The Potential of Total Flavonoids and Phenol Contents in Stem Bark of Gayam (Inocarpus fagiferus Fosb) as an Antioxidant through the Decrease of MDA Level, Increase of SOD Activities and Improvement o.

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Rahayuet al. European Journal of Biomedical and Pharmaceutical Sciences

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THE POTENTIAL OF TOTAL FLAVONOIDS AND PHENOL CONTENTS IN STEM

BARK OF GAYAM (

Inocarpus

fagiferus

Fosb) AS AN ANTIOXIDANT THROUGH THE

DECREASE OF MDA LEVEL, INCREASE OF SOD ACTIVITIES AND IMPROVEMENT

OF LIPID PROFILE IN RATS ATHEROSCLEROSIS HYPERCHOLESTEROLEMIA

*Sri Rahayu Santi, I Made Sukadana and I Made Siaka

Department of Chemistry-Faculty of Science and Maths

Article Received on 10/09/2015 Article Revised on 30/09/2015 Article Accepted on 22/10/2015

I. INTRODUCTION

Medicinal plants are one source of natural antioxidants that can increase endogenous antioxidant and hence, it may reduce the risk of certain diseases such as cancer, heart disease, neurodegenerative diseases, stroke, inflammation, and atherosclerosis.[1,2] Secondary metabolites such as natural phenols, flavonoids, and their derivatives contained in medicinal plants are potentially used as a source of natural antioxidants for free radicals scavenger.[2,3] Gayam (Inocarpus Fagiferus Fosb) or in Bali known as gatep is one of the herbs that empirically in the Fiji Islands is used as a drug to prevent heart disease and atherosclerosis.[4] The stem bark of gayam contains compounds such as triterpenoids, anthraquinone, steroids and flavonoids and phenols as the main content. n-buthanol extract of gayam stem bark contains total flavonoids and phenols respectively by 0.09% and 14.16%, that are able to capture the free radicals DPPH by 50% at a concentration of 20 ppm, and are able to inhibit the formation of lipid peroxide

by 63.03%. The antioxidant activity of n-buthanol extract of gayam stem bark in vitro against DPPH is greater than the antioxidant activity of vitamin E which provides IC50 at a concentration of 25 ppm, as well as its ability to inhibit the formation of lipid peroxides by 50.39 %.[5] Therefore, the potential of antioxidant activity of n-buthanol extract of gayam stem bark is required to be further investigated in vivo by determining the activity of SOD, levels of malondialdehyde (MDA), as well as its effect on lipid profile improvements such as total cholesterol, triglycerides, LDL cholesterol (LDL-cholest) and HDL cholesterol (HDL-cholest) as a marker to prevent the occurrence of atherosclerosis in wistar rats with high fat diet or hypercholesterolemia conditions.[6,7]

This study discusses the differences in the levels of MDA, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and SOD activity of each treatment group compared with the control group of hypercholesterolemia, as the marker of cell

SJIF Impact Factor 2.062 Research Article

ejbps, 2015, Volume 2, Issue 6, 55-58.

European Journal of Biomedical

AND Pharmaceutical sciences

http://www.ejbps.com

ISSN 2349-8870

Volume: 2 Issue: 6

55-58 Year: 2015

*Author for Correspondence: Sri Rahayu Santi

Department of Chemistry-Faculty of Science and Maths

ABSTRACT

This study aimed to demonstrate the antioxidant potential of n-buthanol extract of gayam stem bark in preventing atherosclerosis through the decrease of malondialdehyde (MDA) level, lipid profile improvements such as the decrease of total cholesterol, triglycerides, LDL cholesterol (LDL-cholest) and the increase of HDL cholesterol (HDL-cholest) levels, and SOD activity in Wistar rats that were treated with a high fat diet or hypercholesterolemia conditions within 3 months of observation. The study was initiated by preparing n-buthanol extract of gayam stem bark which was obtained from the partition of 95 g ethanol extract of gayam stem bark. A total of 30 g of n-buthanol concentrate extract was obtained and then was applied to Wistar rats for 3 months with the use of the posttest only control group design. Twenty-five Wistar rats were randomized into 5 groups, K1 (negative control), K2 (positive control, hypercholesterolemia), P1 (hypercholesterolemia + n-buthanol extract in the dose of 50 mg/kg bw), P2 (hypercholesterolemia + n-buthanol extract in the dose of 100 mg/kg bw), and P3 (hypercholesterolemia + n-buthanol extract in the dose of 150 mg/kg bw). The results showed that n-buthanol extract of gayam stem bark in the dose of 50 mg/kg bw was able to decrease the levels of MDA, total cholesterol, triglycerides, and increase the SOD activity, while in the dose of 100 mg/kg bw the extract could decrease the LDL cholesterol and increase of HDL cholesterol levels of blood plasm in wistar rats hypercholesterolemia.

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membrane damage due to lipid peroxidation,[8,9,10] an indirect marker of fatty acid oxidation products,[11] and the marker if n-buthanol extract of gayam stem bark could be SOD inducer to the endogenous antioxidant.

