complaints. It is also effective as inflammatory, diuretic,
to improve fertility and treat syphilis.
5.1.3 Sida rhombifolia
Botanical classification
Kingdom: Plantae
Family: Malvaceae
Genus: Sida
Species: rhombifolia
Fig. 5: Sida rhombifolia
Vernacular names
English: Atibala
Hindi: Jamglimedhi
Malayalam: Kurunthotti
Tamil: Kurunthotti
Telugu: Cirubenda, ciltimulti
Synonym: Arrow leaf, jelly leaf, Malva rhombifolia
Biological origin and parts used: Sida plant is a large genus with about
200 species and distributed as weeds in all parts of India. It is found mainly
as shrubs, with stellate hairs, leaves are toothed. The flowers are axillary
or solitary. Sepals are five, yellow or white. Fruit is globose enclosed in
calyx.
It consists of whole plant of Sida rhombifolia. Petioles have small
spiny stipules at their bases. Flowers are delicate appears singly on flower
stalks and arises from the area between the stem and leaf petioles. They
are made of fine petals which are 4-8 mm (l), creamy to orange-yellow in
colour. Petals are asymmetric. Fruits are capsules that break into 8-10
segments. The plant bears the flowers throughout the year.
Chemical constituents:
Roots or aerial parts contain alkaloid, phenyl ethylamine. Alkaloids –
ephedrine and saponin (roots). The roots also contain choline, pseudo-
ephedrine, beta phenethylamine, vascine and related indole alkaloids.
Uses: The rejuvenating action of this herb extends to the nervous,
circulatory ad urinary systems. It has a diuretic effect and useful in urinary
problems. It is used in inflammations and bleeding disorders and is also
useful in the treatment of rheumatism and gonorrhea. The leaves can be
used as an infusion in the treatment of fever and delirium. The leaves and
roots are used as aphrodisiac.
5.1.4 Ixora brachiata
Botanical classification
Kingdom: Plantae
Family: Rubiaceae
Genus: Ixora
Species: brachiata
Fig. 6: Ixora brachiata
Synonym: Torchwood Ixora
Vernacular names
English: Jungle flame
Hindi: Ravgan
Malayalam: Ethi, Chethi
Tamil: Chethi
Telugu: Manmadibanum
Found in India in evergreen and semi-evergreen forests of Western Ghats.
The species is commonly seen as a small tree which attains a height
of 10-12 m tall with grey bark. Leaves are elliptic, oblong or lanceolate,
attenuated or narrowed base with obtuse apex. Flowers are 9-10 cm long,
with cymose inflorescence and white scent. Calyx, corolla (4), stamens (4),
filaments are short. Ovary has a bilocular style and is clothed with long
white hairs, stigma is fusiform. Fruits are berries with two seeds, which are
hemispherical with a length of 4-5mm. The tree flowers from November to
March.
Ixora brachiata is a plant that is used in the Indian traditional system
of medicine for a variety of ailments. Leaves are used to treat diarrhea,
roots for hiccough, fever, chronic ulcers and skin diseases. Flowers are
used in bronchitis and dysentery.
From a survey that was conducted in the hillocks of Madayipara in
Kannur district of North Kerala it was found that the leaves are being used
by the tribal to treat kidney disorders.
5.1.5 Terminalia bellerica
Botanical classification
Kingdom: Plantae
Family: Apocynaceae
Genus: Terminalia
Species: bellerica
Fig. 7: Terminalia bellerica
Vernacular names
English: Belliric myrobalan
Hindi: Bhaira
Malayalam: Tanni, Tannikka
Tamil: Tanrikkai
Telugu: Teyaku
Distribution: The tree is distributed throughout the Indian forests up to
914 m elevations especially in the foot hills of Himalaya.
Description: The large deciduous tree usually has height of 20-30 m and
bluish or ashy-grey bark with fine longitudinal crack. Leaves are clustered
at the ends of the branches, with simple, alternate, broadly ovate or elliptic
arrangement. Leaves have unequal bases. Glandular petiole is 2.5-7.5 cm
in length. Terminalia have slender, interrupted spikes of 8-15 cm length.
Flowers are 1.25 cm length dirty grey or greenish yellow with offensive
smell. Fruits are 2-5 cm long, ovoid, grey velvety with 5 or more furrows.
Dried fruits have irregular shapes and are available from November to
February. Seeds are thick walled and stone hard.
The tree is propagated through seeds, nursery buds, and coppices
well. It needs 1000-3000 mm of rainfall and is susceptible to frost and
drought.
Terminalia bellerica is considered as a sacred plant. Northern Indian
Hindus consider the plant to be inhabited by demons and avoided sitting
under the shade. Other Indians do not use the timber for building purposes
since they believe that no man can live in it too long. However, Terminalia
bellerica is a well-known medicinal plant and used for different ailments
traditionally. It is commonly used for cough, cold, skin diseases, and
immunity disorders. The plant mainly contains gallic acid, tannins, galloyl
glucose, ellagic acid, etc. It is one of the major constituent of Triphala, a
well-known Ayurvedic formulation used to improve immunity.
Chemical constituents: Main chemical constituents are tannins mainly
include β-sitosterol, gallic acid, ellagic acid, ethyl gallate, galloyl glucose,
and chebulagic acid. Fruit pulp contains cardioglycosides and kernels
contain nonedible oil.
Useful parts and their curative properties
Fruits: Alcoholic extract of the fruit possess bile stimulating activity,
anticancer activity and is used in cough, sores in mouth, diarrhea, fever,
contraceptive, headache, astringent, laxative, asthma, bronchitis, eye
disease, peptic ulcers, hemorrhoids, diabetes, leucorrhea, indigestion and
in general as a tonic, to provide strength, vigor, vitality.
Flower: Stomach disorders, brain tonic, eye lotion, treatment of piles,
leprosy, dropsy, fever
Gum: Urinary disorders
Seeds: Anti-alcoholism, hair tonic, strength, vigor, vitality, purgative
Stem bark paste: Jaundice, gastritis, overdose acts as a narcotic
poison.
5.1.6 Camellia sinensis
Botanical classification
Kingdom: Plantae
Family: Theaceae
Genus: Camellia
Species: sinensis
Fig. 8: Camellia sinensis
Synonyms: Green tea, Matsu-cha
Vernacular names
English: Tea
Hindi: Chai
Malayalam: Theyela
Tamil: Thailayi
Biological origin and parts used: Green tea is a shrub found in Asia. The
most important of the commercial species is Camellia sinensis, is native to
China. It is cultivated on a vast scale and has over 350 cultivars. It was
introduced to Europe in the 17th century. Many species are grown as
ornamental plants for their handsome, glossy foliage and fine flowers.
Description: Small, variable, ever green shrub with leathery elliptic leaves,
white flowers with yellow stamens are born in the axils during winter
followed by capsules containing large oily seeds. Both green tea and black
tea are made from the same plant, but more of the original substances
endure in the less processed green form. Green tea contains high levels of
polyphenols (strong antioxidants).
Plant part used: Dried leaves
Chemical constituents: Tannins, flavonoids, xanthine, lignin, organic
acids, proteins, vitamins.
Uses:
The major action of green tea results from its antioxidant and anticancer
(protective effects against cancer of stomach, intestine, colon, rectum, and
pancreas) actions.
Source of plants
Fresh plants selected for the study were collected from Payangadi
and Madayipara, Kannur in the month of December. They were shade
dried and reduced to a coarse powder. Botanical identities were confirmed
and voucher specimens have been deposited in the Department of
Pharmacognosy, Crescent College of Pharmaceutical Sciences, Kannur,
Kerala, India.
5.2 Extraction of Plant Materials
The plants selected for the study were Ficus bengalensis (leaves),
Hemidesmus indicus (whole plant), Ixora brachiata (leaves), Sida
rhombifolia (roots) Terminalia bellerica (fruits) and Camellia sinensis
(leaves). These were shade dried and powdered to coarse powder and
used for extraction.
The extraction of the plant materials was done on the basis of
selective solvent extraction. First the dried plant materials were defatted
using petroleum ether and then solvents were used in which the
phytoconstituents will be extracted easily and abundantly.
n-Butanol was used as the solvent to extract active constituents from
Ficus and Hemidesmus. Ethyl acetate was used as the solvent for the
extraction of Sida and Ixora and methanol was used for the extraction of
active constituents from Camellia and Terminalia.
About 2 kg each of the plant material was extracted using the
respective solvent in a soxhlet assembly until there was complete
extraction. It was filtered and the filtrate was concentrated in vacuum to
yield the extractive.
5.3 Physicochemical Properties
1. Organoleptic evaluation
Evaluation by sensory characters i.e., taste, appearance, odour, feel of the
drug were carried out on the dried powder.
2. Extractive values (WHO Guidelines 1996; I.P., Vol. II, 1996)
Total soluble constituents of the drug in any particular solvent or
mixture of solvents may be called as its extractive value or percent
extractives. It was studied on wet weight or dry weight basis by using
maceration, percolation, or continuous extraction by means of soxhlet
apparatus. Alcohol soluble extractive values are done for identifying
chemical constituents in the crude drug like tannins, glycosides, resins etc.
Water soluble extractive values are used for identifying the hydrolysable
tannins, mucilage etc.
i. Alcohol soluble extractive value
5 g of crude drug was accurately weighed. The coarsely powdered
and air dried material was macerated with 100 ml of 95% ethyl alcohol in a
stoppered flask for 24 hours. The flask was shaken frequently during the
first six hours. The content was filtered through a filter paper and
evaporated to dryness in a tared, flat bottom, shallow dish. The residue
was dried in a hot air oven at 105˚C. The residue was weighed and kept in
a desiccator.
ii. Water soluble extractive value
5 g of crude drug was accurately weighed. The coarsely powdered
and air dried material was macerated with 100 ml of water in a stoppered
flask for 24 hours. The flask was shaken frequently during the first six
hours. The content was filtered through a filter paper and evaporated to
dryness in a tared, flat bottom, shallow dish. The residue was dried in a hot
air oven at 105˚C. The residue was weighed and kept in a desiccator.
3. Ash values (I.P., Vol. II, 1996; Harborne, 1984, WHO
Guidelines 1996)
The ash of any organic material is composed of their nonvolatile
inorganic compounds, i.e., controlled incineration of crude drug results in
an ash residue consisting of an inorganic material. Determination of ash
value is useful for detecting low grade products, exhausted drugs, earthy
matter and other substances used to improve the appearance of the drugs.
