introduction - upnvj

1
Hypercholesterolemia is caused by overproduction of cholesterol and associated with an increase in the lipid peroxidation product, malondialdehyde (MDA), which indicates high oxygen free radicals level and leads to oxidative stress [1,2]. This condition can contribute to tpathogenesis of cardiovascular diseases, such as coronary artery disease (CAD) [3]. Higher cost and side therapy effect that made by some anti cholesterol medicine which used in long time is the reason why some people change to herb therapy. One of that herb is tamarind leaves (Tamarindus indica). The pulp, in Indonesia has been popular as hypocholesterol and antioxi- dant but no for the leaves. This research aims to determine hypocholesterolemic and antioxidant activity of Ethanol Extract of Tamarind Leaves (EETL). Introduction 2. As Antioxidant : The level of malondialdehyde (MDA) was lower in the serum of ezetimibe and all variance of EETL groups in hypercholesterol fed rats than in the serum of hypercholesterol-fed rats alone. Such as the research before, the tamarindus indica supplementation significantly decreased the oxidative marker MDA in aged rats [1]. . Flavonoid and tannin that contain in EETL supplementation is considered to scavenging of free radicals [2] and activate the antioxidant enzyme activities [9]. The level of MDA was decrease significantly in ezetimibe group than control group. Pandya et al said that ezetimibe especially increase significantly superoxide dismutase (SOD) level [10]. This research also support our research that ezetimibe reduce oxdative stress. 1. As Hyopcholesterolemic : A. Saponin binding with bile acids and become a large mixed micelle. That micelle disturb the absorption in intestinal microvil- lus. And the total cholesterol will decrease from external pathway [5,6,7]. B. Flavonoid will increase the activation of LDL-C receptor in hepar and make the clearance of LDL-C is faster. Finally, total cholester- ol will decrease too [8]. It can be conclude that : 1. All variance of EETL has hypocholesterolemic and antioxidant activity that protect against hypercholesterolemic state. 2. EETL has also similar effect with ezetimibe. 3. Saponin, flavonoid, and tannin that contain in EETL likely have the most contribute to those pharmacology effect. 4. Ezetimibe can be as antioxidant agent. Figure 2. Hypocholesterolemic Activity of Tamarind Leaves (Tamarindus indica) in Hypercholesterol-Fed Rats (1) Shakirin, Faridah Hanim., et al. Antiatherosclerotic Effect of Canarium odontophyllum Miq. Fruit Parts in Rabbits Fed High Cholesterol Diet.Vol 2012. Hindawi Publishing Corporation. 2012. (2) Choi, Chang-Sook., Chung, Hae-Kyung., Choi, Mi-Kyung., Kang, Myung-Hwa. Ef- fects of grape pomace on the antioxidant defense system in diet-induced hypercholes- terolemic rabbits. Nutrition Research and Practice. Korea. 2010 : 4(2):114-120 (3) Deng, Ruitang., Chow, Te-Jin., Hypolipidemic, Antioxidant And Antiinflamatory Activities of Microalgae Spirulina. Cardiovasc Ther. 2010: 28(4) : e33-e45. [PubMed :2907180] (4) Gani, Nanang., Momuat, Lidya I., Pitoi, Mariska M. Profil Lipida Plasma Tikus Wistar yang Hiperkolesterolemia pada Pemberian Gedi Merah (Abelmoschus manihot L.). Jurnal MIPA UNSRAT Online 2 . Indonesia: Universitas Sam Ratulangi, Manado.2013. (5) Francis, Gordon A., et al. Cerebral cholesterol granuloma in homozygous familial hypercholesterolemi. Canadian Medical Association. 2005. (6) Matsuura, H., Saponins in Garlic as Modifiers of the Risk of Cardiovascular Disease. The Journal of Nutrition. 2001. (7) Santoscoy, R.A.C., Uribe, J.A.G., Saldívar, S.O.S., Effect of Flavonoids and Saponins Extracted from Black Bean (Phaseolus vulgarisL.) Seed Coats as Cholesterol Micelle Disruptors. Springer Science + Business Media New York. 2013. (8) Oliveira, T.T., et al. Hypolipidemic Effect of Flavonoids and Cholestyramine in Rat. Latin American Journal of Pharmacy. 