Evaluation of Cardio and Nephro Protective Properties of Leaves extract of
Raphanus sativus Linn.
M. PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE
BY
MANISH SHAH B.Pharm.
UNDER THE GUIDANCE OF
DR. SHIVAKUMAR SWAMY M.Pharm., PhD
H.O.D & PRINCIPAL
MALLIGE COLLEGE OF PHARMACY, BANGALORE
MALLIGE COLLEGE OF PHARMACY
#71 SILVEPURA, BANGALORE 90
Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / Manish ShahS/o Chiranjiwi Shah, Ashok Vatika,
Birgunj-13, Parsa (dist.) ,Narayani zone,
Nepal
02 / Name of the Institution / Mallige College Of Pharmacy
#71, Silvepura,Chikkabanavara Post,
Bangalore -90,Karnataka,
India
03 / Course of the Study Branch / M. Pharm (Pharmacology)
04 / Date of Admission to course / 12/12/2011
05 / Title of the Topic / Evaluation of Cardio and Nephro Protective Properties of Leaves extract of Raphanus sativus Linn.
06 / Brief resume of the intended work
6.1. Need for the Study / Enclosure – I
6.2. Review of the Literature / Enclosure – II
6.3. Objective of the Study / Enclosure – III
07 / Materials and Methods
7.1. Source of data / Enclosure – IV
7.2. Methods of collection of data / Enclosure – V
7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
obtained from your institution
in case of 7.3. / Yes
08 / List of References / Enclosure – VII
09 / Signature of the Candidate / (Manish Shah)
10 / Remarks of the Guide / The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by Mr. Manish Shah under my supervision.
11 / Name and Designation of
(in Block Letters)
11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6.Signature / Dr. SHIVAKUMAR SWAMY
M. Pharm., Ph. D.
Principal & HOD
Mallige College Of Pharmacy,
Bangalore, Karnataka.
Dr. SHIVAKUMAR SWAMY
M. Pharm., Ph. D.
Principal & HOD
Mallige College Of Pharmacy,
Bangalore, Karnataka.
12 / Remarks of the Principal
12.1. Signature / The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.
(Dr. Shivakumar Swamy)
ENCLOSURE: I
06. Brief resume of the intended work:
6.1. Need for the study:
Cardiovascular diseases (CVDs) such as hypertension and myocardial infarction (MI) are the most important cause of mortality in developing countries due to changing lifestyles. MI is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demands, it increases the generation of reactive oxygen species in ischemic tissue, bringing about oxidative damage of membrane lipids, proteins, carbohydrates and DNA and brings changes in the mechanical, electrical, structural and biochemical properties of the heart1.
Nephrotoxic injury is damage to one or both of the kidneys that results from exposure to a toxic material, usually through ingestion. Nephrotoxic injury can lead to acute renal failure, in which the kidneys suddenly lose their ability to function, or chronic renal failure, in which kidney function slowly deteriorates. If unchecked, renal failure can result in death. Chronic exposure to drugs, occupational hazards, or environmental toxins can lead to chronic interstitial renal diseases2. Several drugs causes nephrotoxicity which includes antibiotics, primarily aminoglycosides like gentamicin3 ,sulphonamides, amphotericin B, polymyxin, neomycin, bacitracin, rifampin, trimethoprim, cephaloridine, methicillin, oxy- and chlorotetracyclines, analgesics, including NSAIDS4.Several in vivo and in vitro studies have demonstrated that reactive oxygen metabolites including free radical species, superoxide, hydroxyl radical anion and hydrogen peroxide are important mediators of tissue injury. Oxygen free radicals have been implicated in several biological processes potentially important in glomerular diseases6 and also their role in neutrophil mediated glomerular diseases.
There are many drugs available for cardioprotective and nephroprotective activities. Antioxidants are one of the agents which are extensively used for cardio and nephro protective activities. Antioxidants are the substances which chemically react with free radicals and render them harmless and at the same time break the viscous circle, which involve in the decomposition of fatty acids and proteins, the creation of new free radicals and leads to eventual cell death7.The antioxidant defense system includes both endogenously and exogenously derived compounds, dietary plants based antioxidant have recently received a great attention. Hence many studies have been performed to identify antioxidant compounds with pharmacological activity and a limited toxicity from medicinal plants8.Antioxidants may play an important role in the chronic disease prevention by arresting oxidative damage caused by reactive oxygen species (ROS) to vital molecule such as DNA, lipids and proteins9. There are also many reports that plants possessing free radical scavenging activity are having organ protective effect.
