Effect Of5α-reductase Inhibitors Purified From Shell Of Oiltea Camellia On BPH-1Cells

Benign prostatic hyperplasia (BPH) is a popular disease among old men, which has bad influence on patients’ daily lives.The pathogenesis of BPH is very complicated and5a-reductase plays a very important role during BPH. Our study focused on the shell of oiltea Camellia, and we purified active components from the shell of oiltea Camellia with macroporous resin, polyamide resin and pre-HPLC methods step by step, which was targeted by the inhibition rate on5a-reductase. We detected the effect of active components on BPH-1cells’ proliferation and apoptosis with MTT and Annexin V/PI methods. Furthermore, we detedted its influence on the expression of Bcl-2,Bax and hEGF by qRT-PCR to explore the molecular mechanism.Our main results are as follows:(1) Optimized the HPLC method for detecting the inhibition rate on5a-reductase. Under the optimized1.5mL reaction system, we determined the IC50of finasteride on5α-reductase, which was0.164μg/mL. It was similar to other reports, which meant the optimized reaction system was stable and reliable.(2) Extracted active components from the shell of oiltea Camellia by ultrasonic wave method. The yield of50%ethanol extracts was6.225%.(3) Recovery rate of AB-8macroporous resin purification was89.9%. The yield of40%ethanol phase (OCE) was29.4%and its inhibition rate on5α-reductase was45.31%±0.44%.Got11separate parts by polyamide chromatography, and the major parts were Fr2,Fr4,Fr6and Fr8, which had yield of1.30%,1.51%,1.55%and1.57%. The5a-reductase inhibition rate of Fr8was57.43%±0.39%, which was significantly higher than OCE (P<0.01).Purified Fr8by pre-HPLC method and got6peaks named F1-F6. Their yield were5.93%,3.11%,4.78%,1.09%,6.70%and5.71%. Then purified the key active component F3and got two molecules P1and P2, which both had the same molecular weight as634. And they had similar molecular structure and they were easy to transmute to each other.(4) MTT method showed Fr fractions could inhibit BPH-1cells proliferation, of which100μg/mL Fr8had the top inhibition rate of39.43%±5.77%,which was comparable to positive control feinasteride.F2,F3and F5which were purified from Fr8by pre-HPLC had arisen their inhibition rates on BPH-1cells proliferation signifigcantly (P<0.01). And the inhibition rate was dose-dependent. Their IC50were66.02μg/mL,56.30μg/mL and73.06μg/mL.(5) Annexin V/PI experiment showed F2,F3and F5could induce BPH-1cells apoptosis.100μg/mL F2had the apoptosis rate of12.67%±0.59%which was comparable to positive control feinasteride.(6) qRT-PCR showed F3could regulate up the expression of Bcl-2,Bax and hEGF significantly(P<0.01). And the expression level of F3was significantly high than finasteride (P<0.05). The up-regulation of Bax was more than Bcl-2, which suggested the rise of Bax/Bcl-2maight be the cause of its effect on BPH-1cells apoptosis.F3had the similar effect with finasteride and F3was more effective, which showed F3deserved further research.In summary,5α-reductase inhibitors purified from shell of oiltea Camellia can inhibit BPH-1cells proliferation and induce the cells apoptosis, which may be caused by regulating up the expression of Bcl-2,Bax and hEGF.5α-reductase inhibitors from shell of oiltea Camellia, especially F3which are composed of two similar molecules, has good potential to apply into drugs for BPH.