EVALUATION OF NEPHROPROTECTIVE ACTIVITY OF METHANOLIC
EXTRACT OF TAMARINDUS INDICA LEAVES
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
ATHIRA.K.K.
Under The Guidance of
Mr. ASHOK SHENOY .M.PHARM
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2012 – 2014
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
ANNEXURE-II
BANGALORE, KARNATAKA.
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0
NAME AND ADDRESS OF
THE CANDIDATE
2.0
NAME OF THE
INSTITUTION
3.0
4.0
ATHIRA.K.K.
KARNIKARAM
C.M.C-14
CHERTHALA.P.O
DIST - ALAPUZHA
KERALA
PIN – 688524
SRINIVAS COLLEGE OF PHARMACY
VALACHIL,MANGALORE.-574143
SUBJECT
MASTER OF PHARMACY IN
PHARMACOLOGY.
DATE OF ADMISSION TO
25-6 2012
COURSE OF STUDY AND
COURSE
5.0
TITLE OF THE TOPIC
“EVALUATION OF NEPHRO PROTECTIVE
ACTIVITY OF METHANOLIC EXTRACT OF
TAMARINDUS INDICA LEAVES”
6.0
BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY:
Kidneys play an important part in the maintenance of our endocrine and acid-base balance,
blood pressure, erythropoiesis etc. The main functions of kidney can be categorised as
formation of urine, water and electrolyte balance and production of hormones and enzymes.
Kidneys have some delicate tasks, especially when they have to deal with unwanted
substances, which they have to clear from the system1.
Nephrotoxicity can be defined as renal dysfunction that arises as a direct result of exposure
to external agents such as drugs and environmental chemicals. It has been known from
many years that toxic metals and heavy metals have toxic effects on kidney by accumulating
and producing broad spectrum of morphological and functional effects of kidney. A number
of drugs and antibiotics including penicillins, cephalosporins, tetracycline, sulfonamides and
amino glycosides are known to be potential nephrotoxins2.
There is a growing interest of public in traditional medicine, particularly in the treatment of
nephrotoxicity partly because of limited choice in the pharmacotherapy. Certain Indian
Medicinal plants have been reported to exhibit protective effect of renal tissues against
injuries3. Since there are only few researches made on this field of nephroprotection, this
present study of nephroprotective activity of Tamarindus indica will satisfy the research for
better and cost effective nephroprotection.1
Criteria for selection of the plant :
In Ayurveda plants namely milk thistle, drum stick, ginger, tamarind are used for its
nephroprotective action. The common active constituents present in these plants, responsible
for nephroprotective activity is found to be flavonoids, iso flavonoids, phenolic compounds
like tannins etc. The active constituents present in Tamarindus indica are flavonoids,
phenolic compounds, cardiac glycosides, malic acid, tartaric acid, mucilage, pectin,
arabinose, xylose, galactose, glucose and uronic acid. Also the antioxidants present in the
plant interact with free radicals and may prevent the damage caused by them. Hence an
attempt will be made to explore the nephroprotective activity of methanolic extract of
tamarindus indica leaves.
6.2 REVIEW OF LITERATURE:
6.2.1 Name of the plant : Tamarindus indica (fabaceae)
Other Name: aamlika, tintidi, ambli, amli, puli, vaalan puli, chinch, Hunase, chintachettu
6.2.2 Description :
The tamarind is a long-lived, medium-growth, bushy tree, which attains a maximum crown
height of 12 to 18 metres. Leaves are evergreen, bright green in colour, elliptical ovular,
arrangement is alternate, of pinnately compound type, with pinnate venation and less than
5 cm in length, red and yellow elongated flowers. Tamarind fruit is an indehiscent legume,
sometimes called a pod, 12 to 15 cm (3 to 6 inches) in length, with a hard, brown shell4. The
fruit has a fleshy, juicy, acidulous pulp.
6.2.3 Chemical constituents :
Phytochemical investigation carried out on T. indica revealed the presence of constituents,
such as flavonoids, phenolic compounds5, cardiac glycosides ,malic acid, tartaric acid, the
mucilage and pectin, arabinose, xylose, galactose, glucose, and uronic acid. Ethanolic
extract of T. indica showed presence of fatty acids and various essential elements like
arsenic, calcium, cadmium, copper, iron, sodium, manganese, magnesium, potassium,
phosphorus, lead, and zinc6.
The leaves are fair source of thiamine, riboflavin, niacin, ascorbic acid and β carotene
along with the fat, fibre and mineral content mentioned above. Seeds and pericarp contain
phenolic anti oxidants. Tamarind leaves are also rich in flavonoids, polyphenols, fat, fibre,
and some vitamins such as thiamine, and β-carotene5.
