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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 lifethreatening 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:
 H.Gerhard Vogel’s: Drug Discovery and Evaluation
 Katzung’s : Basic and clinical pharmacology
 Rang and Dale’s: Pharmacology
 Tortora: Human Anatomy and Physiology
 Guyton and Hall’s: A Text Book of Medical Physiology
 Wealth of India
 Materiamedica
 Glossary of Indian Medicinal Plant
2)Internet source:
 Google
 Pubmed
 www.sciencedirect.com
 Wikipedia
 SCOUPS
 Helinet
 Ovid
 Open J-Gate
 DOAJ
 Chemical Abstracts
 CABI
 International Pharmaceutical abstracts
3) Journal sources:
 Indian journal of pharmacology
 Journal of pharmacology and Experimental therapeutics
 Journal of Ethnopharmacology
 Photochemistry
 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.
No of
Animal
group
Treatment
Parameters
Route of
administration
Animals
used
Group- I
Control
Group-II
Gentamicin
Orally
6
Intraperitoneally
6
(80mg/kg)
Group-III
Gentamicin+HACO
Intraperitoneally
80mg/kg+1/10th dose
+
6
Orally
Group-IV
Gentamicin+HACO
Intraperitoneally
80mg/kg+1/20th dose
+
Orally
HACO-hydro-alcoholic leaf extract of Cassia occidentalis.
6
Body weight
determination, Urine
analysis-Sodium,
Potassium, Glucose,
Creatinine estimation.
Blood analysis- Urea,
Creatinine,
Hisopathological
examination, In-vivo
antioxidant parameters
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
Parameters
Animals used
Group –I
Control
Orally
6
Group-II
Paracetamol(2g/kg)
Orally
6
Group-III
Paracetamol+HACO
Orally+Orally
6
Orally+Orally
6
(2g/kg+1/10thdose)
Body weight
determination,
Urine analysisSodium,
Potassium,
Glucose,
Creatinine
estimation.
Blood analysis-
Group-IV
Paracetamol+HACO
(2g/kg+1/20th dose)
Urea,
Creatinine,
Hisopathological
examination, Invivo antioxidant
parameters
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
No of
of administration
Animals used
Group –I
Control
Orally
6
Group-II
Cisplatin
i.p
6
12mg/kg
Group-III
Cisplatin+HACO
i.p+orally
6
i.p+orally
6
12mg/kg+1/10th dose
Group-IV
Cisplatin+HACO
12mg/kg+1/20th dose
HACO-hydro-alcoholic leaf extract of Cassiaoccidentalis.
Parameters
Body weight
determination,
Urine analysisSodium,
Potassium,
Glucose,
Creatinine
estimation.
Blood analysisUrea, Creatinine,
Hisopathological
examination, Invivo antioxidant
parameters
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.
7. Mathew TH. Drug-induced renal disease. Med J 1992; 156:724-28.
8. Ramsammy L, Ling KY, Josepovitz C, et al. Effect of gentamicin on lipid
peroxidation in rat renal cortex. Biochem Pharmacol 1985; 34:3895-900.
9. Yang C, Du X, Han Y. Renal cortical mitochondria are the source of oxygen free
radicals enhanced by gentamicin. Renal Fail 1995;17:21-26.
10. Feldman S, Wang M, Kaloyanides GJ. Aminoglycosides induce a phospholipidosis in
the renal cortex of the rat; An early manifestation of nephrotoxicity. J Pharmacol Exp
Therp 1982; 220:514-20.
11. Leibbrandt ME, Wolfgang GH,MetzAL,OzobiaAA,Haskins JR. Critical subcellular
targets of cisplatin and related platinum analogs in rat renal proximal tubule cells.
Kidney Int 1995;48:761-70.
12. KaushalGP,Kaushal V, Hong X, Shah SV. Role and regulation of activation of
caspases in cisplatin induced injury to renal tubular epithelial cells. Kidney Int
2001;601:1726-36.
13. Sugiyama S,HayakawaM,Kato T, Hanaki Y, Shimizu K,Ozawa T. Adverse effects of
anti-tumor drug,cisplatin on rat kidney mitochondria; Disturbances in glutathione
peroxidase activity. Biochem Biophys Res Commun 1989;159:1121-7.
14. Matsushima H, Yonemura K, Ohishi K, Hishida A. The role of Oxygen free radicals
in cisplatin induced acute renal failure in rats. J Lap Clin Med 1998;131:518-26.
15. Baliga,R., Liu,H. Activation of Caspase 12 by cisplatin (CP) induced
endoplasmicreticulam (ER) stress mediates apoptosis in LLC-PK1 Cells. Am Soc
Nephrol 2004;15:39.
16. Fored CM, Ejerblad E, Lindblad P, Fryzek JP, Dickman PW, Signorello LB, et al.
Acetaminophen, aspirin and chronic renal failure. N Engl J Med 2001; 345: 18011808.
