Evaluation of Cardioprotective Properties of Desmodium
triflorum Herb in various Experimental induced
Myocardial infarction in albino rats
M-PHARM DISSERTATION PROTOCOL
SUBMITTED 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 PHARMACOLOGY
S. C. S. COLLEGE OF PHARMACY
HARAPANAHALLI-583131
2011-12
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BENGALURU, KARNATAKA.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
Name and Address of the
Candidate
Priyanka L.G.
D/o A.L. Naik, Maylara Road,
Harapanahalli.
Pin- 583 131
Davangere (Dt). Karnataka (State)
02
Name of the Institution
T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131
Davangere (Dt). Karnataka
03
Course of the Study
Branch
M. Pharm.,
Pharmacology
04
Date of Admission to course
10.01.2012
Title of the Topic
Evaluation of Cardioprotective
Properties of Desmodium triflorum Herb
in various Experimental induced
myocardial infarction in albino rats
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
Materials and Methods
7.1. Source of data
Enclosure – IV
01
05
06
07
7.2. Methods of collection of data
7.3. Does the study require any
Investigations on animals?
If yes give details
Enclosure – V
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)
List of References (About 4-6)
Enclosure-VII
08
09
10
Signature of the Candidate
Remarks of the Guide
(Priyanka L.G)
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.
Name and Designation of
(In Block Letters)
11.1. Guide
Prof. A. Veerana Goud
M. Pharm
11.2.Signature
11.3.Co-Guide (if any)
Prof. Ittagi Shanmukha
M. Pharm., (Ph.D)
11
11.4.Signature
11.5. Head of the Department
Prof. A. Veerana Goud
Head
P.G. Dept. of Pharmacology
11.6. Signature
Remarks of the Principal
The present study was permitted to work in the
institution and animal ethical committee
permission is granted to it.
12
12.1. Signature
Dr. R. Nagendra Rao
Principal
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 content23: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 content23: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 tannin24: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 mode28: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 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 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 model29:
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.
ENCLOSURE – VII
8.0 List of references:
01. Suchalatha S, Shyamala Devi CS. Effect of arogh - a polyherbal formulation on the
marker enzymes in isoproterenol induced myocardial injury. Indian J. of Clinical
Biochemistry, 2004; 19 (2): 184-189.
02. Vaibhav Patel, Aman Upaganlawar, Rishit Zalawadia, Balaraman R. Cardioprotective
effect of melatonin against isoproterenol induced myocardial infarction in rats: A
biochemical, electrocardiographic and histoarchitectural evaluation. European J. of
Pharmacology 2010; 644:160–168.
03. Sauvetre EJ, Diji CV. Cardiovascular diseases and periodontal treatment. Heart Views
September-November 2007; 8 (3): 100–105.
04. Ashok kumar Kuppusamy, Subhadradevi Varadharajan, Christy Josey, Honey John,
Daphne Sherin, Divia Chirakkan. Cardioprotective effect of Erythrina stricta leaves on
isoproterenol-induced myocardial infarction in rat. Bangladesh J. Pharmacol 2010; 5:1-4.
05. Gheorghe Gluhovschi, Gheorghe Bozdog, Ligia Petrica, Adalbert Schiller, Virginia
Trandafirescu, Silvia Velciov et al. Nephroprotection, part of multiorganprotection. TMJ 2006; 56(2-3): 190-197.
06. Nilesh Shinde, Amit Jagtap, Vaishali Undale, Sujit Kakade, Sachin Kotwal,
Ravindra Patil. Protective effect of Lepidium sativum against doxorubicin-induced
nephrotoxicity in rats. Research Journal of Pharmaceutical, Biological
and Chemical Sciences, July–September 2010; 1(3): 42-48.
07. Maryam Rahimi Balaei, Majid Momeny, Rohollah Babaeikelishomi, Shahram
Ejtemaei Mehr, Seyed Mohammad Tavangar, Ahmad Reza Dehpour. The
modulatory effect of lithium on doxorubicin-induced cardiotoxicity in rat.
European J. of Pharmacology, 2010; 641: 193–198.
08. Devika PT, Stanely Mainzen Prince P. Protective effect of (-)-epigallocatechin-gallate
(EGCG) on lipid peroxide metabolism in isoproterenol induced myocardial infarction in
male wistar rats: A histopathological study. Biomedicine & Pharmacotherapy 2008; 62:
701-708.
09. Thippeswamy BS, Thakker SP, Tubachi S, Kalyani GA, Netra MK, Patil U et al.
Cardioprotective Effect of Cucumis trigonus Roxb on isoproterenol-inducedmyocardial
infarction in rat. American J. of Pharmacology and Toxicology 2009; 4 (2): 29-37.
