Effects of Methanol Extract of Adlay Bran and Adlay Hull on Progesterone Release in Rat Ovarian Granulosa Cells

Abstract

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has long been used as traditional Chinese medicine for the dysfunction of endocrine system and inflammation condition. However, the effect of adlay seed on the endocrine system has not yet been reported. In the present study, the effects and the mechanisms of methanolic extract of adlay bran (ABM) on progesterone synthesis in rat granulosa cell were studied. ABM was further partitioned with different solvent including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions named ABM-Wa (water fraction), ABM-Bu (1-butanol fraction), ABM-EA (ethyl acetate fraction) and ABM-Hex (n-hexane fraction) were obtained. ABM-Bu was further fractionated using Diaion HP-20 resin column chromatography with gradient elution. Granulosa cells were prepared from pregnant mare serum gonadotropin-primed immature female rats and challenged with different reagents including human chorionic gonadotropin (hCG, 0.5 IU/ml), forskolin (10 μM), 8-bromo-adenosine-3’,5’-cyclic monophosphate (8-Br-cAMP, 1 mM), A23187 (10 μM), phorbol 12-myristate 13-acetate (PMA, 0.01 μM), 25-OH-cholesterol (0.1-10 μM) and pregnenolone (0.1-10 μM) in the presence or absence of ABM-Bu (100 μg/ml). The functions of steroidogenic enzyme including protein expression of the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) protein were investigated. Both P450scc and StAR mRNA expressions have also been explored. We found that ABM decreased progesterone production via an inhibition on (1) the cAMP-PKA Uand PKCU signal transduction pathway, (2) P450scc and 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme activity, (3) P450scc and StAR protein and mRNA expressions, and (4) the phosphorylation of ERK1/2 in rat granulosa cells.