2007. 407–410. (9) Martinello.F, et al. Hypolipemic and Antioxidant Activities from Tamarindus indica L. Pulp Fruit Extract in Hypercholesterolemic Hamsters. Food and Chemical Toxicology. Elsevier. 2006. (10) Pandya, N., Santani, D., Jain, S. Antioxidant Activity of ezetimibe in hypercholesterolmic rats. Indian Journal Pharmacology. Vol 38. India. 2006. Reference Figure 3. Antioxidant Activity of Tamarind Leaves (Tamarindus indica) in Hypercholesterol-Fed Rats Conclusion Result Result : The data of hypocholesterolemic activity before and after intervention were analyzed by T-test. All groups except negative control group was significantly difference (p<0.05) Then, the data were also analyzed by one way ANOVA, the significancy of hypocholesterolemic activity is 0.008 and antioxidant activity is 0.001. It was statistically significant (p<0.05). Furthermore, the data of hypocholesterolemic activity were analyzed with Post hoc test and there was a significantly differences between negative control group with Ezetimibe, first, and second dose of EETL group (p<0.05). But there was no significantly differences between Ezetimibe group (p>0.05) all variance of EETL groups. The data of antioxidant activity were also analyzed with Post hoc test, there was a significantly difference between negative control with all interventional groups (p<0.05). But, there was no significantly difference between Ezetimibe group with all variance of EETL groups (p>0.05). Materials and Methods Materials in this experiment are : 1. Rat : Sprague-Dawley male Strain, BW = 150-200 g, 2-3 month from Veterinary faculty of Bogor Agriculture Institute - Indonesia. 2. Tamarind leaves obtained from Herbs and Drugs Reseach Bogor (Balitro, Bogor-Indonesia) and were extracted with 70% ethanol. 3. Ezetimibe 10 mg 4. Carboxymethyl Cellulose (CMC) 0,1% 5. Cholesterol Kit Reagen (DiaSys) 6. Hypercholesterol feed : Standard rat chow, Boiled yolk sac duck’s eggs, fluid of goat’s fat, and water [4]. 7. Spectofotometer 8. TBARS Assay (Thiobarbituric Acid Reactive Substance) : 2.5% trichloroacetic (TCA) and 1% thiobarbituric acid (TBA) [2]. . Discussion Table 1. Hypocholesterolemic and Antioxidant Activity of Tamarind Leaves (Tamarindus indica) in Hypercholesterol-Fed Rats Group Mean MDA (mol/L) Mean Total Cholesterol (mg/dl) Before Intervention After Intervention After Intervention P-Value Negative Control 107,6 ± 14,43 102 ± 9,43 2,8 ± 0,03 0,524 Ezetimibe 99 ± 5,74 71,6 ± 6,58 1,2 ± 0,26 0,006* EETL 0,93 g/BW 109,8 ± 11,69 69,4 ± 6,02 1,3306 ± 0,13 0,003* EETL 1,86 g/BW 103,4 ± 9,96 66,4 ± 10,26 0,8076 ± 0,12 0,001* EETL 3,73 g/BW 99,8 ± 10,18 79 ± 22,62 1,2616 ± 0,02 0,048* Values are Mean ± SE for n = 5. *Sig differences at p < 0,05 a p < 0.05 Compared with negative control. b p < 0.05 Compared with positive control Figure 3. Mechanism of Tamarindus indicca Leaves Vs Ezetimibe –(modified) 25 rats Induce with hyper-cholesterol feed in 2 weeks 1 st Group Negative control (CMC 0,1%) + Hyper cholest feed 2 nd Group Ezetimibe 1,26 g/BW + Hypercholest feed 3 rd Group EETL 1 st dose (0,93g/BW) + Hyper cholest feed 4 th Group EETL 2 nd dose (1,86 g/BW) + Hyper cholest feed 5 th Group EETL 3 rd dose (3,73 g/BW ) + Hyper cholest feed _______________________ For 2 weeks_____________________ Figure 1. Experiment Methods On 29 th day, fasting blood samples were collected from caudalis’ veins rats for post test data and centrifuging at 3,000 rpm for 20 min at 4°C. The serum was examined with spectofotometer to test total cholesterol and MDA. On 15 th day, fasting blood samples were collected from caudalis’ veins rats for pre test data of total cholesterol and the serum was examined with spectofotometer b Contact person: [email protected] [email protected]