6.2.4 Uses :
Tamarindus indica fruit as digestive, carminative, laxative, expectorant and blood tonic,
Hypolipedemic, anti-inflammatory, cytotoxic, against gastrointestinal spasms and modifying
the complement system6. Leaves are used in the treatment of diabetes, asthma, hepatic
disorders(due to their high content of antioxidant polyphenols), abdominal pain, diarrhoea,
dysentery, helminthes infections, wound healing, malaria, fever, constipation, inflammation,
cell cytotoxicity, gonorrhoea, and eye diseases7.
6.3 OBJECTIVES OF THE STUDY:
1) Extraction of Tamarindus indica plant leaves.
2) Preliminary phytochemical screening.
3) To study the nephroprotective activity of extracts using following animal models
a) gentamycin induced nephrotoxicity
b) Cisplatin induced nephrotoxicity
Blood will be collected for the evaluation of the serum biochemical parameters like
Creatinine, urea, cholesterol, and urea nitrogen
Urine will be collected for the evaluation of urinary parameters like
Creatinine, urea, sodium and potassium.
Histopathological examination of kidney will be done to evaluate the nephroprotective
activity.
7.0
MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Experiment will be performed as described in the standard bibliography, literatures and text
books. The reputed journals and publications are obtained from college library and through
web search.
7.2 COLLECTION OF MATERIAL:
7.2.1 COLLECTION OF PLANT MATERIAL & EXTRACTION
The Tamarindus indica leaves will be collected from local region of Mangalore district, and
will be authenticated by a Taxonomist. The collected fresh leaves will be shade dried and
powdered. The powdered drug will be extracted by hot percolation method (Soxhlet
apparatus) using methanol solvent system. The extract will be filtered through a cotton plug
followed by Whattman filter paper No.1 and then concentrated by using a rotary evaporator
at low temperature. The extract will be preserved in airtight containers and kept at 4-5°C
until further use.
7.2.2 DRUGS AND CHEMICALS:
All the drugs & chemicals are of pure analytical grade will be obtained from the local
suppliers.
7.2.3 ANIMALS
Wistar rats (180-200 g) of either sex will be procured from Indian Institute of Sciences.
They will be maintained under standard conditions (temperature 22 ± 2 oC, relative humidity
50±5% and 12 h light/dark cycle).The animals will be housed in sanitized polypropylene
cages containing sterile paddy husk as bedding. They will have free access to standard pellet
diet and water ad libitum. The Institutional Animal Ethics Committee approved the
experimental protocol. All the animals received human care according to the criteria
outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the
“National Academy of Sciences” and published by the “National Institute of Health”. All
the procedures will be performed in accordance with Institutional Animal ethics committee
constituted as per the direction of the committee for the purpose of control and supervision
of experiments on animals (CPCSEA), under ministry of animal welfare division,
Government of India, New Delhi, India.
7.3 EXPERIMENTAL METHODS :
7.3.1 GENTAMYCIN INDUCED NEPHROTOXICITY IN RATS 8,9
EXPERIMENTAL DESIGN :
The Swiss albino rats (180-200g) of either sex will be randomly divided into 5 groups of 6
each. The different groups will be assigned as described below.
Group I
: vehicle control
Group II : Nephro toxic control(Gentamycin 100 mg/kg)
Group III : Reference standard Selenium (6 mg/kg) + Gentamycin (100 mg/kg)
Group IV : T. indica leaf extract low dose(200 mg/kg) + Gentamycin (100 mg/kg)
GroupV
: T. indica leaf extract high dose(400 mg/kg) + Gentamycin (100 mg/kg)
Nephrotoxicity will be induced in all the animals except Group I by injecting 100 mg/kg of
gentamycin intraperitonially. All the treatments will be given orally 24 hrs after intoxication
and continued once daily for three weeks. Body weight will be checked every day.
EVALUATION:
On the 8th day, urine will be collected for urinary estimations. After three weeks, completion
of the treatment, all the animals will be anaesthetized with anaesthetic ether and blood will
be collected by retro-orbital puncture for serum estimation. Kidney were weighed and
processed for histopathological examination10.
7.3.3.: CISPLATIN INDUCED NEPHROTOXICITY IN RATS1,9
EXPERIMENTAL DESIGN:
The Wistar Rats (180-200g) of either sex will be randomly divided into five groups of six
each. The different groups will be assigned as follows.
