Hypolipidemic Effect and Antioxidant Activity of Tamarind Leaves Extract in Hypercholesterol-Fed Rats

Background: Higher cost and side effects made of some anticholesterol drugs used in long time are the reasons why some people change to herbal therapies. Tamarind (Tamarindus indica) leaves is one of the herbal therapies. This research aims to determine hypolipidemic effect and antioxidant activity of extract of tamarind leaves (ETL). Methods: We used 25 rats as samples, divided into five groups of negative control (CMC 0.5%), positive control (Ezetimibe 1.26 g/kgBW)), first, second and third dose of ETL consequently are 0.93, 1.86 and 3.73 g/kgBW. Results: Paired-samples T-test showed ETL significantly decreased total cholesterol (TC), triglyceride (TG) level, and high-density lipoprotein cholesterol (HDL-C) level significantly increased compared with negative control groups (p≤0.05). Low-density lipoprotein cholesterol (LDL-C) level had significant difference only at second dose of ETL (p<0.05). Furthermore, the data’s difference between preand post-intervention were analyzed with one-way ANOVA test in TC, TG, and HDL-C level, ETL had a significant difference (p≤0.05), while there was no significant difference in LDL -C between groups (p>0.05). Data were also analyzed by Post Hoc test. TC, TG, and HDL-C level had a significant difference between all variance ETL’s doses and positive control compared with negative control group (p≤0.05). For antioxidant activity, ETL exhibited the significant reduction in the levels of malondialdehyde (MDA) by pairedsamples T-test (p≤0.05) but there was no significant difference in both of MDA and superoxide dismutase (SOD) level (p>0.05) analyzed by One-way ANOVA test. Conclusion: All variant of ETL’s doses have hypolipidemic effect and antioxidant activity. ETL also has similar effect with Ezetimibe. Saponin, flavonoid, epicatechin, tanin, and polyphenol that is contained likely contribute to these pharmacologic effects.


Introduction
A ccording to World Health Organization, there are around 58 million deaths in the world in 2005, with 17.5 million of them (30%) due to cardiovascular diseases and specifically by heart attacks caused 7.6 million deaths (13%). 1,2Riskesdas 2007 showed that the prevalence for cardiovascular disease in Indonesia is 7.2% while the percentage for ischemic heart disease at all age was
Samples used in this study are albino rats (Rattus norvegicus), wistar strain, male, age 8 weeks, 200-250 gram in weight.Male albino rats obtained from Bogor Agricultural University, matching the inclusion criteria.This study has been approved by the ethics committee of Universitas Pembangunan Nasional "Veteran", Jakarta.
On day 8 through day 22, rats were induced with hypercholesterol feeds and distilled water as their drinking water.Hypercholesterol feeds were done by mixing standard feeds as much as 8 kg added with boiled duck egg yolk (400 g), goat fats (800 g; thawed by boiling), and sufficient amount of hot water for 7 days.One rat was given 50 g of hypercholesterol diet, once per day (modified from Gani, et al's research). 17n day 24, rats were given hypercholesterol feeds and intervention for 2 weeks according to its group, which are: (1)

Biochemical analysis
Biochemical analysis was tested on day 7 (pre-hypercholesterol feed) and day 21 (post-hypercholesterol feed/pre-intervention) by taking 3 cc of blood from caudal vein rats.On day 35 (post-intervention) by taking 6 cc of blood from the heart without anticoagulant for lipid profile test and with anti-coagulant for antioxidant test.Sample then centrifuged with the speed of 1000 rpm for 10 minutes.