17. McCullouh KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ. GADD 153
sensitizes cells to endoplasmic reticulum stress by down regulation of Bcl-2 and
perturbing the cellular redox state. Mol Cell Biol 2001; 21: 1249-1259.
18. Gupta AK. Quality standard of Indian medicinal plants. ICMR 2003;47-48
19. www.ecoplanet.com
20. YadavJP et al .Antimicrobial activity of Cassia occidentalis against various human
pathogen microbes. Life Science &Med Res2010;LSMR – 9:1-11.
21. Dalziel 1937; Keay 1958; Kerhaeo & Adam 1974;Adjanohoun et al.
1985;Burkill;1997
22. Jain SC, Sharma RA, Jain R, Mittal C. Antimicrobial screening of Cassia
occidentalisin vivo and in vitro. Phytother Res1998;12;200-204
23. Saganuwan AS, Gulumbe ML. Evaluation of in vitro antimicrobial activities and
phytochemical constituents of Cassia occidentalis. Animal Res Int 2006;3:566-569
24. Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S, De Bruyne T, Pieters
L, Totté J, Vlietinck AJ. Antimalarial activity of 20 crude extracts from nine African
medicinal plants used in Kinshasa Congo. J Ethnopharmacol 1999; 68: 193-203
25. Jafri MA, Subhani MJ, Javed K, Singh S, Hepatoprotective activity of leaves of
Cassia occidentalisagainst paracetamol and ethyl alcohol intoxification in rats.
J Ethnopharmacol1999; 66: 355-61.
26. Tona L, Cimanga RK, Mesia K, Musuamba CT, De Bruyne T, Apers S, Hernans N,
Miert SV, Pieters L, Totté J, VlietincKAJ. In vitro antiplasmodial activity of extracts
and fractions from seven medicinal plants used in the Democratic Republic of Congo.
J Ethnopharmacol 2004; 93: 27-32.
27. Sharma N, Trikha P, Athar M, RaisuddinS . In vitro inhibition of carcinogen-induced
mutagenicity by Cassiaoccidentalisand Emblicaofficinalis. Drug Chem Toxicol
2000;23:477-84.
28. Naidu MVR, Anwar A, Ratnakar KS. Probucol protects against gentamicin Kumar
induced nephrotoxicity in rats. Indian J Pharmacol 2000;32:108-13.
29. Milind A Khandkar, Dipak V Parmar, Mita Das, Surendra S Katyare. Is activation of
lysosomal enzymes responsible for paracetamol-induced hepatotoxicity and
nephrotoxicity. J Pharm Pharmacol 1996; 48: 437-440.
30. Corcostegui R, Labeaga L, Arteche JK, Orjales A. Protective effect of hidrosmin
against cisplatin induced acute nephrotoxicity in rats. J Pharm Pharmacol 1998; 4:
465-467.
31. Chattopadhyay RR. Possible mechanism of hepatoprotectiveactivity of Azadiractha
indica leaf extract. J Ethnopharmacol 2003;89 : 217-219.
8.
9.
SIGNATURE OF THE
CANDIDATE:
REMARK OF THE GUIDE
“Anti-oxidant and Nephro-protective activities of Cassia occidentalis Linn .” to be
carried out by Ms. M. Gowrisri of M. Pharm has been discussed and worked out
under my directions and supervision as an official guide. The project work envisaged
is of great importance in the field of pharmacology. The work can be carried out in
pharmacology laboratory of Gautham College of Pharmacy for which facilities are
available. Hence the project is viable and is recommended for clearance and
approval.
10.
NAME AND DESIGNATION OF
10.1 GUIDE
Mr. Syed Bilal M.Pharm,
PROFESSOR
Dept. of Pharmacology
10.2 SIGNATURE
10.3 HEAD OF THE
DEPARTMENT
Dr. B.M. Vrushabendra Swamy
M.Pharm, Ph.D, FICCP.
Director / Professor & Head,
Department of Pharmacology.
10.4 SIGNATURE
11.
CLEARENCE FROM INSTITUTIONAL ETHICAL COMMITTEE:
The study is cleared from Animal Ethical Committee of the Institution.
(Approval no: 491/01/c/CPCSEA,) Enclosed Copy. Annexure-1
12. 12.1 REMARK OF THE PRINCIPAL
The Program and the Research work that Ms. M. Gowrisri is undertaking have
potential implication in the field of Pharmacology. The work can be carried out in the
Research Laboratories of Pharmacology Department at Gautham college of
Pharmacy.
Hence the Project is recommended and requested for clearance and approval.
12.2 SIGNATURE
Prof. Archana Swamy. P
M.Pharm (Ph.D.)
Principal
GauthamCollege of Pharmacy
Bhuvaneswarinagar,R,T.Nagar Post,
Bangalore-32