The Separation Of Active Constituents From Shell Of Oiltea Camellia And Its Inhibition On Benign Prostate Hyperplasia

Benign prostatic hyperplasia (BPH) is a common medical problem in nearly80%of geriatric male population severely affecting the quality of life.5a-reductase plays an important role in the development of BPH. This article is oriented by the inhibition rate of5a-reductase, studying extraction and purification methods of active constituents on inhibition of5a-reductase from shell of oiltea Camellia. Then active constituents are researched its effects on BPH through cell and animal models, aiming to provide theoretical basis for developing BPH therapeutic that is efficient and low toxicity. The main contents and results are as follows:(1) Optimized ultraviolet spectrometry method for detecting5a-reductase enzymatic activity, which monitored content variation of testosterone (T) instead of NADPH. The detection system was that T20μmol/L, NADPH40μmol/L, enzyme extract260μg in2mL solution,37℃for4min. The results were similar to high performance liquid chromatography (HPLC) method.(2) Established the process of extracting, purifying, refining and preparation for active constituents from shell of oiltea camellia.i) Extraction:Chosen50%ethyl alcohol (v/v) as the extraction solvent. The inhibition rate of ethyl alcohol extraction on Sa-reductase was36.31%±2.76%and its yield was11.96%.ii) Purification:Chosen40%ethyl alcohol elution (OCE) of AB-8macroporous resin as the column chromatography condition. The inhibition rate of OCE on5a-reductase was57.79%±1.67%and its yield was26.5%.iii) Refining:Selecting developing agent and color reagent as n-butyl alcohol-methanol-water-glacial acetic acid (3:2:2:1) and0.5%(w/v) FeCl3alcoholic solution, OCE were clearly divided into seven fractions, Frl-Fr7, whose Rf was0.85～0.0375through polyamide chromatography method, instead of traditional silica gel chromatography. The inhibition rates of Fr6, Fr5, Fr4on5a-reductase were85.38%±3.71%,76.28%±1.37%and73.01%±2.93%, which were significantly higher than OCE (p<0.01). Their yields were3.79%,2.10%and0.64%. The characteristic polyphenol components of oiltea Camellia extracts were identified as gallic acid (GA), ellagic acid (EA) and3-O-methylellagic acid4’-O-β-D-glucopyranoside (MEAG). Confirmed these polyphenol components were not the key compounds with the inhibiting effect on5a-reductase.iv) Preparation:Isolated Fr5and got active monomers F0and F0’ through semi-preparative HPLC. Their inhibition rates on5a-reductase were76.47%±0.02%and92.08%±0.18%. Their yields were5.17%and1.19%. Inferred their molecular weights were449and335through LC-MS analysis.(3) Using MTT and Annexin V/PI double staining detected active constituents’ effect on BPH-1cell proliferation and apoptosis. At the concentration of50μg/mL, The sequence of antiproliferative effect was Fr6> finasteride> Fr5≈F0> Fr4≈Fr3>OCE>Fr2. Fr6had the strongest inhibitory activity (inhibition rate50.0%±0.7%). Fr5and FO took the second places (inhibition rate43.7%±2.1%and41.4%±2.1%). The inhibiting effect was time and dose-dependent. Apoptosis-promoting effect of FO was dose-dependent. At the concentration of25μg/mL, apoptosis rate induced by Fr6was12.3%±3.8%, which was close to the result of finasteride, suggesting great research and development value.(4) The results of active constituents assessment were similar between BPH-1cell proliferation and5α-reductase enzymatic activity detection model, which verified active constituents’effectiveness on BPH treatment as the5α-reductase inhibitor.(5) Ethyl alcohol extraction could relieve BPH symptom of rats model induced by testosterone propionate. The extraction could significantly reduce prostate and ventral lobe index (p<0.05), alter hyperplasia tissue morphology, suppress the activity of5α-reductase and prostatic acid phosphatase in the prostate (p<0.01) and improve antioxidant level in vivo.In summary, active constituents from shell of oiltea Camellia can prevent and cure BPH through reducing5a-reductase activity, inhibiting cell proliferation, inducing cell apoptosis and scavenging free radicals.