Total ash consists of carbonates, phosphates and silicates.
i. Total ash value
About 2 to 3 g of the ground drug was weighed and spread as a fine
layer at the bottom of a tarred silica crucible. The crucible was incinerated
at a temperature not exceeding 450ºC until free from carbon. The crucible
was then cooled and weighed. The entire procedure was repeated until a
constant weight was observed. The percentage of total ash with reference
to the air-dried sample was calculated.
ii. Acid insoluble ash
To the ash obtained in total ash, 25 ml of 2N hydrochloric acid was
added and boiled for five minutes. The insoluble matter was collected in a
Gooch crucible or on an ashless filter paper and washed with hot water
and ignited to constant weight at a temperature not exceeding 600oC. The
percentage of acid-insoluble ash with reference to the air-dried drug was
calculated.
iii. Water soluble ash
The ash obtained was boiled for 5 minutes with 25 ml of water. The
insoluble matter was collected in a Gooch crucible or on an ashless filter
paper, washed with hot water and ignited for 15 minutes at a temperature
not exceeding 450ºC. The weight of the insoluble matter was subtracted
from the weight of the ash; the difference in weight represents the quantity
of water-soluble ash. The percentage of water-soluble ash with reference
to the air-dried drug was calculated.
4. Moisture content
About 2 g of the powdered material was weighed accurately and
spread as a fine layer in a tared petri dish and heated at 105ºC in an oven
to a constant weight. The petri dish was cooled and weighed. The
percentage loss on drying was calculated.
5.4 Qualitative phytochemical analysis
The plant extracts were subjected to the following tests based on
WHO guidelines for identifying the chemical constituents present.
1. Detection of alkaloids
a. Mayer’s test (potassium mercuric iodide)
To the extract, Mayer’s reagent was added and observed for the
formation of cream color which indicated the presence of alkaloids.
b. Dragendroff’s test (potassium bismuth iodide)
To the extract, Dragendroff’s reagent was added. The mixture was
heated and observed for the formation of reddish orange color precipitate
which indicated the presence of alkaloids.
c. Wagner’s test (iodide in potassium iodide)
To the extract, Wagner’s reagent was added and the mixture was
shaken. Formation of reddish brown color precipitate indicated the
presence of alkaloids.
d. Hager’s test (picric acid)
To the extract, Hager’s reagent was added and observed for the
formation of yellow colored precipitate which confirmed the presence of
alkaloids.
2. Detection of carbohydrates and glycosides
i. Molisch’s test
The extract was treated with a few ml of 2-naphthol solution. A few
drops of sulfuric acid was added through the sides of the test tube and
observed for the formation of violet ring at the junction of the two solutions.
ii. Fehling’s test
The extract was treated with Fehling solution A and B. This mixture
was heated on a water bath and observed for the formation of red
precipitate of cuprous oxide.
iii. Barford’s test
The extract was treated with Barford’s reagent and observed for the
formation of red precipitate.
iv. Benedict’s test
The extract was treated with Benedict’s reagent. This mixture was
heated on a water bath for a few minutes and observed for the formation of
orange red precipitate.
v. Borntrager’s test
The extract was treated with ferric chloride. This mixture was heated
on a water bath for 15 minutes. The mixture was cooled and shaken with
equal volume of benzene. The benzene layer was separated. This
benzene layer was added with half the volume of ammonia solution and
shaken vigorously for the formation of rose pink to cherry red color in the
ammoniacal layer.
3. Detection of fixed oils and fats
i. Spot test
A small portion of extract was pressed in between two filter papers.
Oil stains indicates the presence of fixed oils and fat.
ii. Saponification test
To the extract a few drops of 0.5 N alcoholic potassium hydroxide
and a drop of phenolphthalein was added. The mixture was heated on a
water bath for 1-2 hours and observed for the formation of soap or partial
neutralization of alkali.
4. Detection of phenolic compounds and tannins
i. Ferric chloride test
A few drops of ferric chloride solution was added to the test solution
and observed for the formation of brownish color.
ii. Lead acetate test
Ten percent lead acetate solution was added to the test solution and
observed for the formation of white precipitate.
iii. Gelatin solution test
One percent of gelatin containing sodium chloride solution was
added to the test solution and observed for the formation of white
precipitate.
4. Detection of flavones and flavonoids
i. Aqueous sodium hydroxide test
Aqueous sodium hydroxide solution was added to the test solution
and observed for the formation of yellow-orange color.
ii. Conc. sulphuric acid test
Concentrated sulphuric acid was added to the test solution and
observed for the formation of orange color.
iii. Schinoda’s test
To a small fraction of the extract a piece of magnesium was added
followed by conc. hydrochloric acid, heated slightly and observed for the
formation of dark pink color.
5. Total Phenolic Content
Phenol was extracted by boiling 100 mg of dry sample in 10 ml of
95% ethanol in a water bath for 5 minutes. It is then centrifuged at 2000
rpm and the supernatant is decanted off. The process is repated twice.
The ethanol extract was pooled and evaporated to remove the alcohol and
then diluted to 100 ml with distilled water.
The phenolic extract was allowed to react with Folin Ciocalteu
reagent.
9 ml of distilled water, 1 ml reagent, 1 ml extract was mixed
thoroughly and allowed to stand for three minutes. I ml of 20% sodium
carbonate was mixed with the reaction mixture and heated in a boiling
water bath for 5 minutes. The blue colour developed and the absorbance
was measured at 715 nm by using a colourimeter.
5.5 Preparation of extracts for HPLC quantification
To prepare the samples, 10 gm of dried plant material were
powdered, mixed with methanol (50 ml) for 48 hours and the methanolic
extract was filtered through ultra-cellular nitrate membrane filter and used
for HPLC analysis.
1. Instrument
High performance liquid chromatograph instrument of Shimadzu (VP
series) containing LC-10AT pump, a binary gradient system equipped with
universal Rheodyne injector with injection volume 20 µl fixed loop, variable
wavelength programmable UV/Vis-detector SPD-10AVP, Phenomenex C18
column with 250 x 4.6 mm i.d. and 5 µm particle size were used for the
study. Class VP software was used for data analysis.
2. Standards and chemicals
HPLC grade chemicals were used for the study. The reference standards
lupeol, β-sitosterol and ellagic acid were procured from Sami Labs,
Bangalore.
All determinations were performed at ambient column temperature.
The drug content was calculated by using the formula
Sample area x Standard Weight x Purity % w/w Standard area x Sample weight
5.6 In vitro antioxidant studies
5.6.1 Nitric Oxide Scavenging Activity (Sreejayan, 1997)
Nitric oxide is a very unstable species under the aerobic condition.
It reacts with oxygen to produce the stable product nitrates and nitrite
through intermediates NO2, N2O4 and N3O4. It was estimated by using
Griess reagent. In the presence of the test compound, that acts as a
scavenger, the amount of nitrous acid will decrease. The extent of
decrease will reflect the extent of scavenging which can be measured at
546 nm.
Griess reaction
Griess reagent preparation: Solution A: 1% sulphanilamide in 5%
orthophosphoric acid or 25%v/v hydrochloric acid; Solution B: 0.01%
napthyl ethylene diamine in distilled water. Solution A and Solution B were
mixed in equal volumes within 12 hours of use.
Sodium nitroprusside 5 mM (0.0373 g in 25 ml) was prepared in
phosphate buffer of pH 7.4. To 1 ml of various concentrations of the extract
3 ml sodium nitroprusside was added. The test tubes were incubated at
25ºC for 5 hours. After 5 hours, 0.5 ml of Griess reagent was added. The
absorbance was measured at 546 nm. The experiment was performed in
triplicate.
% Scavenging = Control – Test X 100 Control
5.6.2 Superoxide dismutase scavenging activity (Alkaline DMSO
method)
Alkaline DMSO is used as superoxide generating system. The
generated superoxide will react with NBT to give coloured diformazan.
Diformazan being insoluble in water slowly precipitates out. Therefore, the
spectral measurement must be done immediately after the reaction is
carried out. In the presence of scavenger, reduction of NBT will occur
which is measured at 560 nm.
Reduction of NBT
To 0.5 ml of different concentrations of the extract,1 ml alkaline
DMSO and 0.2 ml Nitro Blue Tetrazolium (NBT) 20mM (50 mg in 10 ml
phosphate buffer pH 7.4) was added. The absorbance was measured at
560 nm. The experiment was performed in triplicate.
% Scavenging = Control – Test x 100 Control
5.6.3 Lipid peroxidation111
Malondialdehyde formed from the breakdown of polyunsaturated fatty
acids serves as a convenient index for determining the extent of
peroxidation reaction. Malondialdehyde reacts with thiobarbituric acid to
form TBARS to give red colour species which was measured at 535 nm.
Formation of TBARS from malondialdehyde
Reagent: Stock TBA-TCA-HCI reagent: 15% w/v trichloroacetic acid,
0.375 % w/v Thiobarbituric acid and 0.25N hydrochloric acid. This solution
was mildly heated to assist the dissolution of TBA.
Preparation of rat liver homogenate: Albino rats (180-200g) of either
sex were used for the study. After decapitation, the liver was removed
carefully. The tissue was immediately weighed and homogenated with ice
cold 1.15% KCI to make 10% homogenate. This homogenate was
immediately used for the in vitro lipid peroxidation study.
Method: 0.5 ml of rat liver homogenate was added to 1ml of various
concentrations of the extract. Then the mixture was incubated for 30
minutes. The peroxidation was terminated by the addition of 2 ml of TBA-
TCA-HCI reagent. The solution was heated for 15 minutes in a boiling
water bath. After cooling, the flocculent precipitate was removed by
centrifugation at 1000 rpm for 10 minutes. The absorbance of the
supernatant was measured at 535 nm. The experiment was performed in
triplicate.
% Scavenging = Control – Test x 100 Control
5.7 Pharmacological Studies
Animal: Healthy adult male albino rats of Wister strain weighing
between 150-200 g was used for the study. The rats were housed in
cages maintained in a temperature regulated and humidity controlled
environment. The rats were fed with food pellets and water ad libitum.
The Institutional Animal Ethical Committee (IAEC No. 282) with Reg.
No. CADD/42 approved the study.
Drugs and chemicals: Cisplatin injection (Biochem Pharmaceutical
Industries, Mumbai), Gentamicin (Biochem Pharmaceutical Industries,
Mumbai), Urea Estimation Kit (Agape Diagnostics, Maharashtra),
Creatinine estimation Kit (Agape Diagnostics, Maharashtra).