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Hypercholesterolemia is caused by overproduction of cholesterol and

associated with an increase in the lipid peroxidation product,

malondialdehyde (MDA), which indicates high oxygen free radicals

level and leads to oxidative stress [1,2]. This condition can contribute to

tpathogenesis of cardiovascular diseases, such as coronary artery disease

(CAD) [3].

Higher cost and side therapy effect that made by some anti cholesterol

medicine which used in long time is the reason why some people change

to herb therapy. One of that herb is tamarind leaves (Tamarindus indica).

The pulp, in Indonesia has been popular as hypocholesterol and antioxi-

dant but no for the leaves. This research aims to determine

hypocholesterolemic and antioxidant activity of Ethanol Extract of

Tamarind Leaves (EETL).

Introduction

2. As Antioxidant :

The level of malondialdehyde (MDA) was lower in the serum of

ezetimibe and all variance of EETL groups in hypercholesterol fed rats

than in the serum of hypercholesterol-fed rats alone. Such as the

research before, the tamarindus indica supplementation significantly

decreased the oxidative marker MDA in aged rats [1]. .

Flavonoid and tannin that contain in EETL supplementation is

considered to scavenging of free radicals [2] and activate the

antioxidant enzyme activities [9].

The level of MDA was decrease significantly in ezetimibe group than

control group. Pandya et al said that ezetimibe especially increase

significantly superoxide dismutase (SOD) level [10]. This research

also support our research that ezetimibe reduce oxdative stress.

1. As Hyopcholesterolemic :

A. Saponin binding with bile acids and become a large mixed

micelle. That micelle disturb the absorption in intestinal microvil-

lus. And the total cholesterol will decrease from external pathway

[5,6,7].

B. Flavonoid will increase the activation of LDL-C receptor in hepar

and make the clearance of LDL-C is faster. Finally, total cholester-

ol will decrease too [8].

It can be conclude that :

1. All variance of EETL has hypocholesterolemic and antioxidant

activity that protect against hypercholesterolemic state.

2. EETL has also similar effect with ezetimibe.

3. Saponin, flavonoid, and tannin that contain in EETL likely have the

most contribute to those pharmacology effect.

4. Ezetimibe can be as antioxidant agent.

Figure 2. Hypocholesterolemic Activity of Tamarind Leaves (Tamarindus indica) in

Hypercholesterol-Fed Rats

(1) Shakirin, Faridah Hanim., et al. Antiatherosclerotic Effect of Canarium odontophyllum Miq. Fruit Parts in Rabbits Fed High Cholesterol Diet.Vol 2012. Hindawi Publishing Corporation. 2012.

(2) Choi, Chang-Sook., Chung, Hae-Kyung., Choi, Mi-Kyung., Kang, Myung-Hwa. Ef-fects of grape pomace on the antioxidant defense system in diet-induced hypercholes-terolemic rabbits. Nutrition Research and Practice. Korea. 2010 : 4(2):114-120

(3) Deng, Ruitang., Chow, Te-Jin., Hypolipidemic, Antioxidant And Antiinflamatory Activities of Microalgae Spirulina. Cardiovasc Ther. 2010: 28(4) : e33-e45.[PubMed :2907180]

(4) Gani, Nanang., Momuat, Lidya I., Pitoi, Mariska M. Profil Lipida Plasma Tikus Wistar yang Hiperkolesterolemia pada Pemberian Gedi Merah (Abelmoschus manihot L.). Jurnal MIPA UNSRAT Online 2 . Indonesia: Universitas Sam Ratulangi, Manado.2013.

(5) Francis, Gordon A., et al. Cerebral cholesterol granuloma in homozygous familial hypercholesterolemi. Canadian Medical Association. 2005.

(6) Matsuura, H., Saponins in Garlic as Modifiers of the Risk of Cardiovascular Disease. The Journal of Nutrition. 2001.

(7) Santoscoy, R.A.C., Uribe, J.A.G., Saldívar, S.O.S., Effect of Flavonoids and Saponins

Extracted from Black Bean (Phaseolus vulgarisL.) Seed Coats as Cholesterol Micelle

Disruptors. Springer Science + Business Media New York. 2013.

(8) Oliveira, T.T., et al. Hypolipidemic Effect of Flavonoids and Cholestyramine in Rat.

Latin American Journal of Pharmacy. 2007. 407–410.

(9) Martinello.F, et al. Hypolipemic and Antioxidant Activities from Tamarindus indica L.

