| Androgenic alopecia is an androgen dependent polygenic genetic disease,which is the most common alopecia disease in clinic.Modern medicine considers that the increase of androgen level and type ? 5?-reductase activity is the key factors leading to androgen induced alopecia.To reduce androgen level by inhibiting type ? 5?-reductase has become an important method to treat androgenic alopecia.Finasteride and other synthetic 5?-reductase inhibitors are often used in the treatment of androgenic alopecia,which has a significant effect,but has side effects such as erectile dysfunction and testicular pain.Similarly,topical minoxidil has a good effect,but it requires long-term medication to maintain it,which will also cause adverse reactions such as increased heart rate,water and sodium retention,and hirsutism.Therefore,it has great potential and broad prospects to find new green and efficient 5?-reductase inhibitors from traditional Chinese medicine and develop new hair products with qualitative basis and clear mechanism.The main purposes of this study are as follows:?1?Based on the experience of traditional Chinese medicine in the treatment of androgenic alopecia,combined with the understanding of modern medicine on androgenic alopecia,taking type ? 5?-reductase as the treatment target,the extract of traditional Chinese medicine with better inhibitory activity was screened from traditional Chinese medicine by establishing a stereoscopic enzyme activity measurement system.?2?Using molecular docking technology to find potential type ? 5?-reductase inhibitions from the chemical components of traditional Chinese medicine.And to verify their activity by in vitro enzyme activity assay system.?3?To further study the effect of type ? 5?-reductase inhibitors on hair growth and hormone level in vivo of androgenic alopecia model mice through animal experiments,which provided valuable theoretical reference for further study of traditional Chinese medicine in the prevention and treatment of hair loss drugs and application in hair products.Methods?1?Establishment of in vitro enzyme activity determination system:The reaction system was composed of type ? 5?-reductase,that was extracted from the prostate and epididymis of male rats,substrate testosterone and hydrogen donor?NADPH?.The substrate concentration,enzyme concentration,NADPH concentration,reaction temperature and time were further optimized.The activity of type ? 5?-reductase was determined by the change of the content of testosterone determined by HPLC before and after the reaction system.The system was validated by finasteride.?2?Screening of inhibitory activity of Chinese herbal extracts on type ?5?-reductase:The effects of water and alcohol extracts?40 samples in total?from 20 kinds of traditional Chinese medicine selected from classical prescriptions,literature reports and clinical experience on the activity of type ? 5?-reductase were determined by using the established in vitro enzyme activity determination system,and the extracts with better inhibitory activity were screened out.?3?Screening of the inhibitory activity of the chemical components of traditional Chinese medicine on type ? 5?-reductase:On the basis of previous experiments,26 kinds of chemical composition were selected from the extracts of traditional Chinese medicine with better inhibitory activity of type ? 5?-reductase,and the type ? 5?-reductase was used as biological target.Based on the docking result of finasteride,to find out the compounds with potential inhibitory activity,the compounds with strong binding ability to ? type 5?-reductase were selected by molecular docking technology and analyzed by molecular dynamics simulation.Finally,the bioactivity of the compounds was verified by the in vitro enzyme activity determination system.?4?Hair growth promoting experiment of ethanol extract of Ligustri Lucidi Fructus:Androgenic alopecia model C57BL/6 mice was induced by depilation cream and testosterone propionate.Hair growth promoting experiment of ethanol extract of Ligustri Lucidi Fructus:Androgenic alopecia model C57BL/6 mice was induced by depilation cream and testosterone propionate.Taking minoxidil as a positive control group,the low,middle and high dose group?1 mg/EAE,2 mg/EAE,4 mg/EAE?of Ligustrum lucidum seed ethanol extract was set.After 30 minutes of subcutaneous injection of testosterone propionate every day,the corresponding drugs were applied every day for 35 consecutive days and observed the changes of skin color and hair growth in depilated area of mice in each group.The hair growth score was made every 7 days.The shape and number of hair follicles were observed by HE staining to promote the hair development of the ethanol extract of Ligustri Lucidi Fructus.In order to inquire into the effect of ethanol extract of Ligustri Lucidi Fructus on the hormone level of androgenic alopecia model mice,the contents of testosterone?T?,dihydrotestosterone?DHT?and estradiol?E2?in skin and serum were detected by ELISA kit.Results?1?Establishment of in vitro enzyme activity determination system:When the concentrations of substrate T,type ? 5?-reductase and NADPH were 0.7 mmol/L,900?g/m L and 1 mg/mL,respectively,the activity of 5type ? 5?-reductase was the highest when it incubated at 37°C for 60 min.The inhibition of 5?R2 by finasteride was dose-dependent.When the concentration of finasteride was 1?mol/L,the inhibition rate was83.21±1.84%and IC50 was 242±2.5nmol/L.?2?Screening of inhibitory activity of Chinese herbal extracts on type ?5?-reductase:40 extracts of traditional Chinese medicine had inhibitory activity on type ? 5?-reductase to some extent?P<0.05?when the concentration is 1mg/mL.Among them,9 extracts with inhibitory rate of type ? 5?-reductase over 60%were ethanol extract of Ligustrum lucidum?81.46±4.04%?,ethanol extract of Platycladus orientalis leaf?77.19±4.15%?,water extract of Ophiopogon japonicus?67.25±5.11%?,water extract of Carthamus tinctorious?64.85±3.37%?,water extract of Dortex dictamni?63.32±1.49%?,ethanol extract of Carthamus tinctorious?62.85±3.45%?,water extract of Garden Balsam Stem?62.36±1.36%?,ethanol extract of Sophora flavescens?61.95±1.76%?,water extract of Ligustrum lucidum?60.45±2.90%?.?3?Screening of the inhibitory activity of the chemical components of traditional Chinese medicine on type ? 5?-reductase:The results of molecular docking showed that 26 chemical components had different binding energy with type ? 5?-reductase.Among them the docking results of ligustroflavone,safflower yellow and hinokiflavone were better than those of other traditional Chinese medicine source chemical components.The results of molecular dynamics simulation showed that the binding of the three compounds with type ? 5?-reductase tended to be stable within 20ns.RMSD values were all kept less than 0.2 nm,and amino acids with less RMSF fluctuation were consistent with key amino acids.These all indicated the rationality and stability of docking of the three compounds with type ?5?-reductase.Ligustroflavone,safflower yellow and hinokiflavone have a certain inhibitory activity on type ? 5?-reductase with the IC50 value of 45.27?mol/L,62.65?mol/L,and 191.46?mol/L,respectively.?4?Hair growth promoting experiment of ethanol extract of Ligustri Lucidi Fructus:The ethanol extract of Ligustrum lucidum has significant effect on hair growth that the skin darkening time,hair growth time,hair quality and the number of hair follicles in the depilated area of mice in each dose group increased significantly?P<0.05?compared with the model group.Compared with the model group,the T content in serum of Ligustrum lucidum in low dose group was significantly lower?P<0.05?.The T and DHT content in serum of middle dose group and high dose group were significantly lower?P<0.05?.The E2 content in serum was not significantly different?P>0.05?.The T and DHT content in skin of Ligustrum lucidum in all dose groups were significantly lower?P<0.05?and the E2 content was not significantly difference?P>0.05?.The ethanol extract of Ligustrum lucidum can significantly reduce the androgen level in serum and skin tissue,especially the DHT level in skin tissue.Conclusion?1?The system established in this study is simple,stable and reproducible,which can be used for the screening of type ? 5?-reductase inhibitors.?2?Through the determination of the inhibitory activity of 40 extracts,Chinese herbal medicines with better inhibitory activity of type ?5?-reductase were screened,including ethanol extract of Ligustrum lucidum,ethanol extract of Platycladus orientalis leaf,water extract of Ophiopogon japonicus,water extract of Carthamus tinctorious,water extract of Dortex dictamni,ethanol extract of Carthamus tinctorious,water extract of Garden Balsam Stem,ethanol extract of Sophora flavescens,water extract of Ligustrum lucidum have the potential to be used as type ? 5?-reductase inhibitors of traditional Chinese medicine.?3?Among the 26 selected chemical components,ligustroflavone,safflower yellow and hinokiflavone have better type ? 5?-reductase inhibitory activity,and have the potential to be used as a new type ?5?-reductase inhibitor in the research and development of androgenic alopecia drugs.?4?The ethanol extract of Ligustrum lucidum,an inhibitor of type ?5?-reductase,can effectively improve the phenomenon of hair reduction and slow growth caused by androgen.One of the main mechanisms of promoting hair growth is to reduce the content of dihydrotestosterone in skin tissue by inhibiting the activity of type ? 5?-reductase. |
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