cardioprotective effect of flavonoids from eggplant
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Cardioprotective Effect of Flavonoids from
Solanum melongena L (Eggplant) Peel Extract on Sprague-Dawley Rats
with Isoproterenol - Induced Acute Myocardial Infarction
DABBAGH, DAGDAGAN, DELA PENA, DIAZ, DOMINGO, DUMALEN,
ENRIQUEZ, FELIPE, GINETE, GUINA, KALAW, LAGGUI
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IntroductionCardiovascular
diseases (CVDs) are the leading causes of death and disability in the world.
In 2008, an estimated 17.3 million people died from CVD, representing 30% of all global deaths.
Of these deaths, 7.3 million were due to coronary heart disease and 6.2 million were due to stroke
Source:“Global Atlas on Cardiovascular Disease Prevention and Control”, World Health Organization, 2011
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Cardiovascular diseases (CVDs) are the leading causes of death and disability in the world. Low and middle-income groups are greatly affected with over 80% of CVD deaths taking place in these groups.
In 2030 CVD will remain as the single leading cause of death with almost 23.6 million people expected to die from it.
Source:“Global Atlas on Cardiovascular Disease Prevention and Control”, World Health Organization, 2011
Introduction
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In the Philippines, the Department of Health
reported that heart diseases remain to be the top leading
cause of mortality in the recent 5-year
average.
Source: Department of Health, Philippineshttp://www.doh.gov.ph
Introduction
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Acute myocardial infarction, commonly known as a heart
attack, is the irreversible necrosis of heart muscle secondary to prolonged ischemia.
- (Zafari M, et al. 2012).
Introduction
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Synthetic drugs have been effective in managing the conditions, but the side effects of the drugs do not weigh their effectiveness.
For example, Aspirin is well known for the prevention of cardiovascular thrombotic events in patients at risk for cardiovascular disease (CVD), however, it carries an increased risk for gastrointestinal (GI) injury (e.g., ulceration).
Introduction
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The use of herbal medicines and research
on newer ones have been encouraged in the country as
part of efforts to find cheaper yet
effective medicines, which will be affordable
to the majority of the population.
Introduction
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flavonoids found in the Solanaceae family, are proven to have:
antioxidant, anti-allergic, anti-inflammatory, anti-microbial and anti-cancer
properties.
Introduction
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Eggplant (Solanum melongena L.) is a nutritious, widely-available, locally-grown, popularly consumed vegetable in the Philippines.
Introduction
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It is ranked as one of the top ten in terms of oxygen radical scavenging capacity due to its phenolic constituents
-(Jung et al., 2011).
Foods rich in antioxidants play an essential role in the prevention of chronic and
degenerative diseases such as cardiovascular diseases and
cancer - (Han et al., 2003).
Introduction
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Studies have also shown that eggplant extracts inhibit inflammation that leads to atherosclerosis -(Han et al., 2003).
Given these facts and because of its abundance in the Philippines, eggplants may
prove to be the safe, effective and economical alternative
treatment to cardiovascular disorders waiting to be discovered.
Introduction
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OBJECTIVES OF THE STUDY
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General Objective
To determine if flavonoids from Solanum melongena L (Eggplant) peel extract have a cardioprotective effect on male Sprague - Dawley rats with
Isoproterenol – induced acute myocardial infarction.
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1. To compare the histopathologic features of the myocardium of male Sprague-Dawley rats
treated with flavonoids from Solanum melongena L. (Eggplant) peel extract from
those treated with Trimetazidine after induction of myocardial infarction with
Isoproterenol.
Specific Objectives
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2. To compare the histopathologic features of the myocardium of male Sprague-Dawley rats treated with Trimetazidine after induction of myocardial infarction with Isoproterenol from
that of the Negative Control.
Specific Objectives
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3. To compare the histopathologic features of the myocardium of male Sprague-Dawley rats
treated with flavonoids from Solanum melongena L. (Eggplant) peel extract from
after induction of myocardial infarction with Isoproterenol from that of the Negative
Control.
Specific Objectives
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4. To compare the mortality rate of Sprague -
Dawley rats with Isoproterenol-induced myocardial
infarction treated with flavonoids from Solanum
melongena L. (Eggplant) peel extract to those
treated with Trimetazidine.
Specific Objectives
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Significance of the Study
Development of an effective, safe and affordable alternative to currently available
cardioprotective drugs.
