HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF ISOFLAVONES AGLYCONE IN INDONESIAN SOYBEAN

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF ISOFLAVONES AGLYCONE IN INDONESIAN SOYBEAN

Mfawwaz ^1*, N Akbar ¹, M Pratama¹, A Saleh ² and Mbaits ¹

Department of Pharmaceutical Chemistry ¹, Faculty of Pharmacy, Universitas Muslim Indonesia, Jl. Urip Sumohardjo Km. 5 Campus II UMI Makassar 90231, South Sulawesi, Republic of Indonesia

Department of Pharmacy ², STIKES Mandala Waluya, Kendari, Republic of Indonesia

ABSTRACT: Soybean is one of the highest isoflavones source. Isoflavones are one type of phytoestrogen that have a chemical structure similar to estradiol, that’s why it can be used not only to inhibite but also to prevent many symptoms related deficiency of estrogen. However in the small intestine, isoflavones like genistin will be hydrolyzed by β-glucosidase and produce aglycone like genistein, this process can be optimized by hydrolysis with enzyme or chemical. The aim of this study was to obtain the extract of soybean that hydrolysed by hydrochloric acid and to obtain scientific data about the content of genistein in the extract by High Performance Liquid Chromatography (HPLC). Analysis of samples by HPLC using C18 reverse phased column. Genistein standard with a concentration 8 ppm, 10 ppm, 12 ppm respectively diluted with methanol: water (8:2) as much as 5 ml. Soybean extract, weighed as much as 3 mg and dissolved in 10 ml of methanol: water (8: 2). Standard and sample then analyzed with the mobile phase used was methanol: water (7:3), a flow rate of 1 ml/min with a temperature of 28 °C, at a wavelength of 254 nm. From the results, show that average of genistein levels contained in the extract of soybean hydrolyzed for Glycine max is 0.5% and Glycine soja is 1.1%.

Soybean, Phytoestrogen,

Isoflavones, Genistein, HPLC

INTRODUCTION: Soybean (Glycinemax (L). Merrill) is a source of complete food with a high nutrient content and good. Soybean seeds are a source of high quality protein, oligosaccharides, dietary fiber, minerals and phytochemicals particularly isoflavones. Levels of isoflavones in soybean seed is the highest among hypocotyledon group (> 20 mg / g) ^{1, 2, 3, 4}.

Actually soybeans are cultivated in two species, namely Glycinemax (yellow, white or green soybean) and Glycinesoja (black soybean). Glycinemax is native to subtropical regions of Asia such as China and Southern Japan, while Glycinesoja is native to tropical Asia in Southeast Asia.

This plant has spread to Japan, Korea, Southeast Asia and Indonesia. According to Indonesia National Standard (SNI)-01-3922-1995, soybeans can be divided into four, namely yellow soybeans, black soybeans, green soybeans and soy mix, while according Sniyeder and Kwon (1987), all originally green soybeans because of their chlorophyll content, but after maturity the chlorophyll becomes invisible ⁵.

Soybean is known to contain very high isoflavone compounds, isoflavones are compounds that have a molecular similarity to estrogen that has been used to the clinical symptoms in women postmenopaus such as diabetes, cardiovascular disease, breast cancer and bone health ⁶. Soybean has a good potential as antimitotic with IC50 = 3.79 x 10^-4 mg/mL⁷.

Isoflavones in soy in the form of glycosides that genistein, daidzein and glisitin. Isoflavones in the form of glycosides are not absorbed by the body, to be absorbed, it is necessary isoflavones hydrolyzed by the enzyme ß-glucosidase in the intestine to break the bonds of it’s glycosides ⁸.

Hydrolysis process can be done by adding a microbiology probiotic bacteria in soymilk. Results of research conducted by Fawwaz showed that the levels of genistein in soy fermented by Lactobacillus acidophilus by 3.46% ⁹, and genistein levels previously obtained through the fermentation of soybean by Lactobacillus bulgaricus by 4.99% ¹⁰. Hydrolysis can also be done chemically by the addition of hydrochloric acid¹¹. Based on previous research, this study was conducted to determine how many levels of genistein contained in the extract of soybean that is hydrolyzed by chemical methods, determined by HPLC (High Performance Liquid Chromatography).

MATERIALS AND METHOD:

Chemical Materials:

Standard genistein G6649 which contains 5 mg was purchased from Sigma Aldrich Chemie GmbH, with purity ≥98%.