5.7.1 Acute toxicity Studies112
Oral acute toxicity studies were carried out using Swiss albino mice
weighing 40-50 gm using three mice per group as per OECD guidelines
423. The rats in four groups were fed with the constituents suspended in
2% gum acacia from 5 mg/kg b.w. – 2000 mg/kg b.w. The animals were
observed for gross behavioral changes intermittently every 2 hours and
finally at the end of 24 and 72 hours for signs of toxicity including death.
LD50 was calculated using 1/10th and 1/5th of the highest dose.
5.7.2 Nephroprotective Activity113
The protective and curative effect of the various extracts of Ficus
bengalensis, Sida rhombifolia and Ixora brachiata on the kidneys was
assessed using cisplatin and gentamicin-induced models. The dose
selected was based upon acute toxicity studies. Eighteen male Wistar rats
were assigned into five groups for the study.
Table 2: Cisplatin treatment regimen to assess Cisplatin Toxicity
Group Treatment Day of Biochemical Estimation
Purpose
I (normal) Vehicle (2%w/v gum acacia)
5, 15, 16 Normal control
II (Toxic) Cisplatin 5 mg/kg (i.p) (1st day) Vehicle (6th - 15th day)
5,16 Curative control
IIIa (Cisplatin + Plant extract + 200 mg/kg)
Cisplatin 5 mg/kg i.p (1st day) Plant extract (6th - 15th day) 200 mg/kg (p.o)
5, 16 To assess curative effect
IIIb (Cisplatin + Plant extract + 400 mg/kg)
Cisplatin 5 mg/kg i.p (1st day) Plant extract (6th - 15th day) 400 mg/kg (p.o)
5,16 To assess curative effect
IV (Preventive control)
Vehicle (1st – 10th day) Cisplatin 5 mg/kg (i.p) 11th day
15 Preventive control
V (Preventive effect)
Plant extract 400 mg/kg (p.o) (1st -10th day) + Cisplatin 5 mg/kg (i.p) (11th day)
15 To assess preventive effect
Table 3: Gentamicin treatment regimen to assess gentamicin toxicity
At the end of experimental period, all the animals were sacrificed
under diethyl ether anesthesia, blood samples were collected through retro
Group Treatment Day of Biochemical Estimation
Purpose
I (normal) Vehicle (2%w/v gum acacia)
9 Normal control
II (Toxic) Gentamicin 80 mg/kg i.p. (8 days)
9 Toxic group
IIIa (Gentamicin + Plant extract 200 mg/kg)
Gentamicin 80 mg/kg i.p. (1st – 13th day) + Plant extract (from 14th - 23rd day) 200 mg/kg [p.o]
24 To assess curative effect
IIIb ( Gentamicin + Plant extract + 400 mg/kg)
Gentamicin 80 mg/kg i.p. (1st – 13th day) + Plant extract (from 14th - 23rd day) 400 mg/kg [p.o]
24 To assess curative effect
IV (Preventive control)
Vehicle (1st – 13th day) + Gentamicin 80 mg/kg i.p. (14th – 23rd day)
24 Preventive control
V (Preventive effect)
Extract (400 mg/kg [p.o]) 1st -13th day + Gentamicin 80 mg/kg i.p. (14th- 23rd day)
24 To observe Preventive effect
orbital vein, and serum was separated by centrifugation and used for
analysis of various biochemical parameters like serum creatinine and
blood urea nitrogen.
Animals were anesthetized by diethyl ether anesthesia and kidneys
were dissected out. The kidneys were perfused in ice-cold saline. The
kidneys were removed, blot dried, weighed and a 10% homogenate was
prepared with ice-cold 1.15% KCI that makes up 10% homogenate using a
homogenizer. The homogenate so prepared was used for the estimation of
SOD, GSH, GST and TBARS.
Histopathological examination
Examination of renal histology was performed according to routine
histology techniques. Kidneys were fixed in 10% formalin, dehydrated
stepwise with increasing concentration of ethanol (50% to 100%) and
embedded in paraffin. Using a microtome, tissue sections of 4 µm
thicknesses were produced and then stained with hematoxylin and eosin
and observed under a light microscope (Olympus BX41, Japan).
Parameters assessed for renal functions
Blood urea: Urea concentration in the blood was estimated by
enzymatic method using urease enzyme kit (Varley, 1980) and modified
Berthelot method.106
Serum creatinine: Creatinine level in serum was estimated by alkaline
picrate method using creatinine kit.106
Determination of in vivo antioxidant activity
Estimation of Lipid peroxidation112
Reagents: TBA-TCA-BHT reagent-2.8% TCA, 0.375% TBA and 50 mg
BHT in 100 ml of double distilled water.
Procedure: 0.5 ml of 10% tissue homogenate was pippeted out into a
10 ml centrifuge tube and 2.5 ml of TBA-TCA-BHT reagent was added
and shaken well. The mixture was incubated for 5 minutes. After
incubation the mixture was heated to 80oC for 10 min in a water bath.
The mixture was centrifuged at 200 rpm for 20 min. Absorbance of the
supernatant was measured at 532 nm.
Estimation of Glutathione-s-transferase [GST]110
The enzyme is assayed by its ability to conjugate GSH and 1-
chloro, 2, 4, dinitrobenzene (CDNB). The extent of conjugation causes
a proportionate change in the absorbance at 340 nm.
Reagents: Phosphate buffer pH 6.5 (1.7418 gm potassium dihydrogen
phosphate in 100 ml distilled water), GSH (30 mM; 16 mg GSH in 2ml
double distilled water, DDW), 62 mg CDNB in 10 ml ethanol.
Procedure
0.1 ml CDNB was added to 0.6 ml of supernatant homogenate and
2.2 ml of phosphate buffer having pH 6.5. This was incubated at 37ºC for 5
minutes and 0.1 ml of 30 mM GSH was added. Absorbance was measured
at 340 nm at intervals of 1, 2, 3, 4, and 5 minutes. Absorbance of blank
was determined in the same manner after omitting the sample.
Estimation of Glutathione [GSH]111
Reagents: 5% Trichloroacetic acid, Phosphate buffer 0.2 M pH 8.0,
0.218 g sodium dihydrogen phosphate was dissolved in 100 ml distilled
water and DTNB-20 mg in 50 ml phosphate buffer (0.6 mM, pH 8.0).
Procedure: Proteins was precipitated by 5% TCA, centrifuged and the
supernatant was collected.0.5 ml of the supernatant was mixed with 3
ml of 0.2 M sodium phosphate buffer and 0.5 ml of 0.6 mM DTNB and
incubated for 10 minutes at room temperature. The absorbance of the
samples was recorded against blank at 412 nm.
Estimation of Superoxide dismutase109
Reagents: 0.067 M Potassium phosphate buffer pH 7.8, 0.1 M
Ethylene diamine tetra acetic acid (EDTA) containing 0.3 mM sodium
cyanide, 0.12 mM Riboflavin (store in cold and dark bottles) and 1.5
mm Nitro Blue Tetrazolium (NBT) (store cold).
Procedure: 0.2 ml EDTA, 0.1 ml NBT 0.1 ml enzyme and phosphate
buffer q.s to 3 ml were pippeted into a series of test tubes. The tubes
were incubated for 5-8 minutes at room temperature. 0.05 ml of
Riboflavin was added at time intervals. All the tubes were incubated in a
light box for 12 minutes and at timed intervals. The absorbances of the
samples were recorded at 560 nm. The amount of enzyme resulting in
one half of maximum inhibition was determined using the following
formula:
Units/mg = 1000/μg enzyme resulting in ½ max inhibition.
5.8 Statistical analysis
The values are expressed as MEAN±SEM. The statistical analysis
was performed using one way ANOVA followed by Bonferroni multiple
comparison tests using statistical software program Instat Graph pad. The
P values less than 0.05 were considered significant.
6.0 Results & Discussion
6.1 Physicochemical Properties
The physicochemical properties of the plant materials were
determined and are shown in Table 4.
Table 4: Physicochemical Parameters of Crude Drugs
Crude drug Total ash (%w/w)
Acid insoluble
ash (%w/w)
Water soluble
ash (%w/w)
Alcohol soluble extractive (%w/w)
Water soluble
extractive (%w/w)
Foreign matter (%w/w)
Ficus bengalensis
6.2 1.8 15.5 8.2 10.3 1.6
Hemidesmus indicus
2.6 0.5 14.6 12.6 9.5 1.4
Ixora brachiata
5.8 0.8 6.2 5.8 6.2 1.5
Sida rhombifolia
4.5 2.2 5.3 3.2 1.5 1.2
Camellia sinensis
13.2 5.6 16.2 6.8 2.2 1.3
Terminalia bellerica
3.1 0.8 6.5 12.2 5.5 1.8
6.2 Percentage Yield of Extracts of Plants
The plant parts were extracted and the percentage yield of the
extract was calculated and tabulated in Table 5.
Table 5: Solvent Extraction of Air Dried Plant Material
Plant Name Parts Used % Yield (w/w) Colour and
Consistency of the extract
Ficus bengalensis Leaf 13.6 Greenish Hemidesmus indicus Whole plant 14.4 Reddish brown Sida retusa Root 9.8 Brownish Ixora brachiata Leaves 10.4 Yellowish green Terminalia bellirica Fruits 8.2 Dark brown Camelia sinensis Leaves 10.6 Greenish black
Phytochemical screening of the extract showed the presence of
various constituents like triterpenoids, phenols, saponins, steroids, tannins
and alkaloids. The phytochemical screening of the extract is tabulated in
Table 6.
Table 6: Phytochemical Screening of Plant Extracts
Crude Drug Alkaloids Glycosides Steroids Tannins Triterpenoids Flavanoids
Phenolic
compounds
Saponins
Ficus bengalensis + - + + + + + +
Hemidesmus indicus + - - + + + - +
Ixora brachiata - + + + + + + -
Sida rhombifolia + _ + + - - - -
Camellia sinensis - + + + + + _ +
Terminalia bellerica + - + + + + + -
+ Present, - Absent
6.2 Total Phenolic Content From the preliminary phytochemical screening of the extract it was
seen that the extracts of Terminalia bellerica, Ficus bengalensis and Ixora
brachiata showed the presence of phenolic compounds. So the extracts of
these plants were analyzed for their total phenolic content.
