The Material Basis And Functioning Mechanism Of Pharmacodynamic Of Platycladus Orientalis Leaves In Promoting Hair Growth

Alopecia（hair loss）is one of the common clinical diseases in modern society.Although it is not a serious disease threatening human life,it brings great mental pressure and psychological burden to patients.Epidemiological investigations revealed that the prevalence rate of alopecia in China is as high as 20%in men and 6%in women.Minoxidil and finasteride are currently recognized clinical therapeutic drugs,but drug dependence and side effects have been reported.Platycladus orientalis（L.）Franco（leaves）is one of the most frequently used herbs in the external prescription of traditional Chinese medicine.Its efficacy to cure hair loss was recorded in many ancient books of traditional Chinese medicine,such as Prescriptions for Emerent Reference,Compendium of Materia Medica and Chinese Medica Herbs.Modern studies showed that both total flavonoids and volatile components of P.orientalis leaves,especially its volatile oil,have the effect of promoting hair growth.However,the material basis and mechanism of such promoting effect have not been fully revealed,which hindered clinical application of P.orientalis leaves as a potential medicine for alopecia cure.Therefore,this study used network pharmacological approach to systematically predict the material basis of the volatile oil in P.orientalis leaves and its potential targets for alopecia.The effects of the volatile oil on hair growth in C57BL/6 mice and the human hair follicle dermal papillary cells were investigated by in vivo and in vitro experiments.Finally,the mechanism of P.orientalis leaves volatile oil on promoting mouse hair growth was further explored at the protein level by proteomics approach.This provided a theoretical basis and potential clinical target for hair loss treatment of P.orientalis volatile oil.The main conclusions of this study were as follows:（1）The volatile oil of P.orientalis leaves were prepared by hydro distillation,subcritical extraction and supercritical CO₂extraction,respectively.GC-MS analysis showed that 58compounds were identified with the relative content greater than 0.5%.The supercritical CO₂extraction method had the highest extraction rate of 6.31±0.10%,followed by subcritical extraction of 3.02±0.20%,and hydro distillation had the lowest extraction of only 0.55±0.15%.The main components of the volatile oil prepared by hydro distillation wereα-pinene（24.34%）,γ-terpinene（9.24%）,β-caryophyllene（7.38%）,α-humulene（5.53%）,myrcene（5.44%）.Monoterpenes（49.5%）and sesquiterpenes（27.46%）are the core components of the volatile oil by hydro distillation.The main components of subcritical extraction were methyl sandaracopimarate（13.88%）,8（14）,ent-isopimara-8（14）,15-diene-3β-ol（9.56%）,isopimara-7,15-dien-3β-ol（7.03%）and germacrene（6.84%）.Alcohol is the main components（25.92%）.The main components of supercritical CO₂extraction were daniellicacid（14.92%）,methyl sandaracopimarate（11.09%）,germacrene（8.13%）.Acids（23.51%）and esters（20.05%）are the main component.The main components of the volatile oil obtained by the three extraction methods are significantly different,among which the chemical composition of the volatile oil prepared by hydro distillation is closest to the volatile components detected in P.orientalis leave powder.Terpenoids are the main components,which have strong skin permeability and are more suitable for external drug.（2）The material basis and the mechanism of the hair growth effect of P.orientalis leaves and the volatile oil were investigated by network pharmacology method.Nineteen potential active compounds from the 34 compounds and 72 common targets of components and alopecia were selected.Compounds such as quercetin,apercenin,kaempferol,myrictin,isopimara-7,15-dien-3β-ol and pinusolide may promote hair growth through the key targets（AKT1,IL6,VEGFA,INS and STAT3）to regulate the PI3K-Akt signaling pathway,AGE-RAGE pathway of diabetes complications,JAK-STAT signaling,EGFR tyrosine kinase inhibitor resistance and etc.（3）The mouse model of hair loss was established using the hair follicle growth cycle activity of the C57BL/6 mice during the second resting phase of hair follicle growth.The results showed that the volatile oil of P.orientalis leaves promoted mouse hair growth in a concentration growth-dependent manner among the concentration range below 15%.Comprehensive hair growth score,hair dry length and skin tissue after H&E staining showed that 10%concentration of P.orientalis leave volatile oil has a significant hair growth promoting effect.By q RT-PCR and WB experiments,the expression levels of genes includingβ-catenin,p-GSK3β,Lef-1,Gli-1,Patched and Smo,were significantly up-regulated in the dorsal skin tissues in the 10%P.orientalis volatile oil-treated group,and the corresponding protein expression increased.This indicates that volatile oil may promote mouse hair growth through Wnt/β-catenin and Shh signaling.（4）The effects of P.orientalis volatile oil on the proliferation capacity,cell cycle and hair growth related signaling were investigated by an in vitro model of human hair papillary cells.The results showed that the volatile oil significantly promoted the proliferation of human papillary cells at 50μg/m L,and enhanced the activity of alkaline phosphatase.The papillary cells in the S and G2 stages significantly accumulated.The expression ofβ-catenin,p-GSK3β,Lef-1,Gli-1,Patched and Smo were up-regulated.It is revealed that P.orientalis volatile oil may promote the proliferation of human hair papillary cells through the activation of Wnt/β-catenin and Shh/Gli-1 signaling,thus regulating the turnover of hair follicle growth period and promote hair follicle development and hair growth.（5）Label-free quantitative proteomic analysis of C57BL/6 mice treated by P.orientalis volatile oil was carried out.In total,4,677 proteins and 33,574 peptides were identified from 15 samples including control groups C₀,C₇,C₁₅and experimental groups EO₇and E0₁₅.A total number of 641 and 744 differential expression proteins were selected from the group EO₇/C₇and EO₁₅/C₁₅,respectively.These differential expression proteins were mainly localized in the cytoplasm,nucleus and secretions,as well as in the mitochondria and plasma membrane.Proteins such as Rpsa,Cct5,Eif3g,Eif2s1,Cct4,Eef1b2,Rpl22l1,Rpl38,Kars,Krt17,and Krt25 may play important roles in promoting hair growth by participating in physiological processes such as post-translational modification,protein turnover,signal transduction,and chaperones.In conclusion,the volatile oil prepared by hydro distillation is mainly terpenoids,with strong skin permeability and is more suitable for topical drugs,to stimulate PI3K-Akt,AGE-RAGE,JAK-STAT,EGFR,Wnt/β-catenin and Shh/Gli-1 signaling pathway,thus promoting hair growth.The results provide a scientific reference for the market products of P.orientalis volatile oil to treat hair loss,and also lay a foundation for the further analysis of the mechanism of P.orientalis volatile oil to promote hair growth.