Group I
: Vehicle control (10 ml distilled water)
Group II : Nephro toxic control(cisplatin 5 mg/kg)
Group III : Reference standard Selenium + (cisplatin 5 mg/kg)
Group IV : T. indica leaf extract low dose(200 mg/kg) +(cisplatin 5 mg/kg)
GroupV
: T. indica leaf extract high dose(400 mg/kg) +(cisplatin 5 mg/kg)
Nephrotoxicity will be induced in all the animals except Group I by injecting Cisplatin (5
mg/kg body weight) intraperitonially. All the treatments will be given orally 24 hrs after
intoxication and continued once daily for three weeks. Body weight will be checked every
day.
EVALUATION:
On the 8th day, urine will be collected for urinary estimations. After three weeks, completion
of the treatment, all the animals will be anaesthetized with anaesthetic ether and blood will
be collected by retro-orbital puncture for serum estimation. Kidney were weighed and
processed for histopathological examination10.
7.4 STATISTICAL ANALYSIS:
The Statistical analysis were carried out using one way analysis variation (ANOVA)
followed by student‘t’ test, p values < 0.05 were considered as significant.
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS
TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
IF SO PLEASE DESCRIBE BRIEFLY.
YES. Study requires investigation on Wistar strain albino rats.
7.6
HAS
ETHICAL
CLEARANCE
BEEN
OBTAINED
FROM
YOUR
INSTITUTION?
YES. Ethical clearance has been obtained. (Copy enclosed),
Ref:SCP/CPCSEA/P13/F150/2012
8.0 LIST OF REFERENCES:
1) Priyadarsini G, Kumar A, Anbu J, Aswini A, Ayyasami S. Nephroprotective activity
of decoction of Indigofera tinctoria against cisplatin induced nephropathy in rats. Int
J life sci & pharm res 2012; Dec 2(4).
2) Tripathi KD, Essentials of medical pharmacology.Newdelhi:Jaypee brothers medical
publishers(P) Ltd;1985: (6) 667-734
3) Rasikh Javaid, Mohd Aslam, Qudsia Nizami, Raheela Javaid. Role of anti oxidant
herbal drugs in renal disorders:an overview. Free Rad Anti ox 2012; 2(1) 2-5
4) Doughari JH. Antimicrobial Activity of Tamarindus indica Linn. Trop J Pharm Res
2006;5 (2): 597–603.
5) Chitra A. Post Infectional Changes in Flavonoids and Phenolic Acids by Fungal
Pathogens in Tamarindus indica L. Res J Chem Envirom 1999 ;3: 305 - 317.
6) Coutino-Rodriguez R, Hernandez-Cruz P, Gillis-Rios H. Lectins in fruits having
gastro-intestinal activity and their participation in the hemagglutinating property of
Escherichia Coli 0157. Arch Med Res 2001; 32:251-9.
7) Samina Kabir Khanzada, Khaikh W, Shahzadi Sofia, Kazi TG, Usmanghani K, et al.
chemical constituents of tamarindus indica L, Medicinal plant in sindh.Pak.J.Bot
2008:40(6):2553-9.
8) Nurhanani Razali, Sarni Mat-Junit, Amirah Faizah Abdul-Muthalib, Senthilkumar
Subramaniam, Azlina Abdul-Aziz. Effects of various solvents on the extraction of
antioxidant phenolics from the leaves, seeds, veins and skins of Tamarindus indica
L. Elsevier 2012;131(2):441-8.
9) N. Kannappan, Madhukar A, Mariymmal, Uma Sindhura P, Mannavalan R.
Evaluation of nephroprotective activity of Orthosiphon stamineus benth extract
using rat model. Int J Pharm Tech Res 2010; 2(1):209-15.
10) Mukesh kumar Meena, HK Kushwah, Manjusha Rajagopala, Ravisankar B. An
experimental evaluation on nephroprotective activity of Nagaradi kashaya. AYU
2009; 30(1):55-61.
9.
SIGNATURE
CANDIDATE
OF
THE
10.
REMARKS OF THE GUIDE
11.
NAME AND DESIGNATION
RECOMMENDED AND FORWARDED
MR.ASHOK SHENOY
ASSISTANT PROFESSOR
11.1 GUIDE
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE – 574143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY)
11.4 SIGNATURE
MR MOSES SAMUEL RAJAN
11.5 HEAD OF THE
ASSOCIATE PROFESSOR
DEPARTMENT
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE – 574143
11.6 SIGNATURE
12.1
12
REMARKS
OF
THE
PRINCIPAL
12.2
RECOMMENDED AND FORWARDED
NAME AND
SIGNATURE OF PRINCIPAL
DR. A. R. SHABARAYA
PRINCIPAL,
SRINIVAS COLLAGE OF PHARMACY
MANGALORE- 574 143.