In a study conducted by Bray & Thorpe, the total phenolic content of
Terminalia bellerica, Ficus bengalensis and Ixora brachiata was 78.6±0.18,
23.2±0.61 and 21.02±0.19 mg/g/GEA respectively. The high phenolic
content in Terminalia bellerica may be due to the presence of tannins in
the plant extract. Phenolic compounds may contribute directly to
antioxidant activity because of their hydroxyl groups which confer free
radical scavenging ability.
6.3 Quantitative Estimation by HPLC Analysis
The major active components in all the six extracts were analyzed
using HPLC. The results of HPLC analysis of three important secondary
metabolites are discussed below. The HPLC profile of sample marker
compounds are given in Fig. 9, and retention times of the marker
compounds are given in table 7.
Table 7: Retention time of Marker compounds
Sl. No. Marker compound Retention time
1. Lupeol 7.3 2. β-sitosterol 1.1 3. Ellagic acid 1.8
Fig. 9: HPLC Chromatogram of marker compounds A – Ellagic acid;
B-Lupeol; C – β-sitosterol
The HPLC chromatogram of secondary metabolites from
F.bengalensis, H.indicus, S.retusa, I.brachiata, T.bellerica and C.sinensis
are given in Fig. 8 to 10. Each compound was identified by overlapping the
HPLC profile of marker compound with a particular retention time with that
of the sample profile. Area of each secondary metabolite in the sample and
its quantity (%weight) was also calculated with the help of the formula
given above.
Table 8: Percentage of Lupeol, β-sitosterol and Ellagic acid in Plant Extracts
Name of the Plant Part of the plant Amount (%w/w) Ficus bengalensis Leaves 8.13
Hemidesmus indicus Whole plant 6.67 Ixora brachiatia Leaves 7.28 Sida rhombifolia Roots 5.07
Camellia sinensis Leaves 3.18 Terminalia bellirica Fruits 8.29
The HPLC chromatogram of samples of F.bengalensis, H.indicus
(Fig. 10), I.brachiata, S.retusa (Fig.11) and T.bellerica, C.sinensis (Fig.12)
are shown. The secondary metabolites present in different samples were
quantified by comparing the area and retention time of standard compound
in HPLC using the equation given in Table 7 & 8.
Lupeol (Fig. 10)
The amount of lupeol was estimated in leaf extract of Ficus
bengalensis and whole plant extract of Hemidesmus indicus. The leaves of
Ficus bengalensis contain 8.13% w/w whereas the whole plant of
Hemidesmus indicus contains 7.6% w/w of lupeol.
β-sitosterol (Fig. 11)
The plants of Sida rhombifolia (roots) and Ixora brachiata (leaves)
was used for the estimation of β‐sitosterol. The roots of Sida rhombifolia
contain 7.07%w/w while the leaves of Ixora brachiata contain 7.28% w/w of
β‐sitosterol.
Ellagic acid (Fig. 12)
The fruits of Terminalia bellerica and leaves of Camellia sinensis
were used for the estimation of ellagic acid. The fruits of Terminalia
bellerica contained 8.2%w/w and the leaves of Camellia sinensis contained
3.17% w/w of ellagic acid.
Fig. 10: HPLC Chromatogram of marker compound from crude
butanol extract of F.bengalensis and H.indicus
Fig. 11: HPLC Chromatogram of marker compound from crude ethyl
acetate extract of S.rhombifolia and I.brachiata
Fig. 12: HPLC Chromatogram of marker compound from crude
methanolic extract of T.bellerica and C.sinensis
6.4 In vitro Antioxidant Activity studies
In vitro antioxidant methods used for the evaluation of antioxidant
activities of the plant extracts include superoxide, nitric oxide and lipid
peroxidase assay. The data shown in figure 15 explains the antioxidant
potential of the extracts. IC50 values were calculated using linear
regression analysis.
The extracts were found to scavenge superoxide generated by
riboflavin. The concentration needed for 50% scavenging of superoxide
was 180.14 µg/ml for T. bellerica, 220.26 µg/ml for C. sinensis, 208.29
µg/ml for I. brachiata, 331.24 µg/ml for S. rhombifolia, 240.17 µg/ml for H.
indicus and 364.43 µg/ml for F. bengalensis.
The extracts were found to scavenge the nitric oxide generated from
sodium nitroprusside at physiological pH. The concentration needed for
50% scavenging of nitric oxide was 136.4 µg/ml for T. bellerica, 176.26
µg/ml for C. sinensis, 184.23 µg/ml for I. brachiata, 261.34 µg/ml for S.
rhombifolia, 184.56 µg/ml for H. indicus, and 287.11 µg/ml for F.
bengalensis.
The generation of lipid peroxides by Fe2+/ascorbate in rat liver
homogenate was found inhibited by the addition of extracts. The
concentration needed for 50% inhibition was 188.04 µg/ml for C. sinensis,
164.18 µg/ml for T. bellerica, 198.7 µg/ml for I. brachiata, 259.18 µg/ml for
S. rhombifolia, 183.57 µg/ml for H. indicus and 208.27 µg/ml for F.
bengalensis.
The present study revealed that the antioxidant activity of the
extract. The free radicals were well scavenged by the extracts in-vitro
which was comparable that of known antioxidant ascorbic acid.
Table 9: In vitro Antioxidant activity of Extracts against Lipid peroxidase Scavenging Assay
Sl. No. Test compounds IC50 values ± S.E
(mcg/ml) Ascorbic acid (IC50 mcg/ml)
1. Ficus bengalensis 183.57
166.17
2 Hemidesmus indicus 208.27 3 Ixora brachiata 198.70 4 Sida rhombifolia 259.18 5 Terminalia bellerica 164.18 6 Camellia sinensis 188.04
Table 10: In vitro Antioxidant activity of Extracts against Nitric Oxide Scavenging Assay
Sl. No. Test compounds IC50 values ± S.E
(mcg/ml) Ascorbic acid (IC50 mcg/ml)
1. Ficus bengalensis 184.56
128.65
2 Hemidesmus indicus 287.11 3 Ixora brachiata 174.70 4 Sida rhombifolia 261.18 5 Terminalia bellerica 136.47 6 Camellia sinensis 176.24
Table 11: In vitro Antioxidant activity of Extracts against Superoxide Radical Scavenging Assay
Sl. No. Test compounds IC50 Values ± S.E
(mcg/ml) Ascorbic acid (IC50 mcg/ml)
1. Ficus bengalensis 240.17
167.33
2 Hemidesmus indicus 269.32 3 Ixora brachiata 208.29 4 Sida rhombifolia 331.42 5 Terminalia bellirica 180.14 6 Camellia sinensis 220.26
Fig. 13: Graph showing IC50 values in antioxidant studies for plant extracts
FB – Ficus bengalensis; HI – Hemidesmus indicus; SR – Sida rhombifolia; IB – Ixora brachiata; TB – Terminalia bellerica; CS – Camellia sinensis;
Std – Ascorbic acid
0
50
100
150
200
250
300
350
400
Lipidperoxidase activity
Nitricoxide activity Superoxide scavenging
IC50
FB
HI
SR
IB
TB
CS
Std
6.5 Pharmacological studies of extracts of Ficus bengalensis, Ixora
brachiata and Terminalia bellerica
6.5.1 Criteria for Selection of Plants for Pharmacological Activity
According to quantification data derived from HPLC studies plant
extracts possessing higher percentage of lupeol, β-sitosterol and ellagic
acid and better IC50 values were chosen for pharmacological studies. Thus
methanolic fruit extracts of Terminalia bellerica, butanolic leaf extract of
Ficus bengalensis and ethyl acetate leaf extract of Ixora brachiata were
further selected from among the six plants initially chosen.
The estimated antioxidant levels were highest in Terminalia
bellerica, Ixora brachiata and Ficus bengalensis compared to Hemidesmus
indicus, Sida retusa and Camellia sinensis. Moreover the IC50 value
according to the three methods of lipid peroxidase, nitric oxide, superoxide
scavenging activity further confirmed that the above selected plants had
the maximum antioxidant effect. Therefore the former plants were selected
for the study in place of the latter.
6.5.2 Acute toxicity studies
Administration of the n-buatnol extract of Ficus bengalensis, ethyl
acetate extract of Ixora brachiata and methanolic extract of Terminalia
bellerica orally, produced no observable side effects including death up to
2000 mg/kg body weight in rats even after 72 hours of observation.
6.6 CISPLATIN TOXICITY
Oxygen free radicals are the main cause of cisplatin-induced acute
renal failure. It has been shown that cisplatin nephrotoxicity in
experimental animal models cause acute renal failure. Cisplatin at the
dose of 8 mg/kg produced renal dysfunction in the present study which
was evidenced by increase in the levels of serum creatinine (1.41±0.462 –
4.28±0.559 mg/dL) and BUN (18.056±0.73 - 45.69±4.14 mg/dL).
Treatment of animals with the methanolic fruit extract of Terminalia
bellerica at a dose of 400 mg/kg b.w showed a significant decrease in the
levels of serum creatinine (4.28±0.559 – 1.24±0.12 mg/dL; P<0.01). The
butanolic leaf extract of Ficus bengalensis at the same dose level also
showed a significant curative activity (4.28±0.559 - 1.46±0.61 mg/dL;
P<0.01). The ethyl acetate leaf extract of Ixora brachiata (4.28±0.559 –
1.92±0.33; P<0.01) also showed a decrease in serum creatinine.
Preventive control animals treated with cisplatin showed a significant
(P0.01) increase in the levels of serum creatinine when compared with
the normal animals. On preventive treatment with 400 mg/kg of the
methanolic fruit extract of T. bellerica, slight decrease in the levels of
serum creatinine (3.36±1.37-3.09±0.14 mg/dL) was observed.
Preventive treatment with 400mg/kg of butanolic leaf extract of F.
bengalensis, slightly decreased the levels of elevated serum creatinine
when compared to preventive control (1.82±0.91 - 1.58±0.155 mg/dL).
