Rajiv Gandhi University of Health s3

A PROTECTIVE ROLE OF AZIMA TETRACANTHA LAM LEAVES ON CARDIO AND NEPHRO TOXICITIES INDUCED IN ALBINO RATS

M-PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, KARNATAKA, BENGALURU

BY

Mr. GOPI KISHOR NAIDU. M

B. Pharm

UNDER THE GUIDANCE OF

PROF. ITTAGI SHANMUKHA

M. Pharm

P. G. DEPARTMENT OF PHARMACOLOGY

S. C. S. COLLEGE OF PHARMACY

HARAPANAHALLI-583131

2011-12

Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

Brief resume of the intended work

Enclosure – I

6.3. Objective of the Study

Materials and Methods

Enclosure – IV

7.2. Methods of collection of data

List of References (About 4 – 6)

Signature of the Candidate

Remarks of the Guide

Name and Designation of

Remarks of the Principal

ENCLOSURE-I

06. Brief resume of Intended Work

6.1 Need for the study.

Plants are exploited as food and medicine since ancient periods. The therapeutic use of medicinal plants has gained a considerable momentum in the past few decades. The over usage of synthetic drug resulting in higher incidence of adverse drug reactions have motivated mankind to go back to nature for safe remedies. Continues exposure to stressful conditions generates free radicals, which may over power the inbuilt protective mechanisms and cause tissue dammage. There are reports that plants possessing free radical scavenging activity are known to have organ/organs protective effect. . Many flowers and green flower are useful as medicines are reported by many scientists1,2. The carotenoids and perhaps non-nutrients like dietary fibers and other phenolic components (falvonoids, tannins and terpenoids) can influence the enzyme involved in the activation and detoxification of xenobiotics including carcinogens3. Similarly, many such herbs are known to protect the organs and organ systems from the environmental, chemical and occupational changes.

Myocardial infarction (MI) is a key component of the burden of cardiovascular disease. The assessment of the incidence and case fatality of myocardial infarction are important determinants of the decline in coronary disease mortality4. Experimental and clinical studies have shown that there is increased generation of reactive oxygen species such as superoxide anion (O2-) and hydroxyl radicals (OH) in heart failure, which involved in the formation of lipid peroxides, damage of cell membrane and destruction of ant oxidative defense system5,6,7. Therapeutic intervention via suppression of free radical generation and/or enhancement of endogenous antioxidant enzymes may limit the infarct size and attenuate myocardial dysfunction8.

Acute kidney injury previously called acute renal failure (ARF) is a rapidly progressive loss of renal function generally characterized by oliguria. The kidneys can often recover from acute failure, allowing the patient to resume a normal life. People suffering from acute renal failure require supportive treatment until their kidneys recover in function and they often remain at increased risk of developing future kidney failure9. Chronic kidney disease is accompanied by cardiovascular involvement; contributing factors to this condition are elevated blood pressure, metabolic disturbances with subsequent coronary atherosclerosis, endothelial damage, hypoxia secondary to anemia, oxidative stress etc. It is assumed that for a patient with chronic kidney disease the probability to reach end-stage renal disease (ESRD) should be lower than the probability to die from cardiovascular complications. Patients with cardiovascular diseases frequently show renal injury, which has not yet been studied in its complexity. Renal involvement has also been involved in many cardiovascular diseases, although there is a relative paucity of data regarding the impact of kidney lesions in cardiovascular diseases10. In addition, it is becoming risk to use synthetic drug because of the adverse drug reaction, toxicity and drug-drug interaction associated with them. Therefore, many researchers are interested to look for alternative system of medicine and herbal drugs. Hence many studies have been performed to identify antioxidant compounds with organ protective property and limited toxicity from medicinal plants11. Plants have been a veritable source of drugs. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive components of the plants are alkaloids, flavonoids, tannins and phenolic compounds12. There are reports that plant possessing polyphenolic compounds, flavonoids and tannins are useful as antioxidants and further it acts as organ protectant13. Keeping this points in view, In our field survey we found as dioecious,highly branched bush, namely Azima tetracantha Lam of family Salvadoraceae commonly known as “mulluchangu” and this species has many uses in an unique folk medicine like Anti rheumatism, diuretic, anti inflammatory, anti microbial, hypoglycemic, antioxidant and hypolipidemic activity. It is also used in the treatment of dyspepsia and chronic diarrhoea14.

The literature survey of this plant revealed that the plant possess dimeric piperidine alkaloids like azimine, azacarpine, carpine. The seeds contain mixture of about 25 flavonoids, fatty acids. Seed oil contains fatty acids like myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, lenoleic acid, ecosonic acid. Friedelin, lupeol, glutinol and β-sitosterol were isolated from the petroleum ether extract of the leaves of Azima tetracantha Lam. It also contains glycosides, triterpenoids, isorhamnetin-3-o-rutinoside, glucosinolates and neo ascorbinogen15.

Hence in the present study an attempt is made to quantify the total polyphenolic, flavonoidal content and to evaluate the cardio and nephro protective activities from the leaves of plant Azima tetracantha Lam.

ENCLOSURE-II

6.2 Review of Literature:

Azima tetracantha Lam (Salvadoraceae) commonly known as “mulluchangu” is a glabrous, rigid, rambling, thorny shrub commonly called Bee sting bush found in Africa, India and Madagascar.

Description:

Azima tetracantha Lam is a species belonging to family of Salvadoraceae commonly found in Africa, India and Madagascar.

The plant is known by different languages16,17:

Sanskrit : Kunthali, kundali, kandaki

English : Needle bush

Hindi : Kanda-gur-kamay

Kannada : Uppimullu

Malayalam : Yasank, Esanku

The plant is dioecious, erect shrub up to 90 cm tall with (1–)2 spines 0.5–5 cm long in each leaf axil, sometimes scandent with stems up to 8 m long; branchlets terete or quadrangular, glabrous to densely hairy. Leaves decussately opposite, simple and entire; stipules absent or rudimentary; petiole short; blade elliptical-oblong to ovate-oblong or orbicular, 1.5–5.5 cm × 0.5–4.5 cm, base rounded or somewhat narrowed, apex mucronate, pinnately veined with one pair of lateral veins from near the base. Inflorescence an axillary, sometimes terminal spike or cyme up to 3 cm long or flowers solitary; bracts ovate, often with long and spinous mucro. Flowers are unisexual, regular, 4-merous, usually sessile; calyx campanulate, 2–4 mm long, with triangular lobes; petals linear-oblong to oblong, greenish to yellowish, the upper part reflexed over the calyx, 2–5 mm long; male flowers with stamens inserted at the base of the rudimentary ovary, exserted; female flowers with staminodes and superior ovary, up to 4.5 mm long with a broad sessile stigma. Fruit a globose berry, 0.5–1 cm in diameter, 1–2-seeded, green turning white, with persistent stigma and seeds disk-like brown to black16,17,18.

Chemical constituents:

There are reports on the phytochemical composition of the plant like presence dimeric piperdine alkaloids azimine, azacarpaine, carpaine19, triterpenoids20, isorhamnetin 3- rutinoside21, neoascorbinogen , glucosinolates , the contain complex mixture of 26 flavonoids predominately as glycosides and acyl glycosides, with traces of aglycones. The core aglycones of these flavonoids were identified as quercetin, isorhamnetin, rhamnetin and rahmnazin22. Friedelin, lupeol, glutinol and β-sitosterol were isolated from the petroleum ether extract of the leaves of Azima tetracantha Lam14. It is well known medicinal plant whose root has been using in various ailments such as dropsy and rheumatism. Leaves possess stimulant, expectorant, antispasmodic, analgesic and anti-inflammatory activities23, it is also reported that, it is having effective hypoglycemic and hyperlipidemic activity in alloxan-induced diabetic albino rats14.

Review of literature regarding Azima tetracantha Lam reveals that:

1) Evaluation of Anthelmintic and Antimicrobial activities of Azima tetracantha Lam15.

2) Antipyretic activity of Azima tetracantha in experimental animals24.

3) In vitro studies on antioxidant and free radical scavenging activities of Azima

tetracantha Lam leaf extracts14.

4) Hepatoprotective activity of Azima tetracantha Lam. in experimental animals17.

5) Genetic diversity in Azima tetracantha Lam assessed through RAPD analysis25.

6) Antiulcer Activity of Azima Tetracantha: A Biochemical Study2

ENCLOSURE –III

6.3 Objectives of the study:

Since there is incomplete phytochemical and pharmacological profile, it is planned to undertake a study on the leaves of the plant Azima tetracantha Lam with the following objectives.

01.  To prepare various extracts (petroleum ether extract, chloroform extract, alcoholic

extract and aqueous extract) by successive extraction technique of the leaves of the Azima tetracantha Lam.

02.  To identify the phytoconstituents present in the leaf extract.

03.  Quantification of phytochemicals such as total poly phenols, falvonoids and tannins.

04.  To assess acute toxicity of the leaf extract

05.  To screen the selected extract for the Cardio and Nephro protective activities on

leaves against experimentally induced cardio (isoproterenol & doxorubicin) and nephro (cisplatin & gentamycin) toxicity in rats.

ENCLOSURE – IV

7. Material & methods:

7.1 Source of data:

Whole work is planned to generate data from laboratory i.e., experiments on animals. The rats and mice will be used for this purpose. Standard analytical procedures will be adopted for estimation of biochemical markers like Lactate dehydrogenase (LDH), Aspartate transaminase (AST), Alanine transaminase (ALT), Creatine kinase (CK), tissue GSH, Lipid per oxidation and SOD etc. and other biochemical estimations like Blood creatinine and Blood urea. It is also planned to use the available literature for interpreting the data.

ENCLOSURE – V

7.2 Method of collection of data:

The whole study is divided into four phases to generate the data as follows.

Phase I: Preparation of extract and Identification of phytoconstituents 26,27 :

The leaves extract will be prepared by successive soxhlation i.e. extracting dried powder with the solvents of increasing order of polarity i.e. Pet. ether (60-80°), chloroform (59.5-61.5°), 70% ethanol (64.5-65.5°) and water. Extracts will be concentrated under reduced pressure.

Phase II: Acute toxicity studies28:

The toxicity of leaves extract of Azima tetracantha Lam will be determined by using albino mice (20-25 g) maintained under standard husbandry conditions. The animals will be fasted for 3 hr. prior to the experiment. Animals will be administered with single dose of extract observed for its mortality up to 48 hr. study period (short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No 423.

Phase III: Experimental design:

Quantitative determination of total phenol, flavonoid and tannin content by Spectrophotometry:

Ø  Quantification of total phenolic content29:-

The total phenolic content of the leaves extract of Azima tetracantha Lam will be determined by taking aliquots of the extracts into 10ml glass tube and the volume will be made up to 3ml with distilled water. Then 0.5ml of Folin ciocalteau reagent (1:1 with distilled water) and 2 ml sodium carbonate (20%) will be added subsequently in each test tube. A blue color will be developed in each test tube because the phenols will undergo complex redox reaction with phosphor molibdic acid in Folin ciocalteau reagent in alkaline medium. This results in a blue colored complex, molybdenum blue. The test solutions will be warmed for 1min, cooled and the absorbance will be measured at 650nm using known concentration of catechol. The concentrations of phenols in the test samples will be calculated from the calibration plot and expressed as mg catechol equivalent of phenol per gram of sample.

Ø  Quantification of total flavonoid content29:-

To determine the total flavonoidal content, the stock solutions of extract will be prepared with ethanol to a suitable concentration for analysis. For determination of total flavonoidal content, aliquots of each extract will be pipetted out in series of test tubes and the volume will be made up to 1ml with distilled water. Sodium nitrite (5%; 0.3ml) will be added to each test tube and incubated for 5minutes at room temperature. Aluminium chloride solution (10%; 0.06ml) will be added and incubated for 5minutes at room temperature. Sodium hydroxide (1M; 0.25ml) will be added and total volume will be made up to 3ml with distilled water. Absorbance will be measured at 510nm against a reagent blank using U.V. spectrometer and concentration of falvonoids in the test sample will be determined and expressed as mg equivalent per gram of sample.

Ø  Quantification of tannins30:-

The tannins will be identified using FeCl3 and gelatin tests. For this purpose, 0.1g of leaves extract will be transferred to a 100ml flask. 50ml of water will be added and boiled for 30min. After filtration with cotton filter, the filtrate will be transferred to a 500ml volumetric flask and the volume will be made up to the mark with distilled water. 0.5 ml aliquots will be transferred to the vials, 1ml 1% K3Fe(CN)6 and 1 ml of 1% FeCl3 will be added and the volume will be made up to 10ml with distilled water. After 5 min the solution will be measured calorimetrically at 720nm. The total content of tannins present in the plant extract will be obtained from standard calibration curve which will be made by taking the tannic acid as standard.