Assessment of hypolipidemic effect from ETL
Lipid profile were measured by using automatic analyzer standard kit. 17Standard serum used for calibration before each parameter was analyzed. 18For the biochemical analysis, the plasma was separated from the blood by centrifugation at room temperature for around 5.1% from the total percentage of cardiovascular disease. 1 Ischemic heart disease or better known as coronary heart disease (CHD) is caused by the formation of plaque, vascular remodeling, both acute and chronic luminal obstruction, blood flow abnormality and decline oxygen supply to target organs. 3ccording to the study of Framingham in Atherosclerosis Risk in Communities, and the study of Honolulu there are risk factors in the occurrence of CHD, i.e.: age, family history, dyslipidemia, cigarette smoking, hypertension, and diabetes. 4The continuity of dyslipidemia formation is correlated with the incident of CHD i.e. increase triglyceride level and decrease cholesterol HDL level will indirectly increase the level of total cholesterol. 5harmacologic therapy usually aimed to lower cholesterol-LDL level, triglyceride, or increase HDL. 6 Drugs from statin groups in Indonesia are the best cholesterol-lowering drugs.Despite statin, there also another group from hypocholesterol agent called Ezetemibe.Ezetemibe therefore has an important role in pharmacological lipid modification. 7However, Ezetemibe having lesser adverse effect than statin. 8astrointestinal disruption and other complaint like headache is the most adverse effect from Ezetemibe, 9 meanwhile statin groups adverse effects are gastrointestinal disruption and hepatotoxic effect. 10Ezetemibe has less number of research for hypocholesterol agent as a control group done than statin in Indonesia.Higher cost and side effects after used in long time are the reasons why some people change to herbal therapy. 11,12ne of the herbal therapy that has been used empirically in lowering blood cholesterol level is Tamarind (Tamarindus indica).Tamarind leaves has been known to contain compound like tannin, alkaloid, saponin, sesquiterpenes, and tannin through phytochemical tests. 12Extract of Tamarind Leaves (ETL) in controlling lipid profile levels is the effect of saponin action to inhibit lipid absorption in intestine. 13,14Saponin levels in ETL (in ethanol 70%) are higher than flavonoid levels. 15Thus, ETL which contained more saponin has a mechanism similar to Ezetemibe as a positive control.Meanwhile, statin inhibit HMG-CoA reductase and has no effect in inhibition of lipid absorption in intestine. 16This research is to compare it with a positive control group which has no difference in their mechanism of action.We aimed at learning more on the hypolipidemic effect and antioxidant activity of ETL in hyper cholesterol-fed rats compared to Ezetimibe.15 min. 19Level of every examination declared in milligram per deciliter (mg/dL). 20

Assessment of antioxidant activities from ETL
Assessment of the antioxidant activities collected from MDA level measurement and SOD.MDA level determine lipid per oxidation level.
Principal measurement using Thiobarbituric Acid-Reacting Substances (TBARS) method on wave length of 532 nm.MDA blood concentration declared as nmol/mL serum. 20SOD level according to the rate of inhibition from ferri-(Fe3+) cytochrome c reductase by superoxide anion produced by xanthine/xanthine oxidase.Xanthine oxidized to uric acid, while superoxide anion which later formed will reduce ferri-(Fe3+) cytochrome c.Reduction of ferri-(Fe3+) cytochrome c was observed according to the increase absorbance on wave length of 550 nm. 21

Statistical analysis
First, the data was analyzed with Shapiro Wilk Test as normally distributed test.If the data is normally distributed then Analysis of Variance (ANOVA) test is proceed to show the significant differences between groups.Furthermore, the data was tested using Post-Hoc Test to show exactly how significant the differences between two groups in a row (confidence degree 95% with score p ≤0.05). 22

Results
All groups were given hypercholesterol feeds for 2 weeks.Mean value lipid profile for 2 weeks shown in Table 1.According to Paired Samples T-Test, there were significant increase of TC, TG, LDL-C level and decrease of HDL-C level in all groups (p≤0.05).The intervention is started by giving hypercholesterol feeds and Ezetimibe 1.26 g/KgBW as positive control and three ETL dose variations for 2 weeks.After 2 weeks, lipid profile examination was repeated.The lipid profile data before and after intervention is shown in Figure 1 and Table 2.
Hypercholesterolemics rats treated with ETL exhibited significant decrease of TC level (p≤0.05) in all group except negative control group (by Pairedsamples Test).Furthermore by One-way ANOVA test also resulted in significant difference between groups compared with negative control groups (p≤0.05).The highest decline was seen in the 1 st dose ETL (0.93 g/ KgBW) as much as 43.3 mg/dL.
There were significant decreases of TG level (p≤0.05) in all groups except negative control group (by Paired-samples Test).One-way ANOVA test also had resulted in significant difference between groups compared with negative control group (p≤0.05).The highest decline was seen in the 1 st dose ETL (0.93 g/ KgBW) as much as 51.6 mg/dL.
Mean value of LDL-C level significantly decreased only in the 2 nd dose ETL group (p≤0.05)(by Paired-samples Test).Further One-way ANOVA test did not found significant difference between groups (p>0.05).And the highest decline was found in 2 nd dose ETL (1.86 g/KgBW) group as much as 11.76 mg/dL.There were significant increases of HDL-C level in all groups (p≤0.05)except negative control groups (by Paired-samples Test).Further One-way ANOVA test also shown significant difference between groups compared with negative control group (p≤0.05).The highest increase was found in the 3 rd dose ETL (3.73 g/KgBW) group as much as 20 mg/dL.
Antioxidant activities showed by MDA and SOD level.MDA level marked the occurence oxidative stress, higher level of MDA similar with higher oxidative stress in the cell.Based on the mean of MDA level before and after intervention, there were significant decrease in MDA level on every groups (p≤0.05)except negative groups and there was no significant difference in all groups (p≥0.05).The lowest MDA decline was found in the 3rd dose of ETL (3.73 g/KgBW), as much as 11.34 µMolar (Table 3).
While for the purpose to fight against advanced oxidation is shown by the presence of activity from SOD.Higher level of SOD simil ar with higher antioxidant activity in the cell.Based on the results of this studies, the highest SOD level was found in the 1 st dose of ETL (0.93 g/KgBW) as much as 183.90 ± 18.02 unit/ml.And there was no significant difference in all groups (p>0.05)(Table 3).

