“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.