II. MATERIALS AND METHODS The Research Materials

The plant material was obtained from Tabanan Bali and its classification has been determined. Chemicals used in this study were ethanol (technical and pa), chloroform (technical and pa), ethyl acetate (technical and pa), n-buthanol, and blood plasm of wistar rat. Chemicals required for plasm analysis were ParameterTM TBARS (R&D Systems, Cat. KGE013), Superoxide Dismutase (SOD) Activity Assay Kit (BioVision, Cat. K335-100). The determination of profile lipid was conducted at UPT. Balai Laboratorium Kesehatan Provinsi Bali. All of the rats were treated in accordance with the rules of the etical clerence of Ethics Committee of Animals Use in Research and Education of the Faculty of Veterinary Udayana University.

Research Instruments

The equipment used in this study was a set of glasses, blender, sieve, extractor, rotary vacuum evaporator, centrifuges, analytical balance, thermometer, micropipette, pipette, volumetric flask, stomach probes, syringes, and UV-vis spectrophotometer.

Researchers procedure

a. Preparation of n-buthanol extract of gayam stem bark

As much as 3 kg dried powder of gayam stem bark was macerated with ethanol for 24 hours. The extract was filtered, while the crude was remacerated several times with ethanol until the compound contained in the sample was extracted out. The filtrate obtained was evaporated with a rotary vacuum evaporator to obtain a concentrate extract of ethanol. The concentrate ethanol extract was further diluted with ethanol-water mixture (7: 3) and then partitioned successively with chloroform, ethyl acetate and n-buthanol. The extract obtained was evaporated and weighed. n-buthanol extract was then applied to the rats.

b. Applications of n-buthanol extract of gayam stem bark on Wistar Rats

n-buthanol extract was applied at various doses i.e 50 mg/kg bw, 100 mg/kg bw, and 150 mg/kg bw. Twenty five wistar rats were randomized into 5 groups with posttest only control group design.[12] as follows:

K1 = group of wistar rats fed with standard diet (negative control) K2 = group of wistar rats fed with a high-fat diet (positive control)

P1 = group of wistar rats fed with a high-fat diet + n-buthanol extract of gayam stem bark in the dose of 50 mg/kg bw

P2 = group of wistar rats fed a high-fat diet + n-buthanol extract of gayam stem bark in the dose of 100

mg/kg bw

P3 = group of wistar rats fed with a high-fat diet + n-buthanol extract of gayam stem bark in the dose of 150 mg/kg bw

After 12 weeks, the blood plasm of all of the rats such as the control groups (K1 and K2) and the treatment groups (P1, P2, and P3) were drawn for MDA, SOD activity, total cholesterol, triglycerides, HDL-cholest and LDL-cholest analyses. The difference of all variables were analyzed by one way Anova with = 0.05.

III. RESULTS AND DISCUSSION 3.1 MDA Blood Plasm

The results of MDA levels of blood plasm analysis of all the rats (K1, K2, P1, P2, and P3) showed a significant decreases in the level of MDA in the rats treated with n-buthanol extract in the doses of 50, 100, and 150 mg/kg bw as described in Figure 1.

Figure 1.

The average of MDA levels of K1 group, treatment groups (P1, P2, and P3) toward K2. The difference mean of groups were: K1 vs K2, p <0.05; P1 vs K2, p <0.05; P2 vs K2, p <0.05; P3 vs K2, p <0.05; P1 vs P2, p <0.05; P1 vs P3, p <0.05; P2 vs P3, p <0.05.

The intake of high cholesterol diet (hypercholesterolemia) caused the increase of MDA level significantly on K2 group 1.162) compared with K1 group or standard diet (K1; 15.623 1.212), treatment group 1 (P1; 12.157

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increase of MDA plasm level in rat groups with high cholesterol diet is a marker of oxidative stress due to hypercholesterolemia,[8,9,10,11,13,14] The results found in this study were in agreement to the research by Han et al.,[15] and,[16] in which the rat that was given a high-fat diet causes hypercholesterolemia condition and an increase in MDA plasm level.

3.2 Blood Plasm Lipid Profile

The illustration of the effect of n-buthanol extract of gayam stem bark at various doses toward total cholesterol, triglycerides, HDL cholesterol (HDL-cholest), and LDL cholesterol (LDL-cholest) contents at the end of the experiment are presented in Figure 2.

Figure 2.