Hypolipidemic effect
There were significant TC and TG level decreases, and HDL-C increases in all groups except negative group.Saponin, flavonoid, epicatechin contained in ETL are likely contributed to hypolipidemic effect..14Flavonoid will increase activation of LDL-C receptor in liver and makes the clearance of LDL-C faster therefore the TC level will decrease because of this mechanism. 26Epicatechin in tamarind leaves will decrease TG level and also increase the clearance of free fatty acid and sterol acid through feces. 27Meanwhile Ezetimibe specifically works as competitive inhibitor in cholesterol absorbtion with cholesterol carrier (protein Niemann-Pick C1 like 1) in epithelial cells of small intestines. 8,10n our study, there was no significant difference between LDL-C level.For the 1 st dose of ETL, the difference between LDL-C levels was lower than the 2 nd dose of ETL.This is because the relationship between pharmacological responses and drug doses can be explained as follows: Increase doses in logarithmic scale followed by increased pharmacological responses. 23inear relationships also occur between drug doses and active concentrations of drugs in serum. 24armacological activity of natural medicinal ingredients (herbal medicines), just like synthetic drugs, is determined by the presence of such drug bonds with receptors.The magnitude of the pharmacological intensity depends on the concentration/number of drugs reaching the receptor and the type of drug-receptor bond, which can be both specific and non-specific.The duration of pharmacological effects depends on the length of the drug remains in the receptor.For natural remedies with large clearances, duration time in the body is shorter than that of natural remedies that have small clearances.It is used as one of the basic doses of natural medicine.The relationship between hypothetical drug concentrations in serum with probability of response and toxicity. 23or the 3 rd dose of ETL, there was no difference in LDL-C reduction with the 2 nd dose of ETL.This may Figure 2. The relationship between drug concentration in serum and probability response or toxicity. 23,24e due to an interaction of a chemical compound in the form of an antagonist mechanism, in which one compounds neutralize the other compound by producing the opposite effect on the same physiological conditions. 25

Antioxidant activity
Flavonoid, polyphenol, and tannin contained in ETL plays a role as antioxidant.These agents work by increasing antioxidant enzymes activity such as glutathione peroxidase (GPx), catalase (CAT), and SOD making lipid difficult to oxidize and prevent the formation atherogenic plaque. 28MDA level will decrease and SOD level will increase because of this mechansim.These facts were supported by RuiLi Yang, M.S et al whom mentions that atherosclerosis decrease by antioxidant correlates with the decrease MDA level in blood. 29his study also found that ezetimibe can decrease oxidative stress as seen on the significant decrease of MDA level before and after intervention.In contrast, Pandaya et al said that Ezetimibe does not significantly decrease the MDA level but increases SOD significantly. 30

Conclusion
All ETL dose variants have hypolipidemic effect and antioxidant activity which play role against hyper-cholesterolemic condition.ETL also have the same effect with Ezetimibe.Saponin, flavonoid and tannin are the contents suspected to have pharmacological effect in improving lipid profile.Ezetimibe may play a role as antioxidant.

Table 1 .
Lipid profile after hypercholesterol feeds for 2 weeks.
TC=total cholesterol; TG=triglyceride; LDL-C=low density lipoprotein-cholesterol; HDL C=high density lipoprotein-cholesterol; ETL=extract of tamarind leaves; a=significance (p≤0.05) in comparison to the negative control group; b=significance (p≤0.05) in comparison to the positive control group.*Significance (p≤0.05)before and after intervention.

Table 3 .
Antioxidant activities of ETL MDA=malondialdehyde; SOD=superoxide dismutase; ETL=extract of tamarind leaves; a=significance (p≤0.05) in comparison to the negative control group; b=significance (p≤0.05) in comparison to the positive control group.*Significance (p≤0.05)before and after intervention.

Table 2 .
Mean of the lipid profile level after intervention ETL=extract of tamarind leaves; a= significance (p≤0.05) in comparison to the negative control group; b= significance (p≤0.05) in comparison to the positive control group; Diff = difference.