Preventive treatment with 400mg/kg ethyl acetate leaf extract of I.brachiata
slight decrease in the elevated levels of serum creatinine was observed
when compared to the preventive control group (1.82±0.12 – 1.44±1.88
mg/dL)[Table 12; Fig.14]
Fig. 14: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of serum creatinine on cisplatin-induced nephrotoxic models
The increased levels of BUN were also decreased in all the extracts
of the plants under investigation at dose levels of 400 mg/kg b.w TB:
(45.69±4.14 – 22.11±0.12 mg/dL; P<0.001). FB (45.69±4.14 - 25.88±0.543
mg/dL; P<0.001) and IB (45.69±4.14 – 38.56±2.68 mg/dL). In the present
study, oral administration of the plant extract to CP–intoxication normalized
serum creatinine and BUN which suggest that the extracts have protective
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
NormalControl T.B FB IB TB FB IB
200 mg/kg
400 mg/kg
Pre ctrl
Prev.act
action against CP–induced nephrotoxicity by attenuating the oxidative
stress induced by administration of cisplatin.
It was observed that there was a significant increase in levels of
blood urea nitrogen in preventive control group 18.056±0.73 - 81.04±0.73
mg/dL (P0.01) when compared to the normal group of animals.
Preventive treatment with 400 mg/kg of the extract of the fruits of T.
bellerica showed a slight decrease in the levels of blood urea nitrogen as
compared to the preventive control group (81.96±0.64 - 64.69±0.63 mg/dL
P�0.001 when compared with the preventive control group). Preventive
treatment with 400 mg/kg leaf extract of F. bengalensis showed a slight
decrease in the levels of BUN when compared to the preventive control
group(81.96±0.64-76.04 ± 0.28 mg/dL P�0.01). However on treatment with
a preventive dose of 400mg/kg of ethyl acetate leaf extract of I. brachiata
showed a slight decrease in the levels of blood urea nitrogen as compared
to preventive control group (81.96±0.026 - 72.62±1.11 mg/dL) Table 12;
Fig.15].
Fig 15: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of blood urea nitrogen on cisplatin-induced nephrotoxic model
CP intoxication causes significant decrease in the levels of renal
antioxidants like GST, SOD and GSH when compared to the normal. The
oxidation of biomolecules in the kidneys is due to generation of ROS, by
CP. The excessive ROs can damage the protein sensitive thiol thereby
inhibiting activities of the antioxidant enzymes SOD which depletes the
thiol cellular content. In the present work, consumption of extracts
improved the levels of antioxidant enzymes GSH and SOD.
The SOD levels of CP intoxicated group was decreased (12.56
±0.97 – 5.16 ± 0.64 µg/mg protein; P<0.001). But on treatment with various
extract of Terminalia bellerica, Ficus bengalensis, Ixora brachiata with
dose of 400 mg/kg b.w the levels of SOD was found to increase
0
10
20
30
40
50
60
70
80
90
Normal Control TB FB IB TB FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Pre.act
significantly (TB: 5.16±0.64 – 9.05±0.44 µg/mg protein; P<0.001, FB -
5.16±0.64 – 8.46±0.44 µg/mg protein; P<0.001 and IB 5.16±0.64 –
6.21±1.33 µg/mg protein; P<0.05).
Preventive control of animals treated with cisplatin decreased the
levels of SOD as compared to the normal group (12.56 ±0.97 – 5.45 ±0.66
µg/mg protein P�0.001). Preventive treatment with 400mg/kg of methanolic
extract of the fruits of T. bellerica slightly increased the levels of SOD as
compared to the preventive control group (5.45±0.66 – 6.01±0.90 µg/mg
protein). Preventive treatment with butanolic leaf extract of Ficus
bengalensis at a dose of 400 mg/kg showed an increase in SOD activity
when compared with the preventive control group (2.38±0.38 to 4.68±1.28
µg/mg protein). Preventive treatment with 400 mg/kg of the ethyl acetate
leaf extract of I. brachiata increased the levels of SOD as compared to
preventive control (5.18±0.66-3.76±1.4 µg/mg protein) [Table 12; Fig.16].
Fig. 16: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of SOD on cisplatin-induced nephrotoxic models
There is a depletion of GSH, due to increased creation of ROS. This
is due to increased consumption of GSH in non-enzymatic removal of
oxygen radicals after kidney toxicity (5.406±0.36 -1.04±0.14 µg/mg protein;
P<0.01). The treatment with plant extracts of fruits of Terminalia bellerica
at a dose of 400 mg/kg showed a significant increase in GSH levels
(1.04±0.14 – 4.18±1.17 µg/mg protein; P<0.001). The extracts of leaves of
Ficus bengalensis and Ixora brachiata also showed an increase in GSH
level as shown by (1.04±0.14 – 3.64±1.37 µg/mg protein; P<0.01,
1.04±0.14 – 5.18±0.62 µg/mg protein; P<0.01). However on treatment with
200mg/kg b.w of the methanolic extract of Terminalia bellerica showed a
significant rise in the levels of GSH when compared with cisplatin
intoxicated group (1.04±0.14 – 4.83±1.28 µg/mg protein; P<0.01).But on
treatment with 200mg/kg leaf extracts of Ficus bengalensis the levels of
0
2
4
6
8
10
12
14
Normal Control TB FB IB TB FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Pre.act
GSH was increased but was not significant (1.04±0.14 – 3.64±1.37 µg/mg
protein) while on treatment with the ethyl acetate leaf extracts of Ixora
brachiata significantly showed an increase in the levels of GSH (1.04±0.14
– 3.62± 1.11 µg/mg protein P�0.05)
There was a decrease in the levels of GSH in the preventive control
group when compared with the normal control( 5.406±0.36 – 2.89±0.80
µg/mg protein P�0.01) Preventive treatment with 400mg/kg of methanolic
leaf extract of T. bellerica showed a slight improvement in the levels of
decreased GSH (2.89±0.80 - 3.68±0.86 µg/mg protein). Preventive
treatment with 400mg/kg of butanol leaf extract of F. bengalensis showed
a slight improvement in the levels of GSH when compared to the
preventive control group (0.89±0.06 to 1.80±0.19 µg/mg protein).
Preventive treatment with 400 mg/kg of the ethyl acetate leaf extract of I.
brachiata showed a slight improvement in GSH levels (2.89±0.80 -
3.06±1.22 µg/mg protein) [Table 12; Fig.17].
Fig 17: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GSH on cisplatin-induced nephrotoxic models
The administration of cisplatin alone in animals significantly
decreased GST activity in tissue to 0.36 ± 0.12 µg/mg protein (P0.001) as
compared to the normal group 0.89 ± 0.18 µg/mg protein. The treatment
with the fruit extract of Terminalia bellerica significantly increased the GST
activity to 0.79±0.84 µg/mg protein (P<0.01) in 400 mg/kg treated group.
There was a slight increase in the levels of GST on treatment with 200
mg/kg methanolic fruit extract of T. bellerica but was not significant
0.36±0.12 to 0.41±0.23 µg/mg protein. The treatment of leaf extract of
Ficus bengalensis significantly and dose dependently increased GST
activity to 0.68 ± 0.17 µg/mg protein (P 0.01) in 400 mg/kg b.w and 0.49 ±
1.12 µg/mg protein (P 0.01) in 200 mg/kg treated animals. The treatment
with methanolic leaf extract of I. brachiata significantly increased the
0
1
2
3
4
5
6
Normal Control TB FB IB TB FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
activity to 0.56 ± 1.25 in 400 mg/kg treated animals when compared to
cisplatin group treated animals 0.36 ± 0.12. µg/mg protein
Preventive control treatment with cisplatin decreased the levels of
GST when compared to the normal control group 0.89±0.18 – 0.84±0.20
µg/mg protein P� 0.001. There was an improvement in the levels of GST of
preventive treatment with 400 mg/kg fruit extract of T. bellerica as
compared to the preventive control group 0.69±0.90 - 0.73±0.14 µg/mg
protein. In the preventive regimen, treatment with 400 mg/kg of the leaf
extract of F. bengalensis showed a decrease in the levels of GST when
compared to the preventive control group 0.65±0.18 to 0.54±0.18 µg/mg
protein [Table 12; Fig.18].
Fig. 18: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GST on cisplatin-induced nephrotoxic models
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Normal Control TB FB IB TB FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
A breakdown of membrane structure and function is seen as a result
of lipid peroxidation. The decomposition of peroxidised lipids yields a wide
variety of end products including MDA. The levels of MDA can be
measured by TBARS assay. There was an increase in TBARS in the
groups of animals intoxicated with cisplatin (1.653±0.03 – 6.95±0.28
mµ/100gm tissue; P<0.001). The treatment with the plant extracts at dose
of 400 mg/kg body weight showed a decrease in the levels of TBARS
which was significant (TB-6.95±0.28 – 2.28±0.127 mµ/100gm tissue;
P<0.01; FB- 6.95±0.28 – 3.63±0.35 mµ/100gm tissue; P<0.01) and IB -
6.95±0.28 - 3.55±0.16 mµ/100gm tissue; P<0.001). It has been observed
that administration of extracts at a dose of 200 mg/kg reduced the levels of
TBARS but was not significant (T.b: 6.95±0.28 = 4.74± 0.52; P<0.05, F.b
(6.95±0.28 – 5.26±0.05; I.b (6.95±0.28 – 4.50±1.12; P<0.05 mµ/100gm
tissue)
A significant increase was observed in the level of TBARS in the
preventive control group treated with cisplatin when compared to normal
control. Preventive treatment with 400 mg/kg of methanolic extract of T.
bellerica comparatively reduced the levels of TBARS (8.76±1.37 –
5.64±0.12 mµ/100gm tissue; P�0.05) respectively. There was however a
decrease in the elevated levels of TBARS in the preventive treatment with
400 mg/kg of the leaf extracts of F. bengalensis when compared to the
preventive control group (1.73±0.28 -2.07±2.27 mµ/100gm tissue P�0.05).
The preventive treatment with 400 mg/kg of ethyl acetate leaf extract of I.
brachiata reduced the levels of TBARS as compared to the preventive
control group (5.74±0.80-4.09±4.09 mµ/100gm tissue; P�0.05) [Table 12;
Fig.19].
Fig. 19: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of MDA on cisplatin-induced nephrotoxic models
0
1
2
3
4
5
6
7
8
9
10
Normal Control TB FB IB TB FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.Act
Table 12: Comparison of Plant Extracts in Cisplatin-induced Renal Damage
a) P<0.001 compared to toxic group; b) P<0.01: compared to toxic; c) P<0.05: compared to toxic
CURATIVE REGIMEN PREVENTIVE REGIMEN
Parameter Normal Toxic Ficus bengalensis Terminalia bellerica Ixora brachiata Ficus
bengalensis Terminalia bellerica
Ixora brachiata
200
mg/kg 400 mg/kg 200 mg/kg 400 mg/kg 200 mg/kg
400 mg/kg
400 mg/kg 400 mg/kg 400 mg/kg
Serum Creatinine (mg/dl)
1.41±0.46 4.28±0.56
(b) 2.12± 0.82
(c) 1.24± 0.61
(b) 2.77± 0.11
1.46 ± 0.33 (c)
2.15± 0.18 1.92± 0.55
(c) 1.58±0.15
(b) 4.80± 0.14
(c) 1.44± 1.88
BUN (mg/dl)
18.05±8.2 45.69±4.1
4(a) 31.55± 0.83 (a)
25.88±0.54 (a)
41.24± 1.22 (b)
22.11±2.68 (a)
41.14±0.53 38.56 ±0.12
76.04±0.28 (a)
64.69±0.80 (b)
72.62± 1.11 (a)
SOD (µg/mg protein)
12.56±0.97
5.16±0.64 (a)
7.64± 0.33 (a)
8.46± 1.33 (a)
6.38± 0.7 7.38± 2.24
(c) 6.21±1.33
(c) 5.21± 0.66
4.68±1.40 a()
7.25± 0.90 (b)
3.76± 1.40
GSH (µg/mg protein)
5.406±0.36
1.04±0.14 (b)
3.64± 1.37 4.25± 0.05
(b) 4.83± 1.28
4.18± 1.17(c)
3.62± 0.62 (c)
5.48 ±0.80 (b)
1.80± 0.19 3.68±
0.86(b) 3.06± 1.22
(b)
GST (µg/mg protein)
0.89±0.18 0.36±0.12
()a 0.49± 1.12 0.68± 0.17 0.41± 0.23
0.75± 0.84 (a)
0.48± 1.25 0.48 ± 0.20
0.56± 0.18 0.62± 0.14
(a) 0.71± 1.9
TBARS (mµ/100g tissue
1.65±0.03 6.95±0.28 4.26± 0.05 3.63±0.35
(b) 4.74±
0.52(c) 2.28± 0.12
(b) 4.50± 0.16
(c) 3.55 ±
0.80 (a) 2.07± 2.27
(a) 5.64±
0.12(a) 4.09±1.9
(b)
6.6.1 Histopathological examination of methanolic fruit extract of
Terminalia bellerica
The sections of rat kidney treated with cisplatin showed marked
congestion of glomeruli associated with desquamation of the epithelium.
Marked peritubular and blood vessel congestion was also exhibited. These
histopathological changes suggest that cisplatin induces acute tubular
necrosis.
However on treatment with the methanolic fruit extract of Terminalia
bellerica at dose of 400 mg/kg showed normalization of kidney with only
mild glomerular congestion. But on treatment with 200 mg/kg the
histopathological changes were very mild. Kidney section of the rats
treated with 400 mg/kg of the extract of the fruits T. bellerica showed mild
normalization of the kidneys .
Table 13: Effect of methanolic extract of the fruits of Terminalia bellerica on histopatholological features of kidney in cisplatin-induced renal damage Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ + _ +++ +
Tubular casts - +++ _ _ +++ _
Peritubular congestion
- +++ + _ +++ _
Epithelial desquamation
- +++ + _ +++ _
Blood vessel congestion
- +++ + _ +++ +
Inflammatory cells
- +++ + _ +++ _
Interstitial edema
- +++ + _ +++ _
(-) normal (+) little effect (+++) severe effect
Fig 20: Histopathological evidences for the nephroprotection of methanolic
fruit extract of Terminalia bellerica in Cisplatin-induced kidney damage
Histopathological examination of butanol leaf extract of Ficus
bengalensis
The sections of rat kidney treated with cisplatin showed marked
congestion of glomeruli associated with desquamation of the epithelium.
There also exhibited marked peritubular and blood vessel congestion.
These histopathological changes suggest that cisplatin induces acute
tubular necrosis.
However on treatment with the extract of Ficus bengalensis at dose
of 400 mg/kg showed normalization of kidney with only mild glomerular
congestion. But on treatment with 200 mg/kg the histopathological
changes were very mild. Kidney section of the rats treated preventively
with 400 mg/kg of the extract of the leaves of F. bengalensis showed mild
normalization of the kidneys.
Table 14: Effect of butanolic leaf extract of Ficus bengalensis on histopatholological features of kidney in cisplatin-induced renal damage
Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ - + +++ +
Tubular casts - +++ - - +++ - Peritubular congestion
- +++ + - +++ -
Epithelial desquamation
- +++ - - +++ -
Blood vessel congestion
- +++ - - +++ +
Inflammatory cells
- +++ + - +++ -
Interstitial edema - +++ + - +++ +
(-) normal (+) little effect (+++) severe effect
Fig. 21: Histopathological evidences for the nephroprotection of butanolic
leaf extract of Ficus bengalensis in Cisplatin-induced kidney damage
Histopathological examination of ethyl acetate leaf extract of Ixora
brachiata
The sections of rat kidney treated with cisplatin showed marked
congestion of glomeruli associated with desquamation of the epithelium.
Marked congestion of peritubular and blood vessel was also exhibited.
These histopathological changes suggest that cisplatin induces acute
tubular necrosis.
However on treatment with the ethyl acetate leaf extract of Ixora
brachiata at dose of 400 mg/kg, normalization of kidney with only mild
glomerular congestion was observed. But on treatment with 200 mg/kg the
histopathological changes were very minimal. Kidney section of the rats
treated with 400 mg/kg of the extract of the leaves of I. brachiata showed
only mild normalization of the kidneys in the preventive group.
Table 15: Effect of butanolic leaf extract of Ixora brachiata on histopatholological features of kidney in cisplatin-induced renal damage
Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ - + +++ +
Tubular casts - +++ - - +++ - Peritubular congestion
- +++ + - +++ -
Epithelial desquamation
- +++ - - +++ -
Blood vessel congestion
- +++ - - +++ +
Inflammatory cells
- +++ + - +++ -
Interstitial edema - +++ + - +++ +
(-) normal (+) little effect (+++) severe effect
Fig. 22: Histopathological evidences for the nephroprotection of
ethylacetate leaf extract of Ixora brachiata in Cisplatin-induced kidney
damage
6.7 GENTAMICIN TOXICITY
Gentamicin (GM) intoxication caused an increase in levels of serum
creatinine and BUN. But there was a significant reversal of the raised
serum creatinine and BUN levels in the curative regimen treated with the
extracts. The extracts showed a dose dependent activity but only highest
dose showed statistically significant activity. Reactive oxygen species
(ROS) are potential mediators of GM induced renal dysfunction. ROS like
hydroxyl radical have been affected in etiology of GM induced
nephrotoxicity. Aminoglycoside-iron complex is formed by Fenton’s
reaction which has been proposed as the major mechanism of gentamicin-
induced acute renal failure. Walker and Shah proved that gentamicin
enhances in vitro generation of hydrogen peroxide by renal cortical
mitochondria and iron chelaters hydroxyl radical scavengers protect
against GM mediated renal toxicity. There is also an important link
between oxidative and nitrosative stress and role between peroxynitrite
and nitric oxide in ensuring acute renal failure. As a therapeutic
intervention, the scavengers of nitric oxide and peroxynitrite are more
effective than iNOS. Gentamicin intoxication causes a significant decrease
in the renal GST, SOD, GSH levels as compared to normal group.
Nephrotoxicity was evident in the rats in the toxic groups since there
was an elevation in the levels of serum creatinine (1.186±0.21-6.32±0.29
mg/dl) and BUN (34.485±0.31-123.01±0.39 mg/dl) levels when compared
to the normal group. The serum creatinine levels were found to increase in
the toxic group. But the elevated levels of serum creatinine and BUN was
found to decrease in the groups treated with the butanolic leaf extract of
Ficus bengalensis at a dose of 400 mg/kg body weight (6.32±0.29-
2.34±0.26 mg/dl), (123.01±0.39-39.65±5.5 mg/dl; P0.05). The methanolic
fruit extract of Terminalia bellerica at a dose of 400 mg/kg also had a
significant effect in decreasing the levels of increased serum creatinine
and BUN (6.32±0.29-1.32±0.38mg/dl; P0.05, 123.01±0.39-68.25±0.53
mg/dl; P0.05). The ethyl acetate leaf extract of Ixora brachiata also
bought down the elevated levels of serum creatinine and BUN (6.32±0.29 -
1.32±-1.81 ± 0.25 mg/dl; P0.05, 123.01±0.39-54.21±0.85 mg/dl; P0.05).
Preventive control animals treated with gentamicin showed an
elevation in the levels of serum creatinine when compared to the normal
control group. Preventive treatment with 400 mg/kg of the methanolic fruit
extract of T. bellerica decreased the increased levels of serum creatinine
as compared to the preventive control group. It was observed that there
was a significant increase in serum creatinine level in the preventive
control group as compared to normal control. Preventive treatment with
400 mg/kg of n-butanolic leaf extract of F. bengalensis decreased serum
creatinine levels when compared to preventive control (7.18±0.45 -
5.63±0.14 mg/dl; P0.05). Preventive control animals treated with
gentamicin produced an elevation in the levels of serum creatinine when
compared to the normal control. Preventive treatment with 400 mg/kg of
ethyl acetate extract of leaves of I. brachiata reduced the levels of serum
creatinine when compared to preventive control (5.67±0.34 - 6.75±0.36
mg/dl; P0.05) [Table 16; Fig.23 & 24].
Fig. 23: Effect of extracts of fruits of T .bellerica and leaves of F. bengalensis and I. brachiata on the levels of Serum creatinine on gentamicin-induced nephrotoxic models
0
1
2
3
4
5
6
7
8
Normal Control T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
Fig. 24: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of blood urea nitrogen on gentamicin-induced nephrotoxic models.
Gentamicin generates superoxide anion (O2-) peroxy nitrate anion,
H2O2 and hydroxyl radical production from renal cortical mitochondria. The
interaction between superoxide anion and H2O2 in the presence of iron as
catalyst forms hydroxyl radical, which is toxic. This induces peroxidation of
lipids of endoplasmic reticulum; as a result the membrane structure and
function are broken down. Further decomposition of peroxidised lipids
leads to formation of a wide variety of end products including
malonaldehyde (MDA), whose increase can be detected by an increase in
levels of thiobarbituric acid species (TBARS) (2.44±1.2 – 6.72±0.94
mµ/100 gm tissue). However the methanolic fruit extract of Terminalia
bellerica was able to attenuate the effect of TBARS (6.72±0.94 -
0
20
40
60
80
100
120
140
NormalControl T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
3.027±6.781 mµ/100 gm tissue) in the curative regimen. But the finding
was not statistically significant. It has also been observed that
administration of extract for sixteen days at a dose of 500 mg/kg prior to
gentamicin administration (80 mg/kg single dose s.c.) in the Preventive
regimen, provided a marked protection against GM-induced renal damage
(6.72±0.94 - 2.72± 1.24 mµ/100 gm tissue).
