EVALUATION OF NEPHROPROTECTIVE ACTIVITY OF HYDRO ALCOHOLIC EXTRACT OF ENSETE SUPERBUM (ROXB)CHEESM SEEDS AGAINST STREPTOZOTOCIN INDUCED DIABETIC NEPHROPATHY IN RATS

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

BY

PRADNYA P. KULKARNI

Under The Guidance of

DR. KARUNAKAR HEGDE M.Pharm, Ph.D.

DEPARTMENT OF PHARMACOLOGY

SRINIVASCOLLEGE OF PHARMACY

MANGALORE – 574143

2013– 2015
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 / NAME AND ADDRESS OF THE CANDIDATE / PRADNYA P. KULKARNI
HUDCO COLONEY MIG-2 HNo:26
AKKOL ROAD, NIPPANI.
BELGAUM (DIST)
KARNATAKA-591237
2.0 / NAME OF THE INSTITUTION / SRINIVAS COLLEGE OF PHARMACY
VALACHIL, MANGALORE- 574 143
3.0 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4.0 / DATE OF ADMISSION TO COURSE / 25-07-2013
5.0 / TITLE OF THE TOPIC / “EVALUATION OF NEPHROPROTECTIVE ACTIVITY OF HYDRO ALCOHOLIC EXTRACT OF ENSETE SUPERBUM (ROXB)CHEESM SEEDS AGAINST STREPTOZOTOCIN INDUCED DIABETIC NEPHROPATHY IN RATS”


