Hypolipidemic effect of Tamarindus indica L fruit on Triton X-100-induced hyperlipidemia in Wistar rats.
The greatest risk factor for coronary heart disease (CHD) is hyperlipidemia.  Of the approximately 9.4 million deaths each year, 51% are caused by stroke and 45% are by CHD.  Tamarindus indica L is widely used as a traditional medicine in Indonesia and is commonly used to treat gastritis, rheumatism, constipation, and fever. [3,4] In India too, this plant is traditionally used to treat fever, stomach disease, diarrhea, and infection.  Several studies have examined the pharmaco logical effect of T. indica L. The results showed that pectin of T. indica L believed to have antioxidants that can reduce serum blood total cholesterol and triglycerides and increase high-density lipoprotein (HDL).  The extract of T. indica L has strong antioxidant effect. [7-9] The methanolic extract of T. indica L seed has antioxidant properties. This extract contains procyanidin.  In vitro, T. indica L fruit pulp has radical scavenging ability that is measured with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals and causes a decrease in serum lipid peroxidation, which is assessed by thiobarbituric acid.  T. indica L has also antibacterial properties. [10,11] T. indica L stem bark and leave were subjected to extraction using aqueous, ethanol, and acetone-inhibited growth of both gram-positive and gram-negative bacteria. These extracts contain tannin, saponin, sesquiterpenes, alkaloids, and phlobatannins.  The extract of T. indica L has hypoglycemic effects. [12,16] Aqueous extract of T. indica L seeds with dose 80 mg/0.5 mL distilled water/100 g/day for 14 days reduces blood glucose level after 7 days in streptozotocin-induced diabetic rat.  The methanolic extract of T. indica seeds with doses 200 and 400 mg/kgbw orally on alloxan-induced diabetic mice reduced blood glucose level in a 5-day study.  The aqueous extract of T. indica L seed (500 mg/kgbw, p.o.) decreases fasting blood glucose and increases plasma insulin.  Aqueous extract of T. indica L. seeds was predicted to restore pancreatic beta cells in streptozotocin-induced diabetic rats.  The methanolic extract of T. indica L seed has cytotoxic activity against sea urchin embryo cells.  The ethanol, chloroform, and aqueous extracts of T. indica L have anti-inflammatory activity on mice and rats after intraperitoneal and topical administration.1191 Dried and pulverized pulp of T. indica L fruits (15 mg/kgbw, p.o.) can reduce total cholesterol level and low-density lipoprotein (LDL)-cholesterol in human subjects.  The methanol extract of T. indica L has spasmolytic effect on yeyunum rabbits.  The aqueous extracts of tamarind leaves, fruits, and unroasted seeds administered for 9 days have hepatoregenerative effect on paracetamol-induced hepatotoxicity in rats.  The aqueous and alcoholic extracts of T. indica L seed coat have anti-inflamatory, anti-arthritic, and anti-nociceptive effects. These extracts inhibit expression of interleukin (IL) and decrease the production of prostaglandin E2.  The methanolic extract of T. indica L seeds with doses of 100 and 200 mg/kgbw administered orally can reduce the total volume of gastric juice and free and total acidity of gastric secretion in rats' pylorus-ligation-induced ulcer model.  The methanolic extract of T. indica L leaves has antiasthmatic activity. Doses of 175, 350, and 700 mg/kgbw (p.o.) of these extracts exhibit mast-cell-stabilizing activity.  T. indica L also has laxative effect. [26-27] The juice of T. indica L leaves with a dose of 40% has laxative properties.  The methanol and butanol extracts of T. indica L leaves have antiemetic properties.  Besides, T. indica L has also anthelmintic activity. The juice of T. indica L (20%, 50%, and 100%, respectively) causes paralysis on pheretima posthuma. 
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
The objectives of this study were to determine the hypolipidemic effects of ethanolic extract of T. indica L fruit flesh and rind on hyperlipidemia in Wistar rats induced by Triton X-100.
MATERIALS AND METHODS
The materials used in this study include fruit tamarind (T. indica L) obtained from Boyolali area in Indonasia, harvested in August 2013, Triton X-100 (Sigma), Simvastatin[R] StarDust FC15 analyzer, silica plate GF254 7 x 6 cm (Merck).
Animals tested were male Wistar rats obtained from Laboratory of Pharmacology of Universitas Muhammadiyah, Surakarta. The age of Wistar rats was approximately 2-3 months and weight was 175-225 g. This research was approved by health research ethics committee of Dr. Moewardi Hospital of Surakarta.
Preparation of Extract
Extracts used were fruit rind and flesh of T. indica L. They were separated, then dried under the sun, and blended into a powder. Each powder was extracted by maceration method by soaking in 70% ethanol for 5 days and then was filtered. Filtrates were evaporated on a water bath temperature of 60-70 [degrees]C as they were stirred and aerated to obtain thick extract.
Methods of Evaluating of Hypolipidemic Effect
Subjects used in the study were male Wistar rats. They were divided into eight groups randomly. Each group consisted of five rats. Group I was the negative control (aquadest); group II was the positive control (Simvastatin 0.72 mg/200 gbw); groups III, IV, and V were treated by 70% ethanolic extract of the fruit flesh with doses of 200, 100, and 50 mg/kgbw; groups VI,VII, and VIII were treated by 70% ethanolic extract of the fruit rind with doses of 250, 200, and 100 mg/kgbw, respectively. The extract was given for 7 days. Plasma total cholesterol and triglyceride levels were measured on days 0, 3, and 10. Cholesterol and triglyceride measurements were carried out by a StarDust FC15 spectrometer with reagent cholesterol oxidase-peroxidase for the measurement of total cholesterol and glycerol phosphate oxidase-peroxidase for the measurement of triglyceride (Diasis).
A total 100 mg of 70% ethanolic extract of the fruit rind and flesh of T. indica L were dissolved in 1 mL methanol p.a. From this solution, 0.5 mL was spotted on thin-layer chromatography (TLC) plates (silica plate GF254). The TLC plate was eluted with mobile phase ethyl acetate/toluene (1:3). The spotting was observed in UV 254 nm and UV 366 nm. The coloring agents were sulfate vanillin for terpenoid, Fe[Cl.sub.3] for polyphenols, dragendorff for alkaloids, and sitoborat for flavonoid. The movement of active compound was expressed by its retention factor ([R.sub.f]). This [R.sub.f] was calculated by the following formula:
[R.sub.f] = Distance traveled by the solute/Distance traveled by the solvent
1. The lowering of total cholesterol and triglycerides Treatment by the extract was done for 7 days. The plasma level of cholesterol and triglycerides was measured on days 0, 3, and 10. The plasma levels of cholesterol on days 0, 3, and 10 are shown in Table 1.
Table 1 and 2 show consequence on the calculation of the percentage reduction of total cholesterol and triglycerides. The result can be seen in Table 3.
2. Thin-layer chromatography
a. The 70% ethanolic extract of T. indica L fruit flesh
The result of TLC of T. indica L fruit flesh can be observed in Figure 1.
The TLC results show the 70% ethanolic extract of T. indica L fruit flesh contains flavonoids, phenolics, terpenoids, and alkaloids
b. The 70% ethanolic extract of T. indica L fruit rind.
The result of TLC of T. indica L fruit rind can be observed in Figure 2.
On the basis of Figure 2, several compounds may be identified and are listed in Table 5.
The TLC results show the 70% ethanolic extract of T. indica L fruit rind contains flavonoids, phenolic, terpenoids, and alkaloid.
Hyperlipidemia is one of the major contributors to coronary heart disease (CHD) and other cardiovascular diseases.  The extract, which has a lipid-lowering effect, is expected to reduce the risk of CHD and other cardiovascular diseases. This study used Triton X-100 to induce hyperlipidemia. The possible mechanism is Triton X-100 blocks TGs-rich lipoproteins from causing acute hyperlipidemia.  Data in this study show that the 70% ethanolic extract of T. indica L fruit rind and flesh was able to lower plasma cholesterol and triglyceride in rats induced by Triton X-100. This study is in line with the previous research. Martinello et al.  found that the pulp of fruit of T. indica L is able to reduce the levels of total cholesterol (50%), non-HDL cholesterol (73%), and triglycerides (60%) but increases HDL 60% on hamster. Research by Jindal et al.  stated the extract of tamarind pulp can decrease total cholesterol and triglycerides significantly in Wistar rats. 
Aqueous extract of T. indica L was able to decrease levels of total cholesterol, LDL, and triglycerides. It also reduced body weight of obesity-induced Sprague-Dawley rats.  Research by Koyagura et al.  showed that extracts of T. indica L decrease levels of total cholesterol, LDL, very low-density lipoprotein, triglycerides, glucose, and increase HDL. Studies in humans indicate that administration of T. indica L fruit (dried and pulverized pulp) with a dose of 15 mg/kgbw lowered the total cholesterol and LDL cholesterol significantly.  Research by Yerima et al.,  showed that extract of T. indica L has effect on hypolipidemia and hypoglycemia.
This result is slightly different from that reported in the study by Ukwuani et al.,  which found that the aqueous extract of pulp of T. indica L decreases levels of total cholesterol and LDL but increases those of HDL and triglycerides. 
Chemical constituents in 70% ethanolic extract of T. indica L fruit rind and flesh in this study were flavonoids, alkaloids, terpenoids, and phenolic. Several other studies mentioned T. indica L contains chemical constituents such as malic acid,  tartaric acid, mucilage and pectin, arabinose, xylose, galactose, glucose, and uronic acid,[38-39] and phenolic compound and cardiac glycosides.  Research by Razali et al.,  showed that T. indica L contains many flavonoids. The leaves of T. indica L contain many polyphenols.  Research by Yerima et al.  stated that the chemical constituents of T. indica L among others are carbohydrates, glycosides, saponins, flavonoids, tannins, alkaloids, and triterpenes.
Hypolipidemic mechanism of this extract is suspected by its polyphenols (flavonoids, limonene, etc.)  and the presence of the antioxidant effect of the extract. [9,15] The content of phenolic in T indica L is a contributor to the antioxidant effects of this plant.  Polyphenols in tamarindus are dominated by proanthocyanidins groups such as procyanidin B2, apigenin, catechin, epicatechin, procyanidin dimers, procyanidin trimers, eriodictyol, taxifolin, and naringenin.  Polyphenols are a group of antioxidants that are most abundant in plant metabolites and are an integral part of both human and animal diets including the simple phenolic molecules.  Hypocholesterolemic and antioxidant effects, presumably through an increase in Apo A1, ABCG5, and LDL receptor gene expression in liver, decrease in HMG CoA reductase and inhibit MTP gene expression. Flavonoids also trigger an increase in the excretion of cholesterol but decrease in the biosynthesis of cholesterol, increase in intake LDL cholesterol from peripheral tissues. They prevent the accumulation of TG in the liver. Treatment by T. indica L fruit pulp in hypercholesterolemic hamsters can protect oxidative damage by increasing hepatic antioxidant enzymes, preventing hepatic lipid peroxidation, and increasing antioxidant activity. 
The 70% ethanolic extract of Tamarindus indica L. fruit flesh and rind can reduce total cholesterol and triglycerides in hyperlipidemia induced by Triton X-100 in rats. The chemical compounds of these extracts are flavonoids, terpenoid, phenolic (tannins), and alkaloids.
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Source of Support: Nil, Conflict of Interest: None declared.
EM Sutrisna, Devi Usdiana, Rizky Maidina Taqwin, Ahmad Roni Rosyidi
Department of Pharmacology, Faculty of Medicine of Universitas, Muhammadiyah Surakarta, Surakarta, Jawa Tengah, Indonesia.
Correspondence to: EM Sutrisna, E-mail: Em.Sutrisna@ums.ac.id or firstname.lastname@example.org
Received March 9, 2015. Accepted March 30, 2015
Table 1: The mean of plasma level of cholesterol [+ or -] SD on days 0, 3, and 10 Cholesterol level (mg/dL) Groups Day 0 Day 3 Negative control 68.4 [+ or -] 15.4 128.2 [+ or -] 43.4 Positive control 84.8 [+ or -] 4.8 150 [+ or -] 33.1 (Simvastatin 0.72 mg/200 gbw) The 70% ethanolic 77.8 [+ or -] 20.7 184.8 [+ or -] 56.7 extract of fruit flesh with dose 200 mg/kgbw The 70% ethanolic 87 [+ or -] 28.20 185.2 [+ or -] 38.6 extract of fruit flesh with dose 100 mg/kgbw The 70% ethanolic 49.2 [+ or -] 13.4 160 [+ or -] 14.9 extract of fruit flesh with dose 50 mg/kgbw The 70% ethanolic 55.00 [+ or -] 22.30 161.16 [+ or -] 28.28 extract of fruit rind with dose 200 mg/kgbw The 70% ethanolic 55.40 [+ or -] 18.86 152.58 [+ or -] 63.39 extract of fruit rind with dose 100 mg/kgbw The 70% ethanolic 59.60 [+ or -] 12.81 153.40 [+ or -] 30.26 extract of fruit rind with dose 50 mg/kgbw Cholesterol level (mg/dL) Groups Day 10 P-value (compared by negative control) Negative control 128.7 [+ or -] 42 Positive control 79.8 [+ or -] 7.8 0.00 (Simvastatin 0.72 mg/200 gbw) The 70% ethanolic 86.02 [+ or -] 19.1 0.00 extract of fruit flesh with dose 200 mg/kgbw The 70% ethanolic 103.4 [+ or -] 37.7 0.01 extract of fruit flesh with dose 100 mg/kgbw The 70% ethanolic 88.8 [+ or -] 14 0.01 extract of fruit flesh with dose 50 mg/kgbw The 70% ethanolic 98.50 [+ or -] 30.62 0.00 extract of fruit rind with dose 200 mg/kgbw The 70% ethanolic 94.50 [+ or -] 18.37 0.01 extract of fruit rind with dose 100 mg/kgbw The 70% ethanolic 76.53 [+ or -] 4.97 0.01 extract of fruit rind with dose 50 mg/kgbw Table 2: The mean of plasma level of triglycerides [+ or -] SD on day 0, 3, and 10 Triglycerides (mg/dL) Groups Day 0 Day 3 Negative control 64.4 [+ or -] 17.6 158 [+ or -] 20.6 Positive control 76.8 [+ or -] 12.4 139 [+ or -] 18.9 (Simvastatin 0.72 mg/200 gbw) The 70% ethanolic 88.4 [+ or -] 16.7 160.8 [+ or -] 11.8 extract of fruit flesh with dose 200 mg/kgbw The 70% ethanolic 101.6 [+ or -] 26.9 160.4 [+ or -] 16.9 extract of fruit flesh with dose 100 mg/kgbw The 70% ethanolic 92.2 [+ or -] 13.9 138 [+ or -] 12 extract of fruit flesh with dose 50 mg/kgbw The 70% ethanolic 86.00 [+ or -] 12.35 145.60 [+ or -] 45.02 extract of fruit rind with dose 200 mg/kgbw The 70% ethanolic 73.80 [+ or -] 16.63 189.40 [+ or -] 60.92 extract of fruit rind with dose 100 mg/kgbw The 70% ethanolic 89.60 [+ or -] 19.32 148.00 [+ or -] 52.24 extract of fruit rind with dose 50 mg/kgbw Triglycerides (mg/dL) Groups Day 10 P-value (compared by negative control) Negative control 208.3 [+ or -] 20.7 Positive control 102.5 [+ or -] 18.9 0.00 (Simvastatin 0.72 mg/200 gbw) The 70% ethanolic 112.5 [+ or -] 11.9 0.00 extract of fruit flesh with dose 200 mg/kgbw The 70% ethanolic 115.2 [+ or -] 16.9 0.01 extract of fruit flesh with dose 100 mg/kgbw The 70% ethanolic 83.6 [+ or -] 12 0.01 extract of fruit flesh with dose 50 mg/kgbw The 70% ethanolic 105.50 [+ or -] 17.25 0.00 extract of fruit rind with dose 200 mg/kgbw The 70% ethanolic 88.12 [+ or -] 23.19 0.00 extract of fruit rind with dose 100 mg/kgbw The 70% ethanolic 101.33 [+ or -] 43.14 0.01 extract of fruit rind with dose 50 mg/kgbw Table 3: The percent reduction of total cholesterol and triglycerides [+ or -] SD The mean of the percent reduction (% [+ or -] SD) Groups Cholesterol Triglycerides Positive control 37.95 [+ or -] 6.1 50.82 [+ or -] 14.9 (Simvastatin 0.72 mg/200 gbw) The 70% ethanolic 33.19 [+ or -] 14.8 41.18 [+ or -] 19.9 extract of fruit flesh with dose 200 mg/kgbw The 70% ethanolic 19.64 [+ or -] 7.2 44.7 [+ or -] 13.9 extract of fruit flesh with dose 100 mg/kg bw The 70% ethanolic 30.99 [+ or -] 10.9 59.89 [+ or -] 10.9 extract of fruit flesh with dose 50 mg/kgbw The 70% ethanolic 23.45 [+ or -] 23.79 49.36 [+ or -] 8.28 extract of fruit rind with dose 200 mg/kgbw The 70% ethanolic 26.56 [+ or -] 14.28 57.70 [+ or -] 11.13 extract of fruit rind with dose 100 mg/kgbw The 70% ethanolic 40.52 [+ or -] 3.85 51.36 [+ or -] 20.71 extract of fruit rind with dose 50 mg/kgbw Table 4: The separation of 70% ethanolic extract of T. indica L fruit flesh Detection No hRf Description of color Chemical compound U[V.sub.254] 1 0 Strong quenching 2 55 Weak quenching 3 57.5 Weak quenching 4 95 Weak quenching U[V.sub.366] 1 0 Weak yellow Flavonoid fluorescence 2 55 Yellow fluorescence Flavonoid Vanilin 1 0 Brown Terpenoid [H.sub.2]S 2 60 Purple-green Terpenoid [O.sub.4] 3 92.5 Blackish Terpenoid Sitroborat 1 0 Weak yellow Flavonoid fluorescence 2 45 Yellow fluorescence Flavonoid 3 55 Yellow fluorescence Flavonoid FeCl3 1 0 Blackish Phenolic 2 55 Blackish Phenolic Dragendorff 1 0 Brown Alkaloid Table 5: The separation of 70% ethanolic extract of T. indica L fruit rind Detection No hRf Description of color Chemical compound U[V.sub.254] 1 0 Strong quenching 2 75 Weak quenching 3 95 Weak quenching U[V.sub.366] 1 0 Weak yellow Flavonoid fluorescence 2 37.5 Blue yellowish Flavonoid fluorescence 3 62.5 Yellow fluorescence Flavonoid 4 90 Blue yellowish Flavonoid fluorescence Vanilin 1 0 Blackish brown Terpenoid [H.sub.2]S 2 75 Purple-green Terpenoid [O.sub.4] 3 95 Blackish Terpenoid Sitroborat 1 0 Weak yellow Flavonoid fluorescence 2 37.5 Blue yellowish Flavonoid fluorescence 3 62.5 Yellow fluorescence Flavonoid 4 90 Blue yellowish Flavonoid fluorescence Fe[Cl.sub.3] 1 0 Blackish Phenolic (Tanin) Dragendorff 1 0 Brown Alkaloid
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|Author:||Sutrisna, E.M.; Usdiana, Devi; Taqwin, Rizky Maidina; Rosyidi, Ahmad Roni|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Jul 1, 2015|
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