Effect Of Cistanche Tubulosa Ethanol Extract On Sex Hormone Regulation In Male Rats And CYP450Enzyme Mediated Mechanism Study

Cistanche tubulosa (Schenk) R. Wight (C. tubulosa) is documented in the ChinesePharmacopoeia since2005, the effect of C. tubulosa on sex hormone levels and mechanism isstill unclear. The present study aimed to investigate the direct effects of C. tubulosa extract(CTE) on levels of progesterone and testosterone, and on expression of two steroidogenicenzymes (CYP11A1and CYP17A1), and the metabolic enzyme (CYP3A4) in male rats. Thethree enzymes belong to the cytochrome P450family, they are located on inner-membranesystem, so the method to prepare the microsomes should be established first. This studycontains three parts:1. Establishment of the method to prepare microsomesThe method to prepare microsomes was established using liver as it contains abundant ofCYP450s. The total protein content of P450was0.55nmol/mg by calculation, within thenormal range (0.4-1.0nmol/mg). In differential spectrophotometry assay, the microsomes hasspecial peak at450nm. In cocktail probe drug method, the activity of CYP2E1was evaluated.All of the results showed that the prepared microsomes were active, demonstrating themethod was established successfully.2. Effect of Cistanche tubulosa on sex hormone levels and its mechanismThe stem of Cistanche was extracted by water extracting method, content ofphenylethanoid glycosides (PhGs) was detected and echinacoside was used as a standard. Therats were randomly divided into four groups:1) control group (treated with normal saline),2)CTE low-,3) middle-and4) high-dose groups (treated with0.2,0.4and0.8g/kg of CTE,respectively). The materials were fed to the rats by intragastric administration for20days.The content of PhGs in CTE was41.52%by calculation. Compared to the control group,middle-and high-dose of CTE caused increase (P<0.001) of progesterone level (1.9fold and1.7fold). Testosterone levels of middle-and high-dose groups exhibited increase of1.5foldand1.3fold, respectively. CYP11A1and CYP17A1levels of low-, middle-and high-dose ofCTE were increased. Besides, expression of CYP3A4were significantly increased by5.3,5.5,and7.0fold.3. Totoxicity effect of Cistanche tubulosa extractTo detect the totoxicity of CTE on rat, liver index, alanine aminotransferase (ALT) level, hepatic and testicular histopathology were assessed. The liver index of the high-dose CTEtreatment group was decreased and the serum ALT was increased compared to the controlgroup. High-dose of CTE caused mild hepatic edema. The testicular structure of each groupwas normal.This study established the CaCl2precipitation method to prepare microsomes, activity ofthe obtained microsome was good enough to do the following assay. Effect of CTE on thelevels of progesterone and testosterone, and the expression of CYP11A1, CYP17A1andCYP3A4were also evaluated. These results provide theoretical basis for the Cistanche relatednew drugs development.