Although modern drugs are effective in preventing the disorders, their use is often limited because of their side effects and adverse reactions. A wide array of plants and its active principles, with minimal side effects, provide an alternate therapy. Number of plants has been evaluated for Cardioprotective and Nephroprotective activities by the virtue of their antioxidant and free radical scavenging properties.
Raphanus sativus Linn (Family - Cruciferae) commonly known as “Radish”, is an aromatic annual herb, which is used in traditional system of medicine to treat various diseases. The roots and leaves of R. sativus have been reported to possess various pharmacological activities like gut-stimulatory effect, hepatoprotective activity, cardioprotective effect, antioxidant activity antiurolithiatic activity. The reported main constituents of R. sativus are alkaloids, proteins, polysaccharides, flavonoids, glycosides, and phenolic compounds10. Roots of the herb have been validated for its cardioprotective activity but none of the work has been done for cardio or nephro protective activities of its leaves, although the leaves were found to possess potent antioxidant and radical scavenging activity11. Hence, the current study has been intended for the evaluation of cardio and nephro protective properties of leaves extract of Raphanus sativus.
ENCLOSURE: II
6.2 Review of literature
The plant family of Cruciferae contains many important vegetables of economic importance. Raphanus sativus L. is originally from Europe and Asia. It grows in temperate climates at altitudes between 190 and 1240 m. It is 30–90 cm high and its roots are thick and of various sizes, forms, and colors. They are edible with a pungent taste12.
Rukhsana Anwar conducted studies of Raphanus sativus (Radish) as hepatoprotective agents. Raphanus sativus leaves powder, its water and ethanol extracts significantly decreased the activity of SGOT, SGPT, SLDH, SAP and serum total bilirubin in paracetamol induced rabbits. Paracetamol produces Hepatotoxicity. Paracetamol significantly increase the SGOT, SGPT, SLDH, SAP and serum total Bilirubin levels both in acute and chornic administration13.
Vanitha Reddy et al., analyzed the antioxidant components, activity and stability of Raphanus sativus leaves. The antioxidant activity of methanol (ME), ethanol (EE) and water (WE) extracts was determined by DPPH radical scavenging, reducing power and in vitro inhibition of lipid oxidation. The extracts showed a dose dependent scavenging of the free radical; DPPH. EE showed higher activity than ME and WE. The extracts also showed varying degree of reducing capacity. All the extracts inhibited the formation of lipid peroxides and their efficacy was in the order of a-tocopherol (80%)>EE (69%)>WE (70%)>ME (65%) 14.
Sravan Prasad Macharla et al. evaluated antidiabetic activity of Raphanus sativus L. Leaves extracts on alloxan induced diabetic rats. The results expressed that aqueous extracts had shown significant protection and maximum reduction in blood glucose was observed in alloxan induced diabetic rats. The results of this comprehensive study revealed that R. sativus leaves showed statistically significant Antidiabetic activity in comparison to the standard glibenclamide15.
Rifat-uz-Zaman made a study on cardioprotective activity of Raphanus sativus fruit powder in the rabbits.its aqueous extract significantly decreased the uric acid and activity of enzyme GOT and LDH in treated rabbits16.
Woo Kyoung et al., investigated the effects of the ethanol extract of aerial parts of Raphanus sativus on breast cancer cell proliferationand gene expression associated with cell proliferation and apoptosis in MDA-MB-231 human breast cancer cells. They suggested that Raphanus sativus, inhibits cell proliferation via the ErbB-Akt pathway in MDA-MB-231 cells17.
Chemical Constituents:
The leaves contain significantly higher amounts of total polyphenols and a-tocopherol followed by ascorbic acid, b-carotene, flavonoids and glutathione14.
According to an article ethanolic and acetone extracts of R. sativus leaves had total polyphenolic content of 86.16 and 78.77 mg/g dry extract, which were comparable to the traditional rich sources such as green tea and black tea. HPLC identification of polyphenolics indicated the presence of catechin, protocatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in leaves and stem. Among the different extraction solvents, methanolic extract of leaves and stem showed potent reductive capacity. Further leaves showed most potent antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content11.
Since most of these constituents have been reported for cardio and nephro protective activities, this forms the rationale for the present study.
ENCLOSURE: III
6.3 Objectives of the study
1. To Collect and authenticate Raphanus sativus leaves.
2. To prepare 70% ethanolic extract of leaves of Raphanus sativus.
3. To analyze the extract for the presence of phytoconstituents.
4. To evaluate Cardioprotective activity of the extract in animal model.
5. To evaluate Nephroprotective activitiy of the extract in animal model.
ENCLOSURE: IV
7. Materials and methods
7.1 Source of data:
The work is aimed to generate data from experiments to be conducted at pharmacology laboratory of our institution. Male albino wistar rats will be used for this purpose.