6.0
7.0
8.0 / BRIEF RESUME OF THE INTENDED WORK:
6.1.NEED FOR THE STUDY:
Kidneys are vital organs, play a critical role in maintaining overall health and it is essential to maintain good kidney health throughout our lifetime. The kidneys are particularly susceptible to various kinds of injuries leads to partial loss of its function; this condition is known as nephropathy. There are many types of nephropathies like drug induced nephropathy, diabetic nephropathy, hypertension nephropathy, contrast induced nephropathy[1] etc., among them diabetic nephropathy is most important one which is a progressive kidney disease caused by angiopathy of capillaries in the kidney glomeruli due to chronic hyperglycemia.
Increased prevalence of diabetic nephropathy worldwide reflects the significant numbers of patients diagnosed with diabetes mellitus with expected global diabetes burden of 380 million by year 2025[2]. Diabetic nephropathy (DN) is the leading cause of end stage renal disease (ESRD), and approximately 30% of patients with type 1diabetes or type 2 diabetes develop DN[3]. Microalbuminuria is the earliest clinical manifestation of DN and is associated with substantial risk for progressive kidney damage. Hyperglycemia activates various inflammatory pathways both directly and via gene transcription to induce oxidative stress, reactive oxygen species, fibrotic factors TGF-β, renin-angiotensin- aldosterone system (RAAS), and advanced glycation end-products, leading collectively to podocyte injury, malfunction, apoptosis, and protein deposition in extracellular matrix of the nephron with albumin leak[4]. This condition is asymptomatic until the later stage of disease and once the symptom occurs, kidney function tends to be 25% lower than normal and is irreversible. Hence there is a great need to prevent the progressive loss of kidney function due to diabetes.
The plants provide a potential source of hypoglycemic drugs because many plants and plant derived compounds have been used in the treatment of diabetes. Many Indian plants have been investigated for their beneficial use in different types of diabetes and reported in numerous scientific journals. Ayurveda and other traditionalmedicinal system described number of plants for the treatment of diabetes as herbal drugs. Hence, they play an important role as alternative medicine due to less side effects and low cost. Oral hypoglycaemic agents like sulphonylureas and biguanides are still the major players in management of the disease but there is growing interest in herbal remedies due to side effects associated with the oral hypoglycaemic agents.
While reviewing the available information for selecting a folklore based remedy for the evaluation of anti-diabetic activity of seeds of wild banana [Ensete superbum (Roxb.)] (Family Musaceae), were found to be used for this purpose in folklore medicine in some parts of the country5. Oral administration of powderof the seeds mixed with milk in case of diabetics in different parts of India. The seeds, stem, and leave of the Ensete superbum plant used for the treatment of the disease like diabetics, chronic fever, urinary tract infections, and kidney disorders by the tribal healers. Ensete superbum has been shown to possess many curative properties in traditional medicine and its several medical applications are well recognized. Medicinal properties like anti-diabetic action, dog bite, leucorrhea, kidney stones, bladder infections, small pox, urinary retention and kidney diseases has been reported6,7,8. Previously Sulakshan (2008)5 carried out antidiabetic activity of this drug against STZ induced diabetes in rats and results were encouraging.However till date no research has been done on this drug for diabetic nephropathy and seeds contained phyto-constituents like flavonoids, terpenes, alkaloids9 which are renowned for anti-oxidant activity. As oxidative stress is the hall mark of diabetic nephropathy,hence this plant may useful in the management of DN due to its anti-diabetic as well as anti-oxidant properties.
Based on these backgrounds,the present study has been designed to evaluatenephroprotective activity of hydro-alcoholic extract of seeds of Ensete superbumagainst experimentally induced diabetic nephropathy.
6.2. REVIEW OF LITERATURE:
6.2.1 Botanical name:
Ensete superbum (Roxb) Cheesm, Family: Musaceae.
Common name: Rock banana, Jungle kela, Wild plantain, bahuja, Western hill banana
Distribution:
Ensete superbum (Roxb) Cheesman is reported as occurring naturally in India mainly in Western Ghats, Anaimalai Hills, Jhadol and Ogna forest ranges in Rajasthan, (North India) and some other South Indian hills in Dindigul and other parts of the peninsular India. It has also been recorded from. There are also reports of a similar species in Thailand6, 7.
