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AIM: Tranditional methods of screening drugs for benign prostatic hyperplasia(BPH) requires senile male animals such as dogs or rats.It consumes a long time to get the results.Over-expression of type Ⅱ5α-reductase in prastate induces BPH.A fast and efficient screening model of type Ⅱ5α-reductase inhibitors for BPH was set up in this paper.METHODS: Microsomes were extracted from male Sprague-Dawley rat livers by gradient centrifugation.Type Ⅱ5α-reductase enzyme-catalyzed reaction was assayed by UV-spectrophotometry using testosterone as a substrate and NADPH as hydrogen donor.The change of enzymatic activity was recorded with a NADPH wavelength of 340 nm by subtracted descending velocity of the control(without 5α-reductase).Effects at different conditions(temperatures,pH,enzyme and testosterone concentrations) on 5α-reductase were assayed.RESULTS: The suitable condition of type Ⅱ5α-reductase reaction was defined as concentration of 109.05 mg protein/L enzyme(pH 6.00) with 2 μmol/L testosterone at 37 ℃.Michaelis’ constant of type Ⅱ5α-reductase was 0.6 μmol/L.Finasteride,a new drug for BPH,significantly inhibited activity of type Ⅱ5α-reductase.IC50 of finasteride was 64.1 nmol/L.As solvent of drugs,concentration of ethanol below 1.1% did not inhibite enzymatic activity(P>0.05).Concentration of ethanol above 1.6 % could obviously suppress enzymatic activity(P<0.01).Daytime difference within five days had no significant difference(P>0.05).CONCLUSION: A handy and fast screening method for type Ⅱ5α-reductase inhibitors has been set up using UV-spectrophotometry.It may be used to screening drugs for BPH treatment.
AIM: Tranditional methods of screening drugs for benign prostatic hyperplasia (BPH) requires senile male animals such as dogs or rats. It consumes a long time to get the results. Over-expression of type II5α-reductase in prastate induces BPH. A fast and efficient screening model of type II5α-reductase inhibitors for BPH was set up in this paper. METHODS: Microsomes were extracted from male Sprague-Dawley rat livers by gradient centrifugation. Type II5α-reductase enzyme-catalyzed reaction was assayed by UV-spectrophotometry using testosterone as a substrate and NADPH as hydrogen donor. The change of enzymatic activity was recorded with a NADPH wavelength of 340 nm by subtracted descending velocity of the control (without 5α-reductase). Effects at different conditions (temperatures, pH, enzyme and testosterone concentrations ) on 5α-reductase were assayed .RESULTS: The appropriate condition of type Ⅱ 5a-reductase reaction was defined as concentration of 109.05 mg protein / L enzyme (pH 6.00) with 2 μmol / L testosterone at 37 ° C. Michaelis’ constant of type Ⅱ 5α-reductase was 0.6 μmol / L.Finasteride, a new drug for BPH, significantly inhibited activity of type II5α-reductase. IC50 of finasteride was 64.1 nmol / L.As solvent of drugs, concentration of ethanol below 1.1% did not inhibite enzymatic activity (P> 0.05) .Concentration of ethanol above 1.6% could obviously suppress enzymatic activity (P <0.01) .Daytime difference within five days had no significant difference (P> 0.05). CONCLUSION: A handy and fast screening method for type II5α-reductase inhibitors has been set up using UV-specific spectrometry. It may be used to screen for drugs for BPH treatment.