The experiments, which involves the following steps:
1. Collection of the leaves of Raphanus sativus.
2. Extraction of leaves of Raphanus sativus with 70% ethanol.
3. Conducting qualitative tests for phytoconstituents of the extract.
4. Evaluation of Cardio protective activity of the extract in rats.
6. Evaluation of Nephroprotective activity of the extract in rats.
ENCLOSURE: V
7.2 Method of collection of data:
The data will be compiled based on results obtained from the animal experiments conducted at our research lab.
1) Collection of raw material:
For this study, Raphanus sativus leaves will be collected from the surrounding farms of Bangalore. The sample will be authenticated by a botanist. Fresh leaves will be cleaned, shade dried at room temperature and powdered.
2) Extraction with 70% ethanol:
The shade dried and coarse powdered material will be extracted with ethanol in a soxhlet apparatus. The extract will be concentrated to a small volume using flash evaporator, further evaporated to dryness in a vacuum desiccators and percentage yield of the same will be recorded18.
3) Phytochemical analysis:
The crude extracts thus obtained will be subjected to phytochemical screening following the standard procedures described in the literature.
4) Determination of Acute toxicity:
Sravan Prasad. Macharla et. al., screened different extracts of Raphanus sativus leaves for acute toxicity by the standard method15. The extract was found safe at the dose of 5000mg/kg by oral route. Thus the dose, 500mg/kg will be used in the present study.
4) Evaluation of Cardio protective activity18, 19
Method:
Male Albino Wistar rats (150-250g.) will be used to evaluate the cardioprotective activity. Rats will be treated with ethanol extract of Raphanus sativus leave (RSLE) orally using an intra-gastric tube daily for 28 days. On 28th day, myocardial injury will be induced in experimental rats by injection of Isoproterenol (ISO) (200 mg kg-1, S.C.) twice at an interval of 24 hr. (i.e., on 28th and 29th day of extract treatment), while normal control and drug control rats will be given an equivalent volume of the vehicle.
Treatment protocol:
The experimental rats will be divided into 4 groups of 6 animals each and treated as follow:
Sl.No. / Group / RSLE
(500mg/kg B.W., p.o.) / ISO
(200mg/kg B.W. s.c)
1. / Normal Control / - / -
2. / Positive control / - / 28th and 29th day
3. / RSLE Control / Daily for 28 days / -
4. / RSLE Pretreated / Daily for 28 days / 28th and 29th day
Biochemical analysis:
24 hr. after treatment period on 30th day blood will be collected from retro-orbital plexus, serum will be separated and used for estimation of marker enzymes, Aspertate Aminotransferase (AST), Alanine Aminotransferase (ALT), Lactate Dehydrogenase (LDH) and Creatine Phosphokinase (CPK) using standard procedure.
Histopathological studies:
At the end of the study, all the rats will be sacrificed by cervical decapitation and the hearts will be dissected out, washed in ice cold saline. Then myocardial tissue will be immediately fixed in 10% buffered neutral formalin solution and will be processed for histopathological studies.
5) Evaluation of Nephroprotective activity 20, 21
Method
Male albino wistar rats (150-250g.) will be used to evaluate the nephroprotective activity. Rats will be treated with ethanol extract of Raphanus sativus leave orally using an intra-gastric tube daily for 14 days. On 12th day, renal injury will be induced in experimental rats with oral dose of Paracetamol 2g/kg b.w. 30 min. after the administration of vehicle or extract, while normal control and drug control rats will be given an equivalent volume of the vehicle.
Treatment Protocol:
Albino rats will be randomly assigned into 4 groups of 6 individuals each and the treatment will be given as per the following procedure-
Sl.No. / Group / RSLE
(500mg/kg B.W., p.o.) / Paracetamol
(2g/kg B.W., p.o.)
1. / Normal Control / - / -
2. / Positive Control / - / On 12th day
3. / RSLE Control / Daily for 14 days / -
4. / RSLE Pretreated / Daily for 14 days / On 12th day
Biochemical estimation:
After 48 hr. of paracetamol administration, blood sample will be collected from the all test animals under anesthesia by cardiac puncture and serum parameters including Creatinine, Blood Urea Nitrogen (BUN) and Uric acid will be estimated using standard procedure.
Histopathological studies:
At the end of the study, all the rats will be sacrificed by cervical decapitation and the kidney will be dissected out, washed in ice cold saline. Pieces of kidney from each group will be fixed immediately in 10% neutral formalin and processed for histopathological studies.