Reduction in morbidity and mortality from cardiovascular diseases.
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Significance of the Study
Optimal utilization of resources.
Providing reference for future research.
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METHODOLOGY
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Research Design
This study utilized an experimental design to evaluate the cardioprotective
effect of flavonoids from S.melongena L
(eggplant) peel extract on Isoproterenol-
induced male Sprague-Dawley rats.
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Research Procedure
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Sample Population
Forty-Five Sprague-Dawley male rats weighing 100-150 grams
The test subjects were equally divided and randomly assigned via the Fishbowl method into three groups with 15 rats per group.
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Experimental Intervention
Collection and Verification of Plant Material
Extraction of Flavonoids
Testing for Cardioprotective Effect
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Collection and Verification of Plant Material
The fruits of Solanum melongena L. or commonly known as eggplant were obtained from Cabugao, Ilocos Sur.
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The fruits were examined and subjected for authentication by the Bureau of Plant and Industry.
Collection and Verification of Plant Material
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Extraction of Flavonoids from Solanum melongena
L.
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Screening of Flavonoids from Solanum melongena L. Bate-Smith and Metcalf Test for
Leucoanthocyanins
Wilstatter Cyanidin Test
Limuaco, O.M. et al, Laboratory Manual in Pharmacognosy. Manila: Centro Escolar University School of Pharmacy, 2008
24-30
Extraction of Flavonoids from Solanum melongena
L.
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Animal Treatment Groups
Fishbowl method
Group 1: Negative Control GroupGroup 2: Positive Control Group
Group 3: Experimental Group
Testing for Cardioprotective Effect of Flavonoids
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Administration of flavonoid extract
Administered via gastric gavage for a period of 14 days.
Vol of extract = Wt. of rat (kg) x dose (mg/kg)
conc. of extract (mg/ml)
Rats were weighed on a weekly basis.
Testing for Cardioprotective Effect of Flavonoids
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Administration of positive control (Trimetazidine)
10mg/kg once a day via gastric gavage for 14 days.
Testing for Cardioprotective Effect of Flavonoids
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Induction of MI with Isoproterenol
Prior to induction, the rats were weighed individually.
85mg/kg was injected subcutaneously on the dorsal part of the body on days 14
and 15 of the study.
Testing for Cardioprotective Effect of Flavonoids
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Cardiac Troponin T
12 hours after all rats were sacrificed via cervical decapitation
Intracardiac aspiration of 2mL of blood from 4 rats from each group
Qualitative evaluation of serum Troponin T levels using Roche Troponin T kit
Testing for Cardioprotective Effect of Flavonoids
Guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) were followed.
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Histopathologic features of myocardium
10% buffered formalin,
subsequently sent to UP Manila Pathology Department for histopathology tissue
preparation.
Five-micrometer thick sections of each rat heart were prepared and stained with
Hematoxylin and Eosin.
Testing for Cardioprotective Effect of Flavonoids
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Histopathologic features of myocardium
Slides were randomly labelled and were sent to a credible pathologist for histopathological examination
Testing for Cardioprotective Effect of Flavonoids
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Histopathologic features of myocardium
Microscopically the lesions of the heart were graded using the following:
Grade-0: No lesion;Grade-1: Fibroblastic swelling or proliferation and
accumulation of histiocyte; Grade-2: Edema mottled staining, fragmentation and segmentation of muscle fiber;
.
Testing for Cardioprotective Effect of Flavonoids
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Histopathologic features of myocardium
Microscopically the lesions of the heart were graded using the following:
Grade-3: Vascular and fatty degeneration, granular disintegration and hyaline necrosis of muscle fibers. Marked capillary dilatation with hemorrhage, extensive edema Grade-4: Confluent lesion throughout the heart. Lesions were similar in characteristics to those in Grade 3 .
Testing for Cardioprotective Effect of Flavonoids
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DATA ANALYSIS
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Data Analysis Chi-square test was used to compare the
mortality proportions between positive control group and the experimental group.
