Comparative Analysis Of Chemical Constituents Of Cultural And Wild Musk

Musk is a secