Evaluation of Cardioprotective Properties of Desmodium triflorum Herb in various Experimental induced

Myocardial infarction in albino rats

M-PHARM DISSERTATION PROTOCOLSUBMITTED TO

THE RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BENGALURU

BY

Priyanka L.G. B.Pharm

Under the Guidance of

Prof. A. Veerana Goud M.Pharm

P. G. DEPARTMENT OF PHARMACOLOGYS. C. S. COLLEGE OF PHARMACY

HARAPANAHALLI-5831312011-12

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BENGALURU, KARNATAKA.

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 Name and Address of the Candidate

Priyanka L.G.D/o A.L. Naik, Maylara Road,Harapanahalli. Pin- 583 131Davangere (Dt). Karnataka (State)

02 Name of the Institution

T. M. A. E. Society’sS. C. S. College of Pharmacy,Harapanahalli – 583 131 Davangere (Dt). Karnataka

03 Course of the StudyBranch

M. Pharm., Pharmacology

04 Date of Admission to course 10.01.2012

05 Title of the Topic

Evaluation of Cardioprotective Properties of Desmodium triflorum Herb in various Experimental induced myocardial infarction in albino rats

06

Brief resume of the intended work6.1. 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 Methods7.1. Source of data Enclosure – IV

7.2. Methods of collection of data Enclosure – V7.3. Does the study require any Investigations on animals? If yes give details

Yes,Enclosure – VI

7.4. Has ethical clearance been Obtained form your institution In case of 7.3.

Yes, Registration No: 157/PO/c/1999 CPCSEA dated 3rd February 2012(Copy enclosed)

08List of References (About 4-6) Enclosure-VII

09 Signature of the Candidate(Priyanka L.G)

10 Remarks of the Guide

The study is highly justifiable and is feasible to work in the institution. This work may thorough light on the therapeutic utility on the Desmodium triflorum Herb.

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

Prof. A. Veerana Goud M. Pharm

Prof. Ittagi Shanmukha M. Pharm., (Ph.D)

Prof. A. Veerana Goud Head

P.G. Dept. of Pharmacology

12

Remarks of the Principal

12.1. Signature

The present study was permitted to work in the institution and animal ethical committee permission is granted to it.

Dr. R. Nagendra RaoPrincipal

ENCLOSURE-I

06. Brief resume of Intended Work

6.1 Need for the study.

Myocardial infarction is a clinical syndrome arising from sudden and persistent

decreasing of myocardial blood supply resulting in the necrosis of the myocardium. This is

followed by numerous pathophysiological and biochemical changes such as lipid

peroxidation, hyperglycemia, hyperlipidemia etc1. It is the common presentation of the

ischemic heart disease, occurs when myocardial ischemia suppresses the critical threshold

level for an extended time resulting in irreversible myocardial cell damage. Myocardial

infarction still remains the leading cause of death worldwide. According to the World Health

Organization it will be the major cause of death in the world by the year 2020. In India,

the number of patients being hospitalized for myocardial infarction, commonly known as

heart attack2. Most of the patients generally suffering from sub sternal pain with radiation to

the neck, jaw, or left arm. The pain may be accompanied by shortness of breath, anxiety,

nausea and sweating. Within 12 hrs highest risk of death occurs in acute myocardial infarction

in which the risk of ventricular fibrillation is higher and within 6 months following an episode

of myocardial infarction, patients are at increased risk of an additional infarction3, 4.

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

etc5. It has been suggested by many investigators that cellular damage induced by

doxorubicin, which is an anticancer drug and belongs to anthracycline antibiotics, it is being

used widely for treatment of various hematological and solid tumor malignancies including

breast cancer, leukemia and sarcomas which leads to irreversible degenerative

cardiomyopathy and congestive heart failure. Angiotensin-converting enzyme activity plays a

major role in arterial hypertension and nephrotoxicity6.

Natural antioxidants are associated with free radical scavenging molecules such as

vitamins, terpenoids, phenolic, lignins, stilbenes, tannins, flavonoids, quinones, coumarins,

alkaloids and betalains7. Such as flavonoid and phenol are the bioactive phytoconstituents

having an important role in control and prevention of tissue damage by activated oxygen

species8. Free radicals are chemically unstable atoms or molecules that can cause extensive damage to cells as a result of imbalance between the generation of reactive oxygen species and the antioxidant enzymes. Molecular oxygen is an essential component for all living organisms, where it helps in the process of oxidation which is a basic component of aerobic life and metabolism9. The free radicals and consequent expression of oxidative damage

have been demonstrated during post-ischaemic reperfusion injury and renal failure in humans.

The multitude of free radicals generated during oxidative stress associated with many

chemically induced myocardial necrosis, which can damage major cellular component

including carbohydrate, lipids, protein and DNA. In such condition uncontrolled injury are

widespread tissue damage and associated contractile dysfunction, arrhythmias, enhanced lipid

peroxidation resulting in increased myocardial injury or myocardial infarction10. Antioxidants

are capable of stabilizing or deactivating, free radicals before they attack cells. Antioxidants

are absolutely critical for maintaining optimal cellular, systemic health and well-being11.

Plants have been used for health and medicinal purpose since thousand years. They are

one of the rich and important sources of medicine since human civilization. Now a day, it is

preferred to use plant based medicines over synthetic medication for the treatment of different

diseases because of their safety and cost effectiveness. Herbal medicines are particularly used

by traditional practitioners since ancient12. In over field survey the plant such as Desmodium

triflorum is a small perennial trailing under shrub belonging to the family; Fabaceae

(papilonaceae). The genus desmodium is rich in alkaloids, trigonelline, β-phenethylamine, 5-

indole-3-alkylamine, indole-3-acetic acid, tyramine, stachydrine (pyrollidine alkaloids),

betaine, choline, hypaphorine, hypaphorine methyl ester, hardenine N,N-dimethyltryptophane

and N,N-dimethyl tryptamine oxide. It contain higher amount of flavonoids like diholosyl

flavones, 2-o-glucosylvitexin, vitexin, isovitexin, apigenin and also polyhydric alcohol like

pinitol13,14, the plant also reported to contain rich content of β-sitoserol, tannins, saponins,

steroids, alkaloids, glycosides, so they are known to antioxidants and antioxidants are

reported to have organ protective role14,15. Hence, in the present study, the plant Desmodium

triflorum Herb have been selected for the evaluating its Cardio protective activity by various

experimentally induced cardio toxicities in animals will be used for the study. Therefore the

study is needed and justifiable.

ENCLOSURE-II

6.2 Review of Literature: Desmodium triflorum belongs to family Fabaceae (papilonaceae), it is known as Three

flower beggar weed in English, Kudaliya in Hindi, Kodalia in Bengal, Ranmeti in Marathi13,15.

Description:-The Desmodium triflorum is a small perennial trailing under shrub belonging to the

family; Fabaceae (papilonaceae) commonly grown in moist locations grassy places, road sides

and lawns in Bangladesh and throughout India, Philippine and extending northward to

Florida. Desmodium triflorum is not sown commercially but it spreads naturally through seed

in dung or by adhering to the coats of grazing animals12,15.

Chemical constituents:The genus desmodium is rich in alkaloids, previous investigation of Desmodium

triflorum revealed the presence of trigonelline, β-phenethylamine, 5-indole-3-alkylamine,

indole-3-acetic acid, tyramine, stachydrine (pyrollidine alkaloids), betaine, choline,

hypaphorine, hypaphorine methyl ester, hardenine N,N-dimethyltryptophane and

N,N-dimethyl tryptamine oxide. Desmodium triflorum is also reported to contain flavonoids

like diholosyl flavones, 2-o-glucosylvitexin, vitexin, isovitexin, apigenin and also polyhydric

alcohol like pinitol13,14,16.

Literature survey reveals that Desmodium triflorum has been reported to possess following

pharmacological activities.

1. Anti-ulcer activity of Desmodium triflorum leaf extract in rats17.

2. Analgesic and anti-inflammatory activity of Desmodium triflorum DC18.

3. Antioxidant and Anti-proliferative Activities of Desmodium triflorum (L.) DC19.

4. An survey report that this herb is used in herbal hepatoprotective formulation in

Lucknow20.

ENCLOSURE –III

6.3 Objectives of the study:

The objective of study is to evaluate the antioxidant and cardio protective properties of

Desmodium triflorum herb.

01. To prepare various extracts (petroleum ether, chloroform, hydro alcoholic and aqueous

extract) by successive extraction technique.

02. To identify the type of phytoconstituents present in the flowers extract 21,22.

03. Quantitative determination of total phenol, flavonoids and tannin content present in the

flowers extract by spectrophometry23,24.

04. To assess the antioxidant property (Super oxide anion scavenging, Hydroxyl radical

scavenging and Reducing power method) 25, 26,27.

05. To assess the cardio protective activity of flowers against experimentally

(Isoproterenol and Doxorubicin) induced myocardial infarction in rats28,29.

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 will be used for this purpose. Standard analytical procedures will be adopted for

estimation of biochemicals like cholesterol, tri-glycerides (TG), LDL, VLDL, HDL, lactate

dehydrogenase (LDH), aspartate transaminase (AST), alanine transaminase (ALT), creatine

kinase (CK) etc. Some in–vitro studies like antioxidant property is also planned to generate

the data. It is also planned to use the available literature for interpreting the data.

ENCLOSURE – V

7.2 Materials and methods

The whole study is divided into Four Phases to generate the data as follows.

Phase I: Preparation of extract and Identification of phytoconstituents21,22:

The extract will be prepared by successive soxhlation i.e. extracting dried powder with

solvents with 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: Experimental design

Antioxidant property:

Super oxide anion scavenging activity25

Hydroxyl radical scavenging activity26

Reducing power27

Nitric oxide radical scavenging activity30

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

Spectrophotometry:

Quantification of total phenolic c ontent 23:-

The total phenolic content of the flowers extract of A. scholaris 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

phosphomolibdic 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.

Quantification of total flavonoid content 23:-

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

flavonoids in the test sample will be determined and expressed as mg equivalent per gram of

sample.

Quantification of tannin 24:-

The tannins will be identified using FeCl3 and gelatin tests. For this purpose, 0.1g of flowers

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.

Cardio protective activity:

Isoproterenol induced cardio toxicity mode 28:-

In the present study, adult albino rats of 150-200g will be divided into five groups of six

animals each as below.

Group 1: Normal saline (2ml/kg/day, p.o for 16days).

Group 2: Normal saline (2ml/kg/day, p.o) for 14 days + Isoproterenol (200mg/kg/day

s.c. on 14th and 15th day).

Group 3: Standard (fluvastatin 20mg/kg, s.c.) + Isoproterenol (200mg/kg/day s.c. on

14th and 15th day).

Group 4: Lower dose of extract of 14 days + Isoproterenol (200mg/kg/day s.c. on 14th

and 15th day).

Group 5: Higher dose of extract of 14 days + Isoproterenol (200mg/kg/day s.c. on

14th and 15th day).

At the end of experimental period (after 24h of second isoproterenol injection or 16 th

day of extract/vehicle treatment) all the rats will anaesthetized with light anaesthetic ether and

blood will be collected from the retro-orbital plexus, the serum was separated and used for the

determination of diagnostic enzymes marker and biochemical estimations like AST, ALT,

LDH, CK, TG, TC, HDL and VLDL. It is also planned to estimate LPO, GSH, CAT and

SOD. The histopathology of heart will also be studied.

Doxorubicin induced cardio toxicity model 29 :

The animals will be randomly divided into five groups of six animals each.

Group 1: Normal saline for 18 days p.o.

Group 2: Normal saline for 18 days p.o. + on 16th day, doxorubicin (10mg/kg i.v.)

Group 3: Analapril 10mg/kg + on 16th day, doxorubicin (10mg/kg i.v.)

Group 4: Lower dose of extract for 18 days + on 16th day, doxorubicin (10mg/kg i.v.)

Group 5: Higher dose of extract for 18 days + on 16th day, doxorubicin (10mg/kg i.v.)

The body weights will be recorded daily. At the end of experimental period (after 24h of

second isoproterenol injection or 16th day of extract/vehicle treatment) all the rats will

anaesthetized with light anaesthetic ether and blood will be collected from the retro-orbital

plexus, the serum was separated and used for the determination of diagnostic marker enzymes

like AST, ACT, LDH, CK, TG, TC, HDL and VLDL. It is also planned to estimate LPO,

GSH and SOD. The histopathology of heart will also be studied.

Phase IV.

Histopathological studies.

Statistical analysis:

The results obtained from the above investigation will be subjected to statistical analysis

using one way ANOVA followed by Tukey- Kramer Multiple Comparison test.

The study design, criteria and plan of work are outlined as below: --

Inclusion criteria for the selection of animals:--

Sex: - Both sex Age- Adult animals.

Weight: -150-240 grams Health condition: - Healthy

Exclusion criteria:- Any animal not conforming with above criteria are not selected for

the experiment.

Study sampling:- Each model of organ toxicity requires five groups of six animals

each.

Since the animal models of toxicity are mortal, there is no need to undergo follow up

observations.

Parameters of study : In antistress (adaptogenic) study, serum glucose, cholesterol,

triglycerides, blood urea nitrogen etc. For assessing cardio protective activity,

aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate

dehydrogenase (LDH), creatine kinase (CK), LPO, GSH, SOD, CAT weight of vital

organs, etc will be assessed.

Duration of the study: Eight months.

ENCLOSURE – VI

7.3 The proposed study requires investigation on albino rats for cardio protective activities.

Experiments are to be conducted on anaesthetized rats.

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