Rajiv Gandhi University of Health Science

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCE

KARNATAKA, BANGALORE

M. PHARM SYNOPSIS

YEAR OF ADMISSION-JUNE 2010

TITLE OF THE SYNOPSIS

“Anti-oxidant and Nephro-protective activities of Cassia occidentalis Linn”

BY

M.GOWRISRI

M.PHARM

DEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Mr. Syed Bilal,M.PHARM

Professor

Department of Pharmacology

INSTITUTION

GAUTHAM COLLEGE OF PHARMACY

SULTHANPALYA, R.T. NAGAR, BANGALORE-560032

KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / Name of The Candidate and Address / Ms. M. GOWRISRI
a. Permanent Address
D/o K.RAJARAO,
D.No:17/698G,
Tilaknagar,
Guntakal,
Andhra Pradesh.,
b.Postal Address
Gautham College of Pharmacy,
Sulthan Palya, R.T. Nagar Post,
Bangalore-560032,
Karnataka.
2. / Name of The Institution / GAUTHAM COLLEGE OF PHARMACY
Sulthanpalya, R.T Nagar.
Bangalore- 560032
3. / Course of study and Subject / Master of Pharmacy in Pharmacology
4. / Date of admission / 10/06/2010
5. / Title of the topic:
“Anti-oxidant and Nephro-protective activities of Cassia occidentalis Linn”

6.BRIEF REVIEW OF THE INTENDED WORK :

6.1 INTRODUCTION:

The term renal failure primarily denotes failure of the excretory function of kidney, leading to retention of nitrogenous waste products of metabolism in the blood. In addition, there is failure of regulation of fluid and electrolyte balance along with endocrine dysfunction. The renal failure is fundamentally categorized into acute and chronic renal failure1,2.

Chronic renal failure (CRF) is an irreversible deterioration in the renal function which classically develops over a period of years, leading to loss of excretory metabolic and endocrine functions. Various causes of renal failure has been attributed like hypertension, diabetes mellitus, antineoplastic agents like cyclophosphamide, vincristin, cisplatin etc.

Acute renal failure (ARF) refers to the sudden and usually reversible loss of renal function which develops over a period of days or weeks. There are many causes of acute renal failure which could be eitherpre-renal (55%), renal (40%), or post renal (5%). Among the renal causes of acute renal failure, acute tubular necrosis is more common accounting for 85% of incidence. Acute tubular necrosis occurs either due to ischemia or toxins. The toxin can be either exogenous or endogenous. The exogenous agents are radiocontrast agents, cyclosporin, antibiotics, chemotherapeutic agents, organic solvents, acetaminophen and illegal abortifacients1,3.

Antioxidant may be defined as radical scavengers which protect the human body againstfree radicals that may cause pathological conditions such as ischeamia, anaemia, asthma,arthritis, inflammation, neurodegeneration, parkinson’s disease, mongolism,ageing process and perhaps dementias4. Experiment evidence suggest that free radicals and reactive oxygen species can be involved in higher number of diseases. Numerous physiological and biochemical processes in the human body produces oxygen-centered free radicals another reactive oxygen species as byproducts. Overproduction of such free radicals can cause oxidative damage to biomolecules eventually leading to many chronic disease such as atherosclerosis, cancer, diabetes, aging and other degenerative disease in humans5.

Gentamicin is an important aminoglycoside antibioticcommonlyused in treating life-

threatening gram-negative infections6.However its usefulness is limited by signs of nephrotoxicity, which may occur in 13-30% of treated patients7. Lipid peroxidation may occur in the course of gentamicin administration8, giving rise to free radicals9,which are highly toxic to tissues10.

Distinct mechanism have been proposed for cisplatin cytotoxicity in renal tubule cells,including direct DNA damage11 ,activation of caspase12 mitochondrial dysfunction13 formation of reactive oxygen species14,effects on the endoplasmic reticulum15 and activation of TNF-α mediated apotopicpathyway.Cisplatininduced nephrotoxicity isclosely associated with an increase in lipid peroxidation in the kidney. In addition,cisplatin has been found to lower the activities of antioxidant enzymes and to induce depletion of GSH.