 Extraction Process:

Soybean seeds crushed (250 g) and added500 ml ethanol70% in the ratio 1:2 (g/ml), the mixture is then heated at a temperature of 90 °C, stirring constantly for 2 hours. Solute mixture is separated using a vacuum filter (Whatman). The filtrate was added 37% hydrochloric acid until the mixture reaches a pH of 3. The mixture is then heated at 90 °C, stirring constantly for 2 hours. The mixture is then added distilled water in the ratio 1:1 (ml/ml) and stirred constantly at room temperature. The precipitate formed is separated using a vacuum filter, the result is stored at 4 °C ¹¹.

 Preparation of Genistein:

Created a standard solution with a concentration of 200 ppm. Genistein weighed as much as 1 mg and dissolved in methanol: water (8: 2) for 5 ml. Pipette 1 ml of the stock solution and add 5 ml of methanol: water (8: 2) to obtain a concentration of 40 ppm.

 Analysis of Genistein:

Analysis of samples was conducted using High Performance Liquid Chromatography (HPLC), used C₁₈ reverse phased column. Genistein standard with a concentration of 40 ppm and then diluted with methanol: water (8:2) to obtain a concentration of 8 ppm, 10 ppm, 12 ppm respectively as much as 5 ml. Series of concentration is then automatically injected into the tool as much as 10 mL. The mobile phase used was methanol: water (7:3), a flow rate of 1 ml/min with a temperature of 28°C, at a wavelength of 254 nm. Data obtained in the form of an area, then determined the values of a, b and r by comparing the sample concentration (ppm) to the area. Created the linear regression equation y = a + bx ^{8, 9}. Soybean extract, weighed as much as 3 mg and dissolved in 10 ml of methanol: water (8: 2). Inserted into the HPLC instrument and then analyzed. The results of the analysis will be obtained area (y) which is in turn embedded in the linear regression equation ⁹.

 RESULT AND DISCUSSION:

Extract of Isoflavone Aglycone:

Isoflavone aglycone could be found if soybean was hydrolyzed; by hydrolyzing glycoside binding will be solved. Soybean hydrolyzed result was extracted with ethanol. We found extract of Glycine max 2.67 g and Glycine soja1.87 g.

 Isoflavone aglycone Analysis by HPLC:

Pure standard genistein was used as isoflavone aglycone to identify and calculate the amount of genistein in extract soybean hydrolyzed. The result of linear regression, we found y = 108918x – 418941, and R² = 0.996.

TABLE 1: CONCENTRATION AND AREA OF GENISTEIN STANDARD

 TABLE 2: WEIGHT OF EXTRACT ANDGENISTEIN LEVEL OF GLYCINE MAX

 TABLE 3: WEIGHT OF EXTRACT ANDGENISTEIN LEVEL OF GLYCINE SOJA

 FIG.1: CHROMATOGRAM OF HYDROLYZED EXTRACT OF GLYCINE MAX

FIG.2: CHROMATOGRAM OF HYDROLYZED EXTRACT OF GLYCINE SOJA

FIG.3: CHROMATOGRAM OF GENISTEIN STANDARD

CONCLUSION: This research show that the level of genistein as isoflavone aglycone of hydrolyzed extract of Glycine maxis 0.5% (w/w) and Glycine sojais 1.1%. The amount of genistein lower than fermented result of soybean by probiotic bacteria. In conclusion enzymatic method more effective than chemical method, however enzymatic need higher cost than chemical method.

ACKNOWLEDGMENTS: This study was carried out with the support of various parties, especially those to the Chairman STIKES Mandala Waluya Kendari (H.M Idrus, SKM, M. Kes), Dean of Pharmacy Faculty Universitas Muslim Indonesia (Rachmat Kosman, S.Si., M. Kes., Apt) and Head of the Laboratory of Pharmaceutical Chemistry Faculty of Pharmacy Universitas Muslim Indonesia Makassar (Zainal Abidin, M.Pharm., Apt).

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    How to cite this article:

    Mfawwaz, Akbar N, Pratama M, Saleh A and Mbaits: High Performance Liquid Chromatographic Analysis of Isoflavones Aglycone in Indonesian Soybean. Int J Pharm Sci Res 2016; 7(10): 4230-33.doi: 10.13040/IJPSR.0975-8232.7(10).4230-33.

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