The profile of total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol of blood plasm of control group (K1 and K2) and the treatment groups (P1,

P2, and P3) after the 3rd month of observation. The results of the difference mean in total cholesterol levels between groups are as follows: K1 vs K2, p <0.05; P1 vs K2, p <0.05; P2 vs K2, p <0.05; P3 vs K2, p <0.05; P1 vs P2, p <0.05; P1 vs P3, p <0.05; P2 vs P3, p <0.05. The results of the difference mean in triglyceride levels between groups are as follows: K1 vs K2, p <0.05; P1 vs K2, p <0.05; P2 vs K2, p <0.05; P3 vs K2, p <0.05; P1 vs P2, p> 0.05; P1 vs P3, p> 0.05; P2 vs P3, p> 0.05. The results of the difference mean in HDL levels between groups are as follows: K1 vs K2, p> 0.05; P1 vs K2, p> 0.05; P2 vs K2, p <0.05; P3 vs K2, p <0.05; P1 vs P2, p> 0.05; P1vs P3, p> 0.05; P2 vs P3, p> 0.05. The results of the difference mean in LDL cholesterol between groups are as follows: K1 vs K2, p <0.05; P1 vs K2, p> 0.05; P2 vs K2, p <0.05; 1.14P3 vs K2, p <0.05; P1 vs P2, p> 0.05; P1 vs P3, p <0.05; P2 vs P3, p> 0.05.

A significant decrease in total cholesterol levels occurred after the groups of rat were fed with high fat diet and n-buthanol extract of gayam stem in the doses of 50 mg/kg bw (P1 1.14, p<0.05), 100 mg/kg bw (P2; 63.3 1.95, p<0.05), and 150 mg/kg bw (P3 compared with the control group K2

The triglyceride levels decreased significantly (p

<0.05) in rat with high cholesterol diet and n-buthanol of gayam stem bark in the doses of 50 mg/kg bw (P1; 237.60 g/dl), 100 mg/kg bw (P2; mg/dl), 150 mg/kg bw _(P3; 228.80 _{mg/dl) compared with K2 (338.0} 2 mg/dl). When the three treatment groups were compared (P1 mg/dl), (P2; mg /dl) and (P3; 228.80 mg/dl) they did not show significant differences. This suggested that n-buthanol extract of gayam stem bark in the dose of 50 mg/kg bw was capable to decrease the triglyceride levels of rats blood plasm.

Theoretically, HDL-cholest levels increase as the doses of the antioxidant compounds given to the rats are higher. The averages of HDL-cholest levels in all groups of rat K1, K2,

P1, P2, and P3 were 50. mg/dl, 47.08 .77 mg/dl, 58.18 6.17 mg/dl, 60.58 g/dl, and 70.26 mg/dl), respectively. The results of difference test were as follows: K1vs K2 (p> 0.05), P1 vs. K2 (p> 0.05), P2 vs K2 (p <0.05), P3 vs K2 (p <0.05), P2 vs P1 (p> 0:05), P3 vs P1 (p> 0.05), and P2 vs P3 (p> 0.05). This indicated that the introduction of n-buthanol extract of gayam stem bark in the dose of 100 mg/ kg bw was able to increase the HDL levels in rat blood.

LDL-cholest is the bad cholesterol. Figure 2 showed the averages of LDL-cholest levels in all groups of rats K1, K2, P1, P2, and P3 which were mg/dl, 1 1.06 mg mg/dl, 10.6 and md/dl, respectively. The results of difference test showed that K1 vs K2, p <0.05; P1 vs K2, p> 0.05; P2 vs K2, p <0.05; P3 vs K2, p <0.05; P1 vs P2, p> 0.05; P1 vs P3, p <0.05; and P2 vs P3, p> 0.05. This suggested that the treatment of P2 and P3 did not give significant differencess (p> 0.05), and therefore this means that the extract in the dose of 100 mg/kg bw has been able to decrease LDL levels in rat blood.

3.3 SOD Blood Plasm

The SOD activities of all rats included two control groups (K1 and K2) and three treatment groups (P1, P2, and P3) were presented in Figure 3.

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The average of Rate Inhibition (%) K1, P1, P2, and P3 toward K2 after the end of experiments. The results of the difference means test between groups are as follow: K1 vs K2, p>0.05; P1 vs K2, p<0.05; P2 vs K2, p<0.05; P3 vs K2, p<0.05; P1 vs P2, p>0.05; P1 vs P3, p>0.05; P2 vs P3, p>0.05.

The analysis results of SOD activity showed that n-buthanol extract of gayam stem bark which was given in each treatment groups (P1, P2, and P3) could increase SOD endogenous antioxidant significantly (p<0.05) toward control group K2. The average of rate inhibition (%) of each treatment groups were P1= 62.2 _{11.841%, P2=} _{and P3=} However, there was no significant differences between treatment groups P1, P2, and P3 (p>0.05), which means that the n-buthanol extract of gayam stem bark in the dose of 50 mg/kg bw had significantly increased the SOD activity(p <0, 05).

IV. CONCLUSIONS

1. n-buthanol extract of gayam stem bark in the dose of 50 mg/kg bw could prevent atherosclerosis by decreasing the plasm malondialdehide (MDA), total cholesterol, and triglyceride levels and increasing the SOD activity in the hypercholesterolemia wistar rast. 2. n-buthanol extract of gayam stem bark in dose

of 100 mg/kg bw could prevent atherosclerosis by decreasin

V. ACKNOWLEDGMENT

We would like to express our gratitude to DIKTI that provided us the fund throughout Hibah Bersaing (2015), the Rector of Udayana University through the Head of LPPM that facilitated our research, and Emmy Sahara, M.Sc(Hons) who has supported the writing of this paper.

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