10. Arya DS, Nandave M, Ojha SK, Kumari S, Joshi S, Mohanty I. Myocardial salvaging
effects of Ocimum sanctum in experimental model of myocardial necrosis: a
haemodynamic, biochemical and histoarchitectural assessment. Current Science
September 2006; 91(5): 667-672.
11. Mark Percival. Antioxidants. Clinical Nutrition Insights, November 1998:1-4.
12. Helena Kaiserova, Tomas Simunek, Wim van der Vijgh JF, Aalt Bast, Eva Kvasnickova.
Flavonoids as protectors against doxorubicin cardiotoxicity: role of iron chelation,
antioxidant activity and inhibition of carbonyl reductase. Biochimica et Biophysica Acta
2007; 1772: 1065–1074.
13. K.Trout, Trout’s notes on the Genus Desmodium: Chemistry, ethnomedicine,
pharmacology, synonyms and miscellary. A better days publication, second 2002
printing with minor revisions, adapted for web viewing march 2004:20-7.
14. Three-flower tick trefoil, lists of Philippine herbal medicinal plants. Philippine
alternative medicine, http://www.stuartxchange.org/index.htm
15. http://www.tropicalforages.info/key/Forages/Media/Html/desmodium_triflorum.htm
16. Chopra RN, Nayr LS, Chopra CI, Glossary of Indian Medicinal Plants: 94p.
17. Singh N, Jain N et al. Anti-ulcer activity of Desmodium triflorum leaves extract.
National Conference on “Recent Advances in Herbal Drug Technology” 2010; O-13: 13
18. Lai SC, Peng WH et al. Analgesic and anti-inflammatory activity of methanol extract
from Desmodium triflorum DC in mice. The American Journal of Chinese Medicine
2009; 37(3): 573-88.
19. Lai SC, Ho YL et al. Antioxidant and Antiproliferative Activities of Desmodium
triflorum (L.) DC. The American Journal of Chinese Medicine 2010; 38(2): 329-42.
20. Pundir R, Singh G, Pandey AA, Saraf SA. Demand of herbal hepatoprotective
formulation in Lucknow – A survey. The Pharma research 2009; 1: 23-33.
21. Kokate CK. Practical Pharmacognosy. 4th Ed. New Delhi: Vallabha prakashan. 1999:
149-56.
22. Khandelwal KR. Practical Pharmacognosy techniques and experiments, 2nd Ed.
Pune: Nirali Prakashan. 2000: 149-56.
23. Shanmukha I, Harshil Patel, Jignesh Patel, Riyazunnisa. Quantification of Total
phenol and Flavonoids of
Delonix regia Flowers. International Journal of
ChemTech Research. 2011;1(3):280-3.
24. Urve Paaver, Vallo Matto, Ani Raal. Total tannin content in distinct Quercus robur
L. galls. Journal of Medicinal Plants Research. 2010;4(8):702-5.
25. Sasanka Chakrabart, Asha Naik S, Gali Reddy R. Phenylhydrazine mediated
degradation of bovine serum albumin and membrane proteins of human
erythrocytes. Biochimica et Biophysica Acta. 1990;1928: 89-94.
26. Barry halliwell, Jhon Gutteridge MC. Formation of thiobarbituric acid reactive
substance from deoxyribose in the presence of iron salts. FEBS Letters. 1981; 128
(2): 347-52.
27. Olayinka A Aiyegoro, Anthony I, Okoh. Phytochemical screening and
Polyphenolic Antioxidant Activity of Aqueous Crude Leaf Extract of Helichrysum
pedunculatum. International Journal of Molecular Sciences. 2009; 10: 4990-5001.
28. Thippeswamy BS, Thakker SP, Tubachi S, Kalyani GA, Netra MK, Patil U et al.
Cardioprotective Effect of Cucumis trigonus Roxb on Isoproterenol-Induced
Myocardial Infarction in Rat. American Journal of Pharmacology and Toxicology.
2009; 4(2): 29-37.
29. Hassanpour Fard M, Naseh G, Bodhankar SL, Dikshit M. Cardioprotective Effect of
Lagenaria siceraria (Molina) Standley (Cucurbitaceae) Fruit Juice on Doxorubicin
Induced Cardiotoxicity in Rats. American Journal of Pharmacology and Toxicology.
2010; 5(2): 103-108.
30. Maria MS. Leaves of glutathione, glutathione reductase and glutathione STransferas activities in rat lung and liver. Biochimica et Biophysica Acta. 1979;
582:67-78.