cardioprotective effect of flavonoids from eggplant

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

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

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

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

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

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

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

flavonoids found in the Solanaceae family, are proven to have:

antioxidant, anti-allergic, anti-inflammatory, anti-microbial and anti-cancer

properties.

Introduction

Eggplant (Solanum melongena L.) is a nutritious, widely-available, locally-grown, popularly consumed vegetable in the Philippines.

Introduction

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

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

OBJECTIVES OF THE STUDY

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.

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

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

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

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

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.

Significance of the Study

Optimal utilization of resources.

Providing reference for future research.

METHODOLOGY

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.

Research Procedure

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.

Experimental Intervention

Collection and Verification of Plant Material

Extraction of Flavonoids

Testing for Cardioprotective Effect


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.


Extraction of Flavonoids from Solanum melongena

L.

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.

Animal Treatment Groups

Fishbowl method

Group 1: Negative Control GroupGroup 2: Positive Control Group

Group 3: Experimental Group

Testing for Cardioprotective Effect of Flavonoids

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.


Administration of positive control (Trimetazidine)

10mg/kg once a day via gastric gavage for 14 days.


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.


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


Guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) were followed.

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.



Slides were randomly labelled and were sent to a credible pathologist for histopathological examination



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;

.



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 .


DATA ANALYSIS

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

Data AnalysisChi-Square Tests

Value df Asymp. Sig. (2-sided)

Exact Sig. (2-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)

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

Data AnalysisChi-Square Tests

Value df Asymp. Sig. (2-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)

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

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.

RESULTS

Figure 3.1 Troponin T Qualitative Test results from randomly selected SD rats

from the three groups

Figure 3.2 Distribution of mortality post induction of Isoproterenol

Figure 3.3 Frequency of myocardial infarction in rats from the different groups by

histopathologic results

Figure 3.4 Comparison of the histopathologic grading for MI in each

group

CONCLUSION

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.

RECOMMENDATIONS

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.

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.

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.

Thank you!DABBAGH, DAGDAGAN,

DELA PENA, DIAZ, DOMINGO, DUMALEN,

ENRIQUEZ, FELIPE, GINETE, GUINA, KALAW, LAGGUI

cardioprotective effect of flavonoids from eggplant

Documents

minimum expected

world health

exact test

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dawley rats

expected count

linear association

male sprague