The butanolic leaf extract of Ficus bengalensis was also found to
have a curative and protective effect on the levels of MDA (6.72±0.94 –
2.15 ± 1.65 mµ/100 gm tissue, 6.72 ± 0.94 – 2.02 ± 0.31 mµ/100 gm
tissue). The ethyl acetate leaf extract of Ixora brachiata was found to have
a significant (P<0.05) action on TBARS in the Preventive regimen
(6.72±0.94 – 2.84 ± 2.12 mµ/100 gm tissue) [Table 16; Fig.25].
Fig. 25: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of MDA on gentamicin-induced nephrotoxic models
0
1
2
3
4
5
6
7
8
9
NormalControl T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
There is an increased consumption of GSH in non-enzymatic
removal of oxygen radical or oxidation of sulphydryl group at the active
site. GSH is depleted due to increased creation of ROS (6.31± 0.96 –
3.63±0.35 µg/mg protein). Methanolic extract of the fruits of Terminalia
bellerica at the dose of 400 mg/kg has shown to significantly increase the
levels of GSH (3.63 ± 0.35 – 5.87 µg/mg protein ± 0.27; P<0.05). The leaf
extract Ficus bengalensis was also found to increase the GSH levels (3.63
± 0.35 – 5.12 ± 0.82 µg/mg protein; P<0.05). Ethyl acetate leaf extract of
Ixora brachiata also showed an increase of GSH levels (3.63 ± 0.35 – 4.76
± 0.28 µg/mg protein; P<0.05). The GSH in the Preventive regimen was
also found to be significant (TB – 2.25 ± 0.88 - 3.48 ± 0.12 µg/mg protein;
P<0.05, FB: 3.56 ± 0.12 – 4.76 ± 1.14 µg/mg protein; P<0.05 & IB: 1.64 ±
0.12 – 2.68 ± 0.30 µg/mg protein; P<0.05) [Table 16; Fig.26].
Fig. 26: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of GSH on gentamicin-induced nephrotoxic models
0
1
2
3
4
5
6
7
Normal Control T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
The administration of GM alone in animals significantly decreased
GST activity in tissue to 0.58±1.12 µg/mg protein (P<0.01) as compared to
the normal group (1.02±0.31 µg/mg protein). The treatment with the fruit
extract of Terminalia bellerica significantly increased the activity to
0.69±0.86 µg/mg protein (P<0.01) in 400 mg/kg and 0.56±0.11 µg/mg
protein in 200 mg/kg treated animals (P<0.01). The treatment with the leaf
extract of Ficus bengalensis significantly increased the activity to 0.61 ±
0.21 µg/mg protein (P<0.01) in 200 mg/kg and 0.81±0.16 µg/mg protein
(P<0.01) in 400 mg/kg treated animals. The treatment with the leaf extract
of Ixora brachiata significantly increased GSH activity to 0.81±0.16 µg/mg
protein (P<0.01) in 400 mg/kg extract and to 0.72±0.21 µg/mg protein
(P<0.05) in 200 mg/kg treated animals.
In the preventive control animals treated with gentamicin, the levels
of GST decreased as compared to the normal group. Preventive treatment
with 400 mg/kg of the methanolic fruit extract of T. bellerica produced a
slight increase in GST levels as compared to preventive control group
(0.47±0.41 - 0.59±1.61 µg/mg protein; P�0.01). Preventive control animals
treated with gentamicin decreased the levels of GST as compared to
normal control groups. Preventive treatment with 400 mg/kg of the extract
of F. bengalensis showed a slight increase in the levels of GST as
compared to preventive treatment group (0.65±0.66-0.92±0.20 µg/mg
protein; P�0.05). Preventive control animals treated with gentamicin
decreased GST levels when compared to normal group. But the level of
GST was increased in the preventive treatment with 400 mg/kg of ethyl
acetate extract of the leaves of I. brachiata when compared to preventive
control (0.77±0.28-0.90±0.20 µg/mg protein; P�0.05) [Table 16; Fig.27].
Fig. 27: Effect of extracts of fruits of T. bellerica leaves of F. bengalensis and I. brachiata on the levels of GST on gentamicin-induced nephrotoxic models
Oxidative stress causes diminished activity of SOD and depletes
thiol cellular content (12.56±5.16±1.46 µg/mg protein). The consumption of
methanolic fruit extract of Terminalia bellerica at the dose of 400 mg/kg,
improved SOD catalase activity (5.16±1.46 – 11.97±1.56 µg/mg protein;
P<0.05). The butanolic leaf extract of Ficus bengalensis at the dose of 400
mg/kg also improved SOD activity (5.16±1.46 – 9.72 ± 1.98 µg/mg protein;
0
0.2
0.4
0.6
0.8
1
1.2
NormalControl T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
P<0.05). There was also an improvement in the SOD activity after the
consumption of ethyl acetate leaf extract of Ixora brachiata (5.16±1.46 –
9.35±0.25 µg/mg protein; P<0.05) at a dose level of 400 mg/kg b.w.
Preventive control animals treated with gentamicin showed decreased
SOD levels as compared to normal control group. The Preventive regimen
also showed a marked protection against gentamicin-induced renal
damage (TB - 5.16±1.46 – 10.25±6.33 µg/mg protein; P<0.01), FB
5.16±1.46 - 10.61 µg/mg protein: IB 5.16±1.46 – 9.84 µg/mg protein:
P<0.05) [Table 16; Fig.28].
Fig. 28: Effect of extracts of fruits of T. bellerica and leaves of F. bengalensis and I. brachiata on the levels of SOD on gentamicin-induced nephrotoxic models
0
2
4
6
8
10
12
14
Normal Control T.B FB IB T.B FB IB
200 mg/kg
400 mg/kg
Prev.ctrl
Prev.act
Table 16: Comparison of Plant Extracts in Gentamicin-induced Renal Damage
CURATIVE REGIMEN PREVENTIVE REGIMEN
Parameter Normal Control Ficus bengalensis Ixora brachiata Terminalia bellerica Ficus
bengalensis Ixora
brachiata Terminalia bellerica
200 mg/kg 400 mg/kg 200 mg/kg 400 mg/kg 200 mg/kg 400 mg/kg 400 mg/kg 400 mg/kg 400 mg/kg
Serum Creatinine (mg/dl)
1.18±0.21
6.32±0.29 (a)
3.81± 1.82 2.34±0.26 2.68±0.25
(c) 1.81±0.25
(c) 2.83±0.71
(c) 1.32±0.38
(c) 5.63±0.14
4.32±0.28 (c)
3.56±0.18 (c)
BUN (mg/dl)
34.48±0.31
123.01±0.39 ()c
56.32±2.2 (a)
39.65±5.5 76.65±1.12
(c) 54.21±0.85
(c) 72.02±0.64
(c) 68.25±0.53
(c) 55.76±0.88
(b ) 97.95±0.86
(c) 84.07±1.66
(c) SOD
(µg/mg protein)
12.56±1.66
5.16±1.46 (c)
8.25±2.17 (a)
9.72±1.98 (a)
8.33±1.35 (a)
9.35±0.25 (a)
10.65±0.65 (a)
11.97±1.56 6.87±0.33 (a) 5.67±0.36
(b) 4.45±1.90
GSH (µg/mg protein)
6.31±0.96
3.63±0.37 (b)
5.68±0.65 6.23±0.82
(a) 4.93±1.13
6.02±0.28 (a)
4.12±1.12 5.21±0.27
(a) 4.76±1.14 (a)
1.64±6.03 (a)
3.48±0.12 (a)
GST (µg/mg protein)
1.02±0.31
0.52±1.12 (b)
0.68±0.25 0.72±0.13
(a) 0.73±0.21
(b) 0.81±0.16
(b) 0.62±0.11
(a) 0.66±0.86
(b) 0.65±0.66 (c)
0.90±0.28 (c)
0.59±1.61 (b)
TBARS (mµ/100g tissue)
2.44±1.27
6.72±0.94 ()b
3.26±1.28 (a)
2.15±1.65 5.82±0.66 3.12±0.25 3.86±0.43
(a) 3.02±0.78
(b) 4.87±0.31 (a) 7.58±2.12
7.34±1.24 (b)
a) P<0.001 compared to toxic group; b) P<0.01: compared to toxic; c) P<0.05: compared to toxic
Histopathological examination of methanolic fruit extract of
Terminalia bellerica
While histopathological section of the control rats showed normal
tubular and glomerular features, the histology of rats treated with
gentamicin showed glomerular, peritubular and blood vessel congestion.
Concurrent treatment with 400 mg/kg methanolic extract of the fruits
of T. bellerica was found to reduce the histopathological changes caused
by gentamicin toxicity. However the section of the rats treated with 200
mg/kg extract of T. bellerica also showed mild glomerular and peritubular
congestion. Sections of the rats treated Preventiveally with 400 mg/kg of
the extract showed only mild regeneration of the toxic kidney.
Table 17: Effect of methanolic extract of the fruits of Terminalia bellerica on histopatholological features of kidney in cisplatin-induced renal damage Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ _ _ +++ +
Tubular casts - +++ _ _ +++ + Peritubular congestion
- +++ - _ +++ _
Epithelial desquamation
- +++ _ _ +++ _
Blood vessel congestion
- +++ + + +++ +
Inflammatory cells
- +++ + + +++ +
Interstitial edema
- +++ _ _ +++ _
(-) normal (+) normal (+++) severe effect
Fig. 29: Histopathological evidences for the nephroprotection of Methanolic
fruit extract of Terminalia bellerica in Gentamicin-induced kidney damage
Histopathological examination of butanolic leaf extract of Ficus
bengalensis
Histopathological section of the control rats showed normal tubular
and glomerular features. Histology of rats treated with gentamicin showed
glomerular, peritubular and blood vessel congestion. Concurrent treatment
with 400 mg/kg butanolic extract of the leaves of F. bengalensis was found
to reduce the changes caused by gentamicin toxicity. Sections of the rats
treated Preventiveally with 400 mg/kg of the extract showed only mild
regeneration of the toxic kidney.
