Study On The Chemical Constituents Of Nuts Oil From Prunus Mira Koehne And The Mechanism Of Promoting Hair Growth

Rosaceae Prunus plant of Prunus mira Koehne is also named(Kangbu,Gamu,Wokan,Kangkang),or wild peach.It is a rare "living fossil group" of peach germplasm resources at home and abroad.It grows up in Sichuan,Yunnan,and Tibet.Because of the kernel of medicine and relative smoothness of nut shell,it is called peach kernel.In the Standard of Chinese Mdicinal Materials(1992 edition),its functions were recorded the same as in TCM,such as activating blood circulation,removing blood stasis,moistening intestines and relaxing the bowels,etc.However,Jing-Zhu Materia Medica(1745 years),a classic Tibetan medicine work,recorded "oil smear of seed extract for hair,eyebrows,beard and other abscission diseases",but the clinical experience has not been promoted.Objective:1.Analyze fat-soluble components of nuts oil from P.mira to discover specific chemical components.2.Mensurate chemical composition content of nuts oil from P.mira to improve quality control level.3.Reveal the dose-effect relationships of nuts oil from P.mira to promote hair regrowth and mechanism.Methods:1.The nuts oil from P.mira was extracted by hot pressing method and then was isolated and identified by GC-MS,and meanwhile the relative content of various compounds was determined.Afterward,The results were analyzed by cluster analysis and principal component analysis.2.The content of composition in nuts oil from P.mira in a total of 14 batches was determined by HPLC method with the column temperature at 38℃,the flow rate of 1.0 m L·min-1,the detection wavelength at 210 nm,and the mobile phase consisting of methanol、acetonitrile and water as isometric elution.3.Set the eight doses of nuts oil from P.mira to study dose-effect relationship with two models including Na2 S and depilatory paste for 7days on end,in the way of new born hair growth status score,length and weight indexes.Explore preliminary the effective dose range of nuts oil from P.mira to promote hair growth.4.With Na2 S leading to hair removal model in C57BL/6 mice,daub the alopecia part of the mice with nuts oil from P.mira continuation 21 d,in the way of hair growth status score,length、growth rate、weight、number of hair follicles and dermis thickness indexes evaluate.By real-time quantitative PCR and Western blotting methods,the expression of Wnt10 b,GSK-3β and β-catenin m RNA or protein in skin were detected to study the mechanism of nuts oil from P.mira.Results:1.By means of GC-MS,forty-one compounds were isolated from nuts oil from P.mira,with main contents of oleic acid、β-sitosterol、trans-squalene、γ-tocopherol、Vitamin E and five endemic compounds(4-methyl nonane 、 cyclohexane,1,2,4-trimethyl-、 trans-2,4-decadienal and octacosanal 、 twenty-eight aldehyde 、2,4-decadienal).2.By means of HPLC,the precision,repeatability,stability and the sample recovery rate of the three chemical components namely α-tocopherol、Vitamin E andβ-sitosterol in nuts oil from P.mira meet the requirements with RSD value less than3%,the linear range varying from 72.00~1256.00 μg·m L-1,7.60~187.00μg·m L-1 and100.00~904.00 μg·m L-1,which shows a good linear relationship within the scope.3.The dose-effect relationships experiment results showed that,nuts oil from P.mira and diluent(15.06~60.26 mg /cm2/d)increased the growth status of new hair,extended the hair length and increased hair weight for eighth days.More importantly,the dose was proportional to the effect.But nuts oil from P.mira shows no effect in promotings hair growth with a dose of 0.47~7.53 mg medicine /cm2/d.4.C57BL/6 mice experienced a shorter period of skin blackening,on the other hand they underwent a clear growth in hair length,growth rate,weight,and number of hair follicles as well as dermis thickness.Nuts oil from P.mira diluent 2 times group obviously improved Wnt10 b and β-catenin m RNA expression,and aslo improved Gsk-3β m RNA expression in C57BL/6 mice skin.Meanwhile,the obviously raised Gsk-3β and β-catenin protein expression also improved Wnt10 b protein expression in C57BL/6 mice skin.Conclusion:1.The identification of Fat-soluble and specific chemical components of nuts oil from P.mira provides references for the quality control level research and a study on pharmacophore basis of from P.mira.2.The methods for determining content of 3 kinds of chemical composition in nuts oil from P.mira was established,which is simple,accurate and reproducible,and can be used for improving the quality control of nuts oil from P.mira.3.Nuts oil from P.mira can promote alopecia mouse model hair growth in the does of 15.06~60.26 mg medicinal materials/cm2/d.4.Nuts oil from P.mira can preferably promote hair growth in 30.13 mg medicinal materials/cm2/d.The mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway in the skin.