Description:
It is characterized by a strongly swollen at the base apparent strain as 2.5m circumference can measure up and narrows to 1m circumference. The trunk is about 2m high. It does not produce suckers. The leaves are bright green on both sides with a short, red and deeply grooved petiole. They are on average 150 cm long and 40-50 cm wide. The leaf sheaths are persistant and leave closely set scars in the corm. Inflorescence is first spherical, about 30 cm long, later drooping, up 1/3 of the length of the pseudo-stem. The bracts are round, dark brown-red, 1m long and broad, dense rows with each 10 to 15 flowers. The outer perianth is whitish; the inner perianth is shorter than the outer. The fruit is 15 cm long and roughly triangular in section with dark brown spherical seeds. They are edible. The plant grows on limestone cliffs, on edges or in crevices it dies back in winter or dry season and resumes next spring from the corm. Remarkably, when Ensete superbum grows on barren land, it goes through a life cycle of four years but in humid conditions it is evergreen and has a life cycle of only three years7.
Part used: root, stem, leaves, Flower, seed.
Active constituents of Ensete superbum seed:
There are different types of phytochemicals they are alkaloids, tannins, glycosides, flavanoids, carbohydrates, amino acids, triterpenoids, saponins, steroids, phenols and Non-steroidal phytosterol (4-hydroxy-3-methyl-hex-5-enyl) isolated from seeds 6, 10.
Medicinal Uses:
It is used in the Ayurveda to treat diabetics, leucorrhea, kidney stones, bladder infections, dog bite, small pox, urinary retention, vesicle calculi, burns, scalds, odema, and menorrhagia, antifertility, anti-vaccinia, anti-variola and general weekness7.
6.3. OBJECTIVES OF THE STUDY:
The main objectives of the present study are as follows:
1) Authentication and collection of the plant Ensete superbum (Roxb) Cheesman seeds.
2) Extraction of Ensete superbum seeds using 1:1 water and ethanol.
3) Preliminary phytochemical investigation.
4) Acute toxicity evaluation to derive the doseand safety.
5) To study the activity of extract on diabetic nephropathy using streptozotocin induced diabetic nephropathy.
6) To evaluate of the serum biochemical parameters like creatinine, glucose, proteins, urea, cholesterol and urea nitrogen.
7)To evaluate the urinary parameters like creatinine, glucose, urea, sodium and potassium using urine sample.
8)To evaluate the histopathological status of kidney.
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 are obtained from college library and electronic media.
7.2. COLLECTION OF MATERIAL:
7.2.1 DRUGS AND CHEMICALS:
All other chemicals and reagents will be of pure analytical grade and obtained from local suppliers.
7.2.2 PLANT MATERIAL AND EXTRACTINON:
For the present study the seeds will be collected from authentic source. The seeds will be shade dried, powdered mechanically and sieved by using a mesh no. 10/44. Extraction will be done using 1:1 water and ethanol solvent in a soxhlet apparatus. The concentrated extract will be dried to a thick mass using rotary flash evaporator. The weight of dried extract will be recorded, preserved in airtight containers and kept at 4-5°C until further use.
7.2.3 ANIMALS:
Wistar Albino rats (150 to 200 g) and albino mice (20-25 g) of either sex will be used for this study. They will be maintained under standard conditions (temperature 22 ± 2°C, relative humidity 60±5% and 12 h light/dark cycle) and have free access to standard pellet diet and water ad libitum. The animals will be housed in sanitized polypropylene cages containing sterile paddy husk as bedding. The Institutional Animal Ethics Committee reviewed and approved the experimental protocol. 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).
7.3. EXPERIMENTAL METHODS
7.3.1Acute toxicity evaluation:
Acute toxicity study of the extract of seeds of the plant Ensete superbum (Roxb)Cheesmanwill be performed as per the OECD guidelines 425 at a limit dose of 2000 mg/kg or 5000 mg/kg. The doses will be administered by oral route in albino mice (20-25g). Animals will be observed individually at least once during the first 30 minutes after dosing, periodically during the first 24 hours (with special attention given during the first 4 hours), and daily thereafter, for total 14 days for sign of toxicity and/or mortality if any.
7.3.2STREPTOZOTOCIN INDUCED DIABETIC NEPHROPATHY IN RATS
EXPERIMENTAL DESIGN :
The Wistar albino rats (150-200g) of either sex will be selected and the diabetes will be induced by administration of a single dose of STZ (50 mg/kg i.p). After 72 hours of STZ injection the blood glucose level (BSL) will be assessed and the animals having BSL>200 will be selected for the study. These diabetic animals will be divided into different groups as mentioned below. Development of nephropathy will be assessed in terms of significant increase in proteinuria, serum creatinine, blood urea nitrogen (BUN) periodically11, 12.
The diabetic animals will be divided into following groups containing 6 animals each.
Group I : Vehicle control
Group II : Diabetic Nephropathy control
Group III : Diabetic animals will be treated with Ensete superbum seed extract
(Lower dose)
Group IV: Diabetic animals will be treated with Ensete superbumseed extract (Higher dose)
7.3.3 EVALUATION:
Parameters to be studied:
Biochemical estimations:
(1)Measurement of serum glucose, creatinine, proteins, urea, cholesterol and urea nitrogen.
(2)Evaluation of oxidative stress by measuring Serum glutamate oxalate transaminase (SGOT) and SGPT.
(3) Measurement of Urinary glucose, creatinine, urea, sodium, and potassium.
Histopathological studies:
The kidney will be allowed to fix in 10% formalin and processed through graded ethanol, xylene and impregnated with paraffin wax. Sections will be made using microtome. After staining with haematoxylene and eosin, the different histopathological indices will be determined.
7.4. STATISTICAL ANALYSIS:
The Statistical analysis will be carried out using analysis variation (ANOVA) test.
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 albino rats and mice.
7.6. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
REFERENCES:
  1. Jose M, Lopez-Novoa, Carlos Martinez-Salgado, Ana B Rodriguez-Pena, Francisco J.Lopez Hernandez.Common pathophysiological mechanisms of chronic kidney disease: Therapeutic perspectives. Pharmacol Ther 2010;128:61-81.
  2. Guilbert JJ. The World Health Report 2006: Working Together with Health. Educ Health (Abingdon) 2006;19:385-7.
  3. Dalla Vestra M, Saller A, Bortoloso E, Mauer M, Fioretto P. Structural involvement in type 1 and type 2 diabetic nephropathy. Diabetes Metab 2000;26(4):8-14.
  1. Suma Dronavalli, Irena Duke, George L Bakris. The pathogenesis of diabetic nephropathy. NatClin Pract Endocrinol Metab 2008;4(8):444-51.
  2. Chavan Sulakshan S. Pharmacological Evaluation of Wild Banana Seed. EnseteSuperbum (Roxb.) with special reference to anti-hyperglycaemic and anti-diabetic activity. M.Sc. Dissertation submitted to Gujarat Ayurved University Jamnagar, Gujarat, India. 2008.
  3. Saroj Kumar Vasundharan, Jaishanker Raqhuanathan, Annamalai Arunachalam, Sunil Kumar Koppala Narayana. Investigation into the pharmacognostical and phytochemical features of seeds of Ensete superbum (Roxb.) Cheesman: An unexplored medicinal plant of India. Pharmacognosy Journal 2013;5(4):163-9.
  4. Jungle garden.blogspot.com/2010/08/Ensete-superbum.html.
  5. RejukrishnanR. An experimental evaluation on Ashmari bhedana property of Ensetesuperbum(Roxb)CheesmanandCocos nuciferaL- A comparative study. “Ayurveda Vachaspati”M.D (Ayurveda) Dissertation submitted to Rajiv Gandhi University of Health Sciences Karnataka, Bangalore 2011.
  6. Pushpak Patidar, Gaurav Raghuwanshi, Nirmal Dongre. Preliminary pharmacognostic and phytochemical investigation ofEnsete superbum (Roxb.) Cheesman (Musaceae). Environ Conserv Journal 2010;11(1&2):61-3.
  7. Pranjal Kumar, Snehal Kishor Badgujar, Nathar VN. Prelimanary screening of different phytochemicals from Ensete superbum(Roxb)Cheesman: A highly medicinal plant of Indian origin.Int JRes Phytochem Pharmacol 2013;3(1):57-60.
  8. Singh J, Budhiraja S, Lal H, Arora BR. Renoprotection by telmisartan versus benazepril in streptozotocin induced diabetic nephropathy. Iranian J Pharmacol Ther2006;5:135-9.
  9. Pitchal Balakumar, Vishal Jain Chakkarwar, Vijay Kumar, Akash Jain, Jayarami Reddy, Manjeet Singh. Experimental models for nephropathy.J Renin-Angiotensin-Aldosterone Syst 2008;9:189-95.

9. / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF THE GUIDE / Recommended and Forwarded.
11. / NAME AND DESIGNATION
11.1 GUIDE / DR. KARUNAKAR HEGDE
ASST. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE- 574 143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY) / Not Applicable
11.4 SIGNATURE CO-GUIDE / -
11.5HEAD OF THE
DEPARTMENT / DR. KARUNAKAR HEGDE
HEAD OF DEPARTMENT
DEPARTMENT OF PHARMACOLOGY
SRINIVAS COLLEGE OF PHARMACY
MANGALORE- 574 143
11.6SIGNATURE
12 / 12.1 REMARKS OF THE PRINCIPAL / Strongly recommended and forwarded for favorable action.
12.2NAME AND SIGNATURE OF PRINCIPAL / DR. A. R. SHABARAYA
PRINCIPAL
SRINIVAS COLLAGE OF PHARMACY
MANGALORE- 574 143.

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