Pérez L Yohani, Menéndez Roberto, Má Rosa, González Rosa M
Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba.
Curr Ther Res Clin Exp. 2006 Nov;67(6):396-405. doi: 10.1016/j.curtheres.2006.12.004.
D-004, a lipid extract of the fruit of the Cuban royal palm (Roystonea regia), has been found to reduce prostatic hyperplasia (PH) induced with testosterone (T), but not PH induced with dihydrotestosterone (DHT), in rodents, suggesting the inhibition of prostate 5α-reductase activity.
The aims of this study were to assess whether D-004 inhibits prostate 5α-reductase activity in vitro and to examine the effects of D-004 on enzyme kinetics.
This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Soluble rat prostate preparations were used as the source of 5α-reductase, and ((3)H)-DHT production was measured to determine prostate 5α-reductase activity. Cell-free rat prostate homogenates were pre-incubated with carboxymethyl cellulose 2% alone (control tubes) or D-004 (0.24-125 μg/mL) suspended in the vehicle (treated tubes) for 10 minutes prior to adding the labeled substrate ((3)H)-T Once the reaction was stopped, sterols were extracted with chloroform and aliquots were applied on silica gel plates developed in benzene-acetone (4:1, v/v). Areas containing DHT were scraped and radioactivity was counted. The median inhibitory concentration (IC50) was determined by measuring the conversion of T to DHT The apparent Michaelis-Menten constant (Km) and Vmax values before and after adding D-004 were determined in kinetic studies using labeled T (0.5-25 μmol/L).
Compared with controls, D-004 significantly and dose-dependently inhibited the enzymatic reaction at doses of 1.95 to 125.0 μg/mL) (all, P < 0.05). The IC50 of D-004 required to inhibit 5a-reductase activity was 2.25 μg/mL. Enzyme inhibition was noncompetitive, since D-004 lowered the Vmax from 15.3 to 10.0 nmol DHT/min · mg(-1) protein, while the Km (4.54 μmol/L) was almost unaffected.
D-004 dose-dependently and noncompetitively inhibited in vitro 5α-reductase activity in soluble fractions of rat prostate. Although the extent of the maximal inhibition was high and the value of IC50 was low, the relevance of such inhibition requires further study in vivo.
D - 004是古巴王棕（大王椰子，Roystonea regia）果实的脂质提取物，已发现其可减轻啮齿动物中由睾酮（T）诱导的前列腺增生（PH），但不能减轻由二氢睾酮（DHT）诱导的PH，提示其对前列腺5α - 还原酶活性有抑制作用。
本研究旨在评估D - 004在体外是否抑制前列腺5α - 还原酶活性，并研究D - 004对酶动力学的影响。
本实验研究在古巴哈瓦那国家科学研究中心天然产物中心药理系进行。使用可溶性大鼠前列腺制剂作为5α - 还原酶的来源，通过测量（³H）- DHT的生成来确定前列腺5α - 还原酶活性。将无细胞大鼠前列腺匀浆分别与单独的2%羧甲基纤维素（对照管）或悬浮于溶媒中的D - 004（0.24 - 125μg/mL）（处理管）预孵育10分钟，然后加入标记底物（³H）- T。反应终止后，用氯仿提取甾醇，并将等分试样点样于以苯 - 丙酮（4:1，v/v）展开的硅胶板上。刮下含有DHT的区域并计数放射性。通过测量T向DHT的转化来确定半数抑制浓度（IC50）。在使用标记T（0.5 - 25μmol/L）的动力学研究中，测定加入D - 004前后的表观米氏常数（Km）和最大反应速度（Vmax）值。
与对照组相比，D - 004在1.95至125.0μg/mL的剂量下显著且呈剂量依赖性地抑制酶促反应（所有P < 0.05）。抑制5α - 还原酶活性所需的D - 004的IC50为2.25μg/mL。酶抑制是非竞争性的，因为D - 004将Vmax从15.3降低至10.0 nmol DHT/min·mg⁻¹蛋白，而Km（4.54μmol/L）几乎未受影响。
D - 004在体外剂量依赖性且非竞争性地抑制大鼠前列腺可溶性组分中的5α - 还原酶活性。尽管最大抑制程度较高且IC50值较低，但这种抑制作用在体内的相关性仍需进一步研究。