Table 18: Effect of butanolic extract of the leaves of Ficus bengalensis on histopatholological features of kidney in gentamicin-induced renal damage Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ + _ +++ +
Tubular casts - +++ + + +++ + Peritubular congestion
- +++ + + +++ +
Epithelial desquamation
- +++ _ _ +++ _
Blood vessel congestion
- +++ _ _ +++ _
Inflammatory cells
- +++ + + +++ +
Interstitial edema
- +++ + + +++ +
(-) normal (+) little effect (+++) severe effect
Fig. 30: Histopathological evidences for the nephroprotection of butanolic
leaf extract of Ficus bengalensis in Gentamicin-induced kidney damage
Histopathological examination of ethyl acetate leaf extract of Ixora
brachiata
While histopathological section of the control rats showed normal
tubular and glomerular features, the histology of rats treated with
gentamicin showed glomerular, peritubular and blood vessel congestion.
Concurrent treatment with 400 mg/kg methanolic extract of the
leaves of I. brachiata showed a normalization of the kidneys in the curative
group. However the section of the rats treated with 200 mg/kg extract of I.
brachiata also showed mild glomerular and peritubular congestion
following gentamicin administration. Sections of the rat kidneys in the
preventive treatment with 400 mg/kg of the extract showed only mild
regeneration of the toxic kidney.
Table 19: Effect of ethyl acetate extract of the leaves of Ixora brachiata on histopatholological features of kidney in gentamicin-induced renal damage
Histopathological features
Normal control
Toxic control
Extract 200 mg/kg
Extract 400 mg/kg
Preventive control
400 mg/kg extract
Glomerular congestion
- +++ + _ +++ +
Tubular casts - +++ + + +++ + Peritubular congestion
- +++ + + +++ +
Epithelial desquamation
- +++ _ _ +++ _
Blood vessel congestion
- +++ _ _ +++ _
Inflammatory cells
- +++ + + +++ +
Interstitial edema - +++ + + +++ +
(-) normal (+) little effect (+++) severe effect
Fig 31: Histopathological evidences for the nephroprotection of
ethylacetate leaf extract of Ixora brachiata in Gentamicin-induced kidney
damage
7.0 Conclusion
Phytoconstituents present in plants are responsible for the
therapeutic effectiveness of the particular plants. This study mainly focuses
on the role of plant constituents such as ellagic acid, lupeol and β-sitosterol
as antioxidants for treatment of nephrotoxicity. The plants selected for the
study are a major source and are also traditionally used by the tribals of
Kannur district in Kerala for the treatment of kidney disorders. The plants
under study were Ficus bengalensis (leaves), Hemidesmus indicus (whole
plant), Sida rhombifolia(roots), Ixora brachiata(leaves), Camellia sinensis
(leaves) and Terminalia bellerica (fruits). The plants were collected and
authenticated. The physicochemical analysis of the dried plant materials
were carried out. The active constituents from the dried plant parts were
extracted, concentrated and quantified using High Performance Liquid
Chromatography.
The presence of phytoconstituents in each extract was identified and
was found to contain triterpenoids, flavonoids, tannins, alkaloids, phenolic
compounds, and steroids. The percentage of phytoconstituents under
study in the dried plant extract, i.e, lupeol in Ficus bengalensis was 8.13%
w/w and in Hemidesmus indicus was 6.67%, β-sitosterol in Ixora brachiata
was 7.28% w/w and Sida rhombifolia was 5.07% w/w and ellagic acid in
Terminalia bellerica was 8.29% w/w and in Camellia sinensis, 3.17% w/w.
The amount of phenolic phytochemicals was then determined as they were
identified as natural antioxidants which provide immense health benefits by
several researchers.119 The total phenolic content was estimated was
estimated by Folin Catechu Method and found that Terminalia bellerica,
Ficus bengalensis and Ixora brachiata contains 78.6±0.18, 23.2±0.61, and
21.02±0.19 mg/gGAE compared to the other plants under study.
In vitro antioxidant studies like nitric oxide scavenging, super oxide
scavenging and lipid peroxidase scavenging were conducted on the
extracts by comparing with standard ascorbic acid and their IC50 was
calculated using liner regression analysis. From the results obtained it was
concluded that methanolic fruit extract of Terminalia bellerica, butanolic
leaf extract of Ficus bengalensis and ethyl acetate leaf extract of Ixora
brachiata possessed comparable antioxidant activity in all the methods
tested.
Plant extracts possessing higher percentage of lupeol, β-sitosterol
and ellagic acid, higher phenolic content and IC50 values comparable to
Vitamin C were chosen for the further pharmacological studies. Thus
methanolic fruit extracts of Terminalia bellerica, butanolic leaf extract of
Ficus bengalensis and ethyl acetate leaf extract of Ixora brachiata were
further selected from among the six plants initially chosen.
The current study focused on the use of Terminalia bellerica, Ficus
bengalensis and Ixora brachiata in the treatment of cisplatin and
gentamicin-induced nephrotoxicity. Renal dysfunction arises as a result of
production of RNS and ROS by the body in response to external agents
like drugs and environmental chemicals.
Acute toxicity studies of the plant extracts was done under OECD
guidelines 423 and the extracts produced no toxic symptoms up to the
dose of 2000 mg/kg body weight even after 24 and 72 hours. 1/10th and
1/5th of the largest dose was used for screening of nephroprotective activity
using cisplatin and gentamicin nephrotoxic models. Doses of 200mg/kg
b.w and 400 mg/kg b.w were used for the pharmacological studies. The
study revealed that the plant extracts at doses of 200 and 400 mg/kg body
weight had an ameliorating effect on the kidneys. The extracts were able to
normalize the gentamicin and cisplatin-induced renal parameters. The
histopathological section of rat kidney showed tubular congestion and
glomerular atropy which indicates acute renal necrosis.120 There was a
marked reduction in glomerular congestion and degenerative necrotic
epithelial cells which shows that the extracts of Terminalia bellerica, Ficus
bengalensis and Ixora brachiata (400 and 200 mg/kg b.w) had effective
curative protection against renal damage. The histopathological sections of
the preventive regimen showed only partial protection characterized by
some regenerative changes in tubular epithelial cells. Among the various
classes of phytoconstituents, the most effective in the treatment of
nephrotoxicity are polyphenol compounds and pentacyclic triterpenoids.
The extracts enhanced the effectiveness by their antioxidant
defence systems. In the preventive study, slight improvement in renal
parameters and effect on antioxidants systems were observed. The
rationale behind protective study is to show the effectiveness of the
protective agent on the renal tissue before the occurrence of nephrotic
damage
Nephrotoxicity is a major side effect of different classes of drugs
such as NSAIDS and antibiotics which are used on a daily basis.
Nephrotoxins are chemicals displaying nephrotoxicity. The relationship
between oxidative stress and nephrotoxicity has been well demonstrated in
experimental animal models. So antioxidants and free radical scavengers
of natural or synthetic origin might prove as good antioxidants.
Nephrotoxicity in the case of aminoglycosides is caused by its
accumulation in the renal cortex. Nephrotoxicity caused by gentamicin and
cisplatin is through oxidative stress. One mechanism by which cisplatin
induces free radical damage is by increasing the activity of calcium
independent nitric oxide synthetase. It is proved that flavonoids can act as
potent scavenger of free radicals. An increase in the number of phenolic
group in the molecule increases its nephroprotective activity. Literature
survey suggests that pentacyclic triterpenoids, lupeol, alkaloids and β-
sitosterol exhibit biological functions such as analgesic, hepatoprotective,
and anti-inflammatory activities.
Cisplatin at a dose of 5 mg/kg produced significant toxic symptoms
which are characterized by an increase in serum creatinine and BUN.
Antioxidant defence system was also impaired as indicated by an increase
in TBARS and decrease in GST, GSH and SOD levels in the renal tissues.
All these signs of toxicity were also evidenced by histopathological
changes.
The process of kidney damage occurs mainly through free radicals
like superoxide, hydroxyl and lipid peroxides that are produced from
leukocytes, macrophages and mesangial cells. The in vitro antioxidant
scavenging activity suggests a potent property against nitric oxide, super
oxide and lipid peroxidase free radicals.
Among the various phytoconstituents the most effective in the
treatment of nephrotoxicity are polyphenol compounds and pentacyclic
triterpenoids. The present study proved that Terminalia bellerica and Ficus
bengalensis can act as nephroprotective agents. From a further analysis of
the activity, it can be concluded that Terminalia bellerica containing ellagic
acid, a polyphenol is a better nephroprotective when compared to Ficus
bengalensis and Ixora brachiata. From this study it can be proved that
plants with more amounts of phenolic groups act as good nephroprotective
agents. Phenolic compounds function as high level antioxidants since they
possess the ability to absorb and neutralize free radicals as well as quench
the reactive oxygen species. Further research is needed to investigate the
exact mechanism of nephroprotective activity by studying the reaction of
natural agents towards various enzymes indirectly involved in ROS
production and nephrotic damage.
As nephrotoxicity is caused mainly by oxidative stress, it can be
deduced that higher amounts of phytoconstituents which act as
antioxidants can mitigate the deleterious effect of drugs on kidneys through
modification of oxidant antioxidant balances. The selected plants also
contained tannins which are complex polyphenolic compounds exhibiting
good antioxidant activity whereby it acts as nephroprotectives. Thus it may
be concluded that the nephroprotective activity of the plants selected for
the study is due to the presence of phytochemicals capable of producing
antioxidant and free radical scavenging activity which may act individually
or synergistically.
The findings of the study confirm the ancient wisdom of the tribals -
Pachiayals and Uchiyamas of Kannur district, Kerala for their use in kidney
diseases. A further investigation of individual compounds, their
characteristics and interrelationships with other bioactive compounds is
needed to fix responsibility for their significant efficacy.
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APPENDIX - A
APPENDIX - B
List of Publications
1. Mathew A, Panayappan, Divakar MC. HPLC Quantification of Lupeol
and Nephroprotective Activity of N- butanol extract of Hemidesmus
indicus. Inventi Rapid: Planta Activa, 2013(2):1-4.
2. Mathew A, Divakar MC, Philip S. Antioxidant Activity of Some
Common Medicinal Plants. International Journal of Pharmaceutical
and Clinical Research 2013; 5(2): 43-46.