Treatment * Dead Cross tabulation
Count
DeadTotal
Alive Dead
TreatmentGroup 2 (Positive Control) 11 4 15
Group 3 (Experimental) 12 3 15
Total 23 7 30
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Data AnalysisChi-Square Tests
Value df Asymp. Sig. (2-sided)
Exact Sig. (2-sided)
Exact Sig. (1-sided)
Pearson Chi-Square .186a 1 .666
Continuity Correctionb .000 1 1.000
Likelihood Ratio .187 1 .666
Fisher's Exact Test 1.000 .500Linear-by-Linear Association .180 1 .671
N of Valid Cases 30
a. 2 cells (50.0%) have expected count less than 5. The minimum expected count is 3.50.
b. Computed only for a 2x2 table
There is no significant difference between the proportion of dead and living mice for the positive control and experimental group
(X2(df=1, n=30) = 0.186, p = 0.50 > 0.05, ns)
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Data Analysis Chi-square test was used to compare the
frequency of myocardial infarction in rats from the different groups by histopathologic results
Treatment * MI Crosstabulation
Count
MITotal
.00 1.00
Treatment2.00 0 15 15
3.00 3 12 15
Total 3 27 30
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Data AnalysisChi-Square Tests
Value df Asymp. Sig. (2-sided)
Exact Sig. (2-sided)
Exact Sig. (1-sided)
Pearson Chi-Square 3.333a 1 .068
Continuity Correctionb 1.481 1 .224
Likelihood Ratio 4.493 1 .034
Fisher's Exact Test .224 .112
Linear-by-Linear Association 3.222 1 .073
N of Valid Cases 30
a. 2 cells (50.0%) have expected count less than 5. The minimum expected count is 1.50.
b. Computed only for a 2x2 table
(X2(df=1, n=30) = 3.333, p = 0.112 > 0.05, ns)
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Data AnalysisOne-way ANOVA , Duncan’s and Tukey’s
test histopathologic features of the three groups.
Tests of Between-Subjects Effects
Dependent Variable: Grade
Source Type III Sum of Squares df Mean Square F Sig.
Corrected Model 14.444a 2 7.222 8.096 .001
Intercept 153.089 1 153.089 171.612 .000
Treatment 14.444 2 7.222 8.096 .001
Error 37.467 42 .892
Total 205.000 45
Corrected Total 51.911 44
a. R Squared = .278 (Adjusted R Squared = .244)
F(2,42)=8.096,p=.001<.01
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Data AnalysisGrade
Treatment NSubset
1 2
TukeyHSDa,b
1.00 15 1.0667
3.00 15 2.0667
2.00 15 2.4000
Sig. 1.000 .602
Duncana,b
1.00 15 1.0667
3.00 15 2.0667
2.00 15 2.4000
Sig. 1.000 .339
Means for groups in homogeneous subsets are displayed. Based on observed means. The error term is Mean Square(Error) = .892.
a. Uses Harmonic Mean Sample Size = 15.000.b. Alpha = 0.05.
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RESULTS
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Figure 3.1 Troponin T Qualitative Test results from randomly selected SD rats
from the three groups
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Figure 3.2 Distribution of mortality post induction of Isoproterenol
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Figure 3.3 Frequency of myocardial infarction in rats from the different groups by
histopathologic results
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Figure 3.4 Comparison of the histopathologic grading for MI in each
group
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CONCLUSION
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Conclusion
There is no significant difference between the mortality rates and the histopathologic grading of myocardial infarction in the two
treatment groups, therefore the cardioprotective effect of flavonoids from eggplant (Solanum melongena L.) peel
extract is comparable to that of Trimetazidine.
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RECOMMENDATIONS
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Recommendations
1) To increase the sample size and perform the experiment on another animal species.
2) To isolate and use pure flavonoids of the S. melongena L. peel extract.
3) To determine the optimum dosage or concentration of the S. melongena L. peel extract by comparing different dosages.
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Recommendations4) To extend the length of Trimetazidine and
S. melongena L. peel extract treatment.
5) To include another group to be induced with Isoproterenol but not treated with Trimetazidine or S. melongena L. flavonoid peel extract, as much as possible do the Isoproterenol induction in the same facility to reduce variables.
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Recommendations 6) To consult at least 3 pathologists in
reading the histopathologic slide specimens,and;
7) To observe the possible adverse effects of S. melongena L. peel extract.
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Thank you!DABBAGH, DAGDAGAN,
DELA PENA, DIAZ, DOMINGO, DUMALEN,
ENRIQUEZ, FELIPE, GINETE, GUINA, KALAW, LAGGUI