5α-还原酶抑制剂体外评价体系的建立及其应用

The establishment and application of an in vitro evaluation system for 5α-reductase inhibitors

建立了用于筛选5α-还原酶抑制剂的体外评价体系,采用SD大鼠肝脏制备5α-还原酶,以度他雄胺作为阳性对照,使用高效液相色谱法测定反应前后体系中睾酮含量的变化,计算相对抑制率以表观样品对5α-还原酶的抑制作用。方法学研究结果表明,反应液中其他组分对睾酮的测定无干扰,睾酮在0.059~9.860μg/mL范围内线性关系良好,相关系数r=0.9997,定量限(S/N≥10)为0.059μg/mL;精密度试验RSD为0.3%,重复性试验RSD为0.7%。采用本评价体系对多种活性成分进行测定的结果显示,包括透明质酸锌在内的多种水溶性锌盐对5α-还原酶具有较强的抑制作用。该体系可用于筛选抑制5α-还原酶的活性成分,为开发控油、抗痤疮功效的化妆品提供依据。

5α-reductase(5AR)is a microsomal enzyme that relies on reducing coenzymeⅡ(NADPH)as a hydrogen donor to irreversibly convert testosterone to dihydrotestosterone(DHT).There are three isoenzymes in the alpha-reductase family,namelyⅠ(5AR1),Ⅱ(5AR2)andⅢ(5AR3).Two distinct isozymes are found in mouse,rat,monkey,and human:5AR1 and 5AR2.In human,5AR1 is predominant in the sebaceous glands of most regions of skin,including scalp,and liver.5AR1 is responsible for approximately one-third of the circulating DHT.5AR2 isozyme is primarily found in prostate,seminal vesicles,epididymites,hair follicles as well as liver,and is responsible for two-thirds of the circulating DHT.5AR1 in sebaceous gland cells plays a certain role in local androgen metabolism,stimulation of excessive sebum secretion and the occurrence of acne.The high level of DHT is easy to cause acne and other skin problems.According to the above reaction principles,an in vitro model was established to screen 5AR inhibitors.The reaction system was divided into four groups:Blank control,Negative control,Positive control and Sample group.5AR was prepared from the liver of SD rats.The content of testosterone was determined by HPLC method using Dutastamide,which had strong inhibition on both 5AR1 and 5AR2,as the positive control.Specificity test shows that other components have no interference on the determination of testosterone,and the method has good specificity.Correlation coefficient r is 0.9997,testosterone has a good linear relationship in the range of 0.059-9.860μg/mL,and the limit of quantitation(S/N≥10)is 0.059μg/mL.RSDs of the precision test are 0.3%,and the precision is good.RSDs of the repeatability test are 0.7%,indicating that the method has good repeatability.Several samples were screened using this screening model,and several samples such as zinc hyaluronate shows strong inhibition of 5AR.This model can effectively screen 5AR inhibitors,and provide reference for the development of oil-control,anti-acne and other products.Compared with other existing androgen antagonists,5AR inhibitor selectively blocks DHT synthesis while not affecting the normal level and physiological function of testosterone,and inhibits 5AR as an important method for the treatment of benign prostatic hyperplasia,male alopecia and other androgen-dependent diseases.With the inhibition of 5AR activity as the target,steroid and non-steroid 5AR inhibitors from various sources are becoming a hot spot in drug development.