Pulp Fruit Extract in Hypercholesterolemic Hamsters. Food and Chemical Toxicology.

Elsevier. 2006.

(10) Pandya, N., Santani, D., Jain, S. Antioxidant Activity of ezetimibe in

hypercholesterolmic rats. Indian Journal Pharmacology. Vol 38. India. 2006.

Reference

Figure 3. Antioxidant Activity of Tamarind Leaves (Tamarindus indica) in

Hypercholesterol-Fed Rats

Conclusion

Result

Result :

The data of hypocholesterolemic activity before and after intervention

were analyzed by T-test. All groups except negative control group was

significantly difference (p<0.05)

Then, the data were also analyzed by one way ANOVA, the significancy

of hypocholesterolemic activity is 0.008 and antioxidant activity is

0.001. It was statistically significant (p<0.05).

Furthermore, the data of hypocholesterolemic activity were analyzed

with Post hoc test and there was a significantly differences between

negative control group with Ezetimibe, first, and second dose of EETL

group (p<0.05). But there was no significantly differences between

Ezetimibe group (p>0.05) all variance of EETL groups.

The data of antioxidant activity were also analyzed with Post hoc test,

there was a significantly difference between negative control with all

interventional groups (p<0.05). But, there was no significantly

difference between Ezetimibe group with all variance of EETL groups

(p>0.05).

Materials and Methods

Materials in this experiment are :

1. Rat : Sprague-Dawley male Strain, BW = 150-200 g, 2-3 month from

Veterinary faculty of Bogor Agriculture Institute - Indonesia.

2. Tamarind leaves obtained from Herbs and Drugs Reseach Bogor

(Balitro, Bogor-Indonesia) and were extracted with 70% ethanol.

3. Ezetimibe 10 mg

4. Carboxymethyl Cellulose (CMC) 0,1%

5. Cholesterol Kit Reagen (DiaSys)

6. Hypercholesterol feed : Standard rat chow, Boiled yolk sac duck’s eggs,

fluid of goat’s fat, and water [4].

7. Spectofotometer

8. TBARS Assay (Thiobarbituric Acid Reactive Substance) : 2.5% trichloroacetic

(TCA) and 1% thiobarbituric acid (TBA) [2]. .

Discussion

Table 1. Hypocholesterolemic and Antioxidant Activity of Tamarind Leaves

(Tamarindus indica) in Hypercholesterol-Fed Rats

Group Mean MDA

(mol/L) Mean Total Cholesterol (mg/dl)

Before

Intervention

After

Intervention

After

Intervention P-Value

Negative Control 107,6 ± 14,43 102 ± 9,43 2,8 ± 0,03 0,524

Ezetimibe 99 ± 5,74 71,6 ± 6,58 1,2 ± 0,26 0,006*

EETL 0,93

g/BW 109,8 ± 11,69 69,4 ± 6,02 1,3306 ± 0,13 0,003*

EETL 1,86

g/BW 103,4 ± 9,96 66,4 ± 10,26 0,8076 ± 0,12 0,001*

EETL 3,73

g/BW 99,8 ± 10,18 79 ± 22,62 1,2616 ± 0,02 0,048*

Values are Mean ± SE for n = 5. *Sig differences at p < 0,05

a p < 0.05 Compared with negative control. b p < 0.05 Compared with positive control

Figure 3. Mechanism of Tamarindus indicca Leaves Vs Ezetimibe –(modified)

25 rats

Induce with hyper-cholesterol feed in 2 weeks

1st Group

Negative control

(CMC 0,1%)

+ Hyper cholest

feed

2nd Group

Ezetimibe 1,26

g/BW +

Hypercholest

feed

3rdGroup

EETL 1st dose

(0,93g/BW) +

Hyper cholest

feed

4th Group

EETL 2nd dose

(1,86 g/BW) +

Hyper cholest

feed

5th Group

EETL 3rd dose

(3,73 g/BW ) +

Hyper cholest

feed

_______________________ For 2 weeks_____________________

Figure 1. Experiment Methods

On 29th day, fasting blood samples were collected from caudalis’ veins rats for post test

data and centrifuging at 3,000 rpm for 20 min at 4°C. The serum was examined with

spectofotometer to test total cholesterol and MDA.

On 15th day, fasting blood samples were collected from caudalis’ veins rats for pre test data

of total cholesterol and the serum was examined with spectofotometer

b

Contact person:

[email protected]

[email protected]