DEVELOPMENT OF SPHEROID PELLET OF ISOFLAVONE AGLYCONE FROM TEMPEH EXTRACT AND ACTIVITY EVALUATION IN A RAT POLYCYSTIC OVARY SYNDROME (PCOS) MODEL

• Main Author: Raihanah, Cut
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/87281
• Institution: Institut Teknologi Bandung

Polycystic ovary syndrome (PCOS) is a hormonal disorder in productive age women, with the main symptoms being irregular menstrual cycles, excess androgen hormone levels, and polycystic ovary morphology. Studies have demonstrated the ability of isoflavones from soybeans to alleviate the symptoms of PCOS. Isoflavone aglycone is a form of isoflavone that has a better biological activity as a phytoestrogen than its glycoside form. Isoflavone aglycone is abundant in tempeh as a result of the fermentation process. Although it has biological activity, isoflavone aglycone is poorly soluble in water, so it has low bioavailability and reduces its effects. Making a pellet spheroid form is one method to increase the solubility of isoflavone aglycone. The purpose of this study was to design and develop a spheroid pellet form of isoflavone aglycone from a tempeh extract, evaluate its activity against PCOS in vivo, and predict its mechanism of action through an in silico approach. This study began with the analysis of isoflavone aglycone levels of three soybean varieties (Detap 1, Devon 1, and Argomulyo). The results of the analysis of daidzein and genistein levels by HPLC in the Detap 1, Devon 1, and Argomulyo varieties were 53.02±3.16 mg/100 g and 6.05±0.20 mg/100 g; 45.67±12.07 mg/100 g and 9.93±1.50 mg/100 g; 40.48±1.39 mg/100 g and 6.55±0.48 mg/100 g, respectively. The statistical analysis of each variety showed no significant difference between the daidzein levels of Detap 1 and Devon 1, while there was a significant difference between the genistein levels of Detap 1 and Devon 1 (P<0.05) so that the Devon 1 variety soybean had the highest daidzein and genistein levels compared to the other two varieties. Based on the previous experimental results, tempeh was made from Devon 1 soybeans with a fermentation time of 61 hours. However, the isoflavone aglycone content of Devon 1 tempeh after 61 hours of fermentation was still lower than the Devon 1 soybean extract hydrolyzed with HCl, so an effort was made to increase the isoflavone aglycone content of Devon 1 tempeh by heating. The results of daidzein and genistein levels in Devon 1 tempeh with heating at temperatures of 40 C, 50 °C and 60 °C were 605.39±3.00 mg/100 g and 153.82±0.65 mg/100 g; 529.82±10.59 mg/100 g and 128.56±2.64 mg/100 g; 625.42±13.71 mg/100 g and 149.30±2.67 mg/100 g, respectively, where there was a significant increase in daidzein and genistein levels (P<0.05) under all heating temperatures compared to Devon 1 tempeh without heating and Devon 1 soybean extract hydrolyzed with HCl. Heating the Devon 1 tempeh sample at a temperature of 50 °C resulted in a significant decrease in daidzein and genistein levels compared to a temperature of 40 °C, while at a temperature of 60 °C the daidzein and genistein levels increased again but were not significantly different from heating at a temperature of 40 °C. Therefore, the heating method at a temperature of 40 °C was chosen to increase the daidzein and genistein levels of Devon 1 tempeh after 61 hours of fermentation followed by defatization with n-hexane and extraction using ethyl acetate solvent. The daidzein and genistein levels of the tempeh extract were 607.90±15.58 mg/100 g and 220.98±8.22 mg/100 g, respectively. The results of the in vivo activity test of tempeh extracts on PCOS showed that administration of this preparation at a dose of 250 mg/kgbw for 14 days in PCOSinduced rat was able to restore the persistence of the diestrus phase to a normal cycle better than the metformin group. In addition, the tempeh extract at a dose of 250 mg/kgbw also reduced serum testosterone levels and percentage of body weight gain that were 24.26±8.47 nmol/L dan 21.12±3.91%, respectively. The best formula in formulating tempeh extract into spheroid pellet form included active ingredients (tempeh extract 20%) and excipients consisting of aerosil (10%), Ac-Di-Sol (3%), avicel PH 101 (16%) and water (51%). Based on the evaluation, the resulting tempeh extract spheroid pellet fulfilled the requirements in terms of their spherical shape, drying shrinkage of less than 5%, particle size of 0.5-2.0 mm and short disintegration time. The results of the activity test after formulation showed that the tempeh extract spheroid pellet form showed better activity than the tempeh extract in terms of reducing serum testosterone levels as 23.79±4.87 nmol/L and improving the estrus cycle. Molecular docking results showed that both daidzein and genistein have affinity for amino acid residues responsible for androgen receptor inhibition, but the bonds are weak. In contrast, only genistein has a high binding energy to the amino acid residues responsible for the 17-?-HSD enzyme inhibition, so the possibility of an antiandrogenic mechanism of this isoflavone aglycone is as an androgen receptor inhibitor. Overall, the tempeh extract spheroid pellet form has the potential to be further developed as an alternative treatment for PCOS by exploring the possibility of its mechanism from other pathways, such as balancing the hormones LH, FSH, progesterone, and estrogen.