Paracetamol overdose cause acute renal failure and chronic exposure to paracetamol is linked to chronic renal failure16. Information about the specific molecular pathways that lead to apoptosis of tubular cell during nephrotoxic injury is incomplete.Paracetamol induces apoptosis by upregulating the death receptor Fas expression. Fas expression increases in tubular cells upon paracetamol treatment.

Paracetamol treatment leads to activation of caspase-9 and caspase-3 in renal tubular epithelial cells. Caspase-12 cleaves caspase-9 in-vitro in the absence of cytochrome c. Caspase-12 is the apical caspase in paracetamol induced apoptosis in tubular epithelial cells. However, the possibility that other factors (released or not from the mitochondria) responsible for paracetamol induced caspase-9 activation cannot be excluded. Paracetamol causes endoplasmic reticulum (ER) stress in tubular cells, leading to GADD 135 (a transcription factor that promotes apoptosis)17 upregulation and translocation to the nucleus, as well as caspase-12 cleavage.

6.2NEED FOR THE STUDY:

Natural products are playing a vital role in health care for decades. Often different sources of natural products, plants have been a source of chemical substance, which serves as drugs in their own right or key ingredients in formulation containing synthetic drugs. The selection of the plant species is a crucial factor for the ultimate success of investigation. Through random selection gives some hint, targeted collection based on chemotaxonomic relationships and ethnomedical information derived from Tradition Medicine are more likely to yield pharmacologically active compounds.

Although the advances in modern medicines are significant, there remains an ever increasing demand for herbal medicines. Effective and potent herbal medicines require evaluation by standard scientific methods so as to be validated for the treatment of diseases.

Drug induced Nephrotoxicity is major health problem that challenges not only healthcare professionals but also the pharmaceutical industry and also drug regulatory agencies. The inhibition of free radical generation can serve as facile model for evaluating the activity of Nephro-protective agents.

6.3REVIEW OF LITERATURE:

Botanical name: Cassia occidentalisLinn

Synonym: Kasonda (Hindi),Kasmard (Sanskrit) ,Coffee senna, Stinging weed(English)

Distribution: West Bengal, SouthIndia, Burma, Srilanka18

Family: Fabaceae

Plant description:

It is an erect annual herb or under shrub. The leaves are lanceolate or ovate lanceolate,the leaf lets 3-paired memberanousglaucous ovate or lanceolate, flowers are yellow, in short raceme ,the pods are recurved, glabrous & compressed ;the seeds dark olive green, ovoid compressed, hard smooth&shining19.

Traditional & Folk uses:

Wound healing,sores, itch, cutaneous diseases, bone fracture, fever, ring worm, skin disease & throat infections20. It is also used in treatment of kidney disorders21.

Reported literature:

Antibacterial22,23

Antimalarial24

Antimutagenic2

Antiplasomodial26

Anticarcinogenic27

Hepatoprotective activities20

6.4 OBJECTIVES OF THE STUDY:

The objective of the proposed study is to investigate the antioxidant nephro-protective activity of Cassia occidentalishydro-alcoholic extracts of leaf by using rats.Toxicological studies(LD50) using mice. The whole study is divided into three phases

Phase I:

1) Collection & authentication of plant material.

2)  Preparation of Cassia occidentalis hydro-alcoholic extracts of leaf using Soxhlet apparatus.

3)  To investigate preliminary phytochemical constituents present in the extracts.

4)  Determination of LD50 value and dose selection for the Nephro-protective activity(i.e., selection of two appropriate doses from the LD50 value) those doses considered as low and high doses respectively.

Phase II:

To evaluate the Nephro-protective activity of Cassia occidentalis of leaf extracts in various experimental animals models like:

1)Gentamicin induced nephrotoxicity in rats

2)Cisplatin induced nephrotoxicity in rats

3)Paracetamol induced nephrotoxicity in rats.

Parameters to be studied:

1)  Body weight determination,

2)  Urine analysis-Sodium, Potassium, Glucose, Creatinine estimation.

3)  Blood analysis-Urea, Creatinine, Histopathological examination

In vivo anti-oxidant studies

1)  Lipid peroxidation (LP)

2)  Superoxide dismutase (SOD)

3)  Catalase

4)  Glutathione

Phase III:

To evaluate in vitro antioxidant activity of Hydro alcoholic of leaf extract of

Cassiaoccidentalis. It is also planned to evaluate the following parameters.

In vitro antioxidant studies:

1)Superoxide anion radical scavenging

2)Nitric oxide inhibition assay

3)Reducing power assay

4)Hydroxyl radical scavenging activity

5)DPPH assay.

7.0 MATERIALS AND METHODS:

7.1 SOURCE OF DATA:

The sources of data will based on laboratory experiments on animals and also the data obtained from the literature.

1)Standard Books:

ü  H.Gerhard Vogel’s: Drug Discovery and Evaluation

ü  Katzung’s : Basic and clinical pharmacology

ü  Rang and Dale’s: Pharmacology

ü  Tortora: Human Anatomy and Physiology

ü  Guyton and Hall’s: A Text Book of Medical Physiology

ü  Wealth of India

ü  Materiamedica

ü  Glossary of Indian Medicinal Plant

2)Internet source:

ü  Google

ü  Pubmed

ü  www.sciencedirect.com

ü  Wikipedia

ü  SCOUPS

ü  Helinet

ü  Ovid

ü  Open J-Gate

ü  DOAJ

ü  Chemical Abstracts

ü  CABI

ü  International Pharmaceutical abstracts

3) Journal sources:

ü  Indian journal of pharmacology

ü  Journal of pharmacology and Experimental therapeutics

ü  Journal of Ethnopharmacology

ü  Photochemistry

ü  Phytomedicine.

7.2 METHOD OF COLLECTION OF DATA:

Materials:

1)  Identification andauthentication ofthe Plant byPharmacognogist.

2)  Chemicals used(paracetamol,cisplatin, gentamicin) use will be in analytical grade.

Extraction of plant material:

About 150 g of powdered shade dried leaf of Cassia occidentaliswill be extracted with 70% v/v of ethyl alcohol by continuous heat extractionin a soxhlet extractor for 24 hours, extract will be concentrated to small volume underreduced pressure and evaporated to dryness. The extract will be stored in a refrigerator at 40c in a small and sterile bottle20.

7.3 EXPERIMENTAL ANIMALS

Albino Wistar rats of either sex weight about 200-225g will be used. Toxicological studies (LD50) by using mice.

7.4 PLANT MATERIAL

Naturally available plant Cassia occidentalis will be collected, identified and extracted with hydro-alcoholic solution.

7.5 DOSE

A dose of hydro-alcoholic leaf extract of Cassiaoccidentaliswill be taken as per thework.

7.6 NEPHRO-PROTECTIVE MODELS:

Nephro-protective activity will be studied using three models viz. Gentamicin, Cisplatin,paracetamol nephrotoxicity models in rats.

Study of protective effect of hydro alcoholic leaf extract of Cassia occidentalis in Gentamicin induced nephrotoxicity in rats28

Nephrotoxicity in rats is to be induced with the administration of gentamicin(80mg/kg i.p) for 8 days. The extract will be administered orally 3 days before the administration of gentamicin and continued for another 8 days along with gentamicin. The rats will be selected and randomized into 4 groups, each group consisting of 6 animals. The leaf extract will be administered orally and gentamicin willbeadministeredintraperitoneally.

Animal group / Treatment / Route of administration / No of
Animals used / Parameters
Group- I / Control / Orally / 6 / Body weight determination, Urine analysis-Sodium, Potassium, Glucose, Creatinine estimation.
Blood analysis- Urea, Creatinine, Hisopathological examination, In-vivo antioxidant parameters
Group-II / Gentamicin
(80mg/kg) / Intraperitoneally / 6
Group-III / Gentamicin+HACO
80mg/kg+1/10th dose / Intraperitoneally
+
Orally / 6
Group-IV / Gentamicin+HACO
80mg/kg+1/20th dose / Intraperitoneally
+
Orally / 6

HACO-hydro-alcoholic leaf extract of Cassia occidentalis.

Study of protective effects of hydroalcoholic leaf extract of Cassia occidentalisin paracetamol induced nephrotoxicity in rats29

Nephrotoxicity in rats is to be induced with the administration ofparacetamol (2g/kg oral)for 2 days. The extract will be administered by orally 3 days before administration of paracetamol and continued for 5days along with paracetamol. The rats will be selected and randomized into 4 groups each groupconsisting of6 animals. Theleaf extract will be administered orally and paracetamol will be administered orally.

Animal group / Treatment / Route of administration / No of
Animals used / Parameters
Group –I / Control / Orally / 6 / Body weight determination, Urine analysis-Sodium, Potassium, Glucose, Creatinine estimation.
Blood analysis- Urea, Creatinine, Hisopathological examination, In-vivo antioxidant parameters
Group-II / Paracetamol(2g/kg) / Orally / 6
Group-III / Paracetamol+HACO
(2g/kg+1/10thdose) / Orally+Orally / 6
Group-IV / Paracetamol+HACO
(2g/kg+1/20th dose) / Orally+Orally / 6

HACO-hydro-alcoholic leaf extract of Cassiaoccidentalis.

Study of protective effects of hydro alcoholic leaf extract of Cassia occidentalisinCisplatin induced nephrotoxicity in rats30.

Nephrotoxicity in rats is to be induced with the administration ofcisplatin(12mg/kg).The extract will be administered by orally 1 hr before the administration of cisplatin and at 24h and 48h after cisplatin injection .The parameters will be studied after 72h after cisplatin administration. The normalcontrol group will not be administered with either extract or cisplatin. The rats will be selected and randomized into 6 groups, each group consisting of 6animals.

Animal group / Treatment / Route
of administration / No of
Animals used / Parameters
Group –I / Control / Orally / 6 / Body weight determination, Urine analysis-Sodium, Potassium, Glucose, Creatinine estimation.
Blood analysis- Urea, Creatinine, Hisopathological examination, In-vivo antioxidant parameters
Group-II / Cisplatin
12mg/kg / i.p / 6
Group-III / Cisplatin+HACO
12mg/kg+1/10th dose / i.p+orally / 6
Group-IV / Cisplatin+HACO
12mg/kg+1/20th dose / i.p+orally / 6

HACO-hydro-alcoholic leaf extract of Cassiaoccidentalis.

In-vivo antioxidant Parameters:

o  Lipid peroxidation (LP)

o  Superoxide dismutase (SOD)

o  Catalase

o  Glutathione 31

In-vitro antioxidant parameters:

o  Superoxide anion radical scavenging

o  Nitric oxide inhibition assay

o  Hydroxyl radical scavenging activity

o  Reducing power assay

o  DPPH assay

7.7 Statistical Analysis:

All the values that are generated out of this study execution will be expressed as mean ± SEM from six animals. Statistical difference in mean will beanalysed using one way ANOVA (Analysis of Variance) followed by Dunnett’s ‘t’ test. P values less than 0.05 were considered as indicative of significance.

7.8Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please mention briefly.

Yes, the above study requires In vivo screening techiniques on Wistar rats

7.9 Has ethical clearance been obtained from your institution

The copy of ethical clearance certificate is enclosed.

REFERENCES

1.  Herfindal, Gourley. Text book of therapeutic drug and disease management. 7thEdn. Charcil Livingstone, London; 2000; 425-36.

2.  Barry M, Brenner, Floyd C, Rector. The kidney 6th Ed. Vol I, W.B. Saunders company, Philadelphia; 2000; 3-67.

3.  Paul L Munson. Principles of pharmacology, Basic concepts and clinical applications. Chapman and Hall ITP an international thomson publishing company New York; 685.

4.  NazninAra, HasanNur. In Vitro antioxidant activity of methanolic leaves and flowers extracts of Lippia Alba. Res J Med and Med Sci 2009; 4(1):107-110.

5.  Jain PK, Agrawal RK. Antioxidant and free radical Scavenging Properties of developed mono- and polyherbal Formulations Asian J Exp Sci 2008;22 (3): 213-220.

6.  Ali BH. Gentamicin nephrotoxicity in humans and animals: Some recent research Gen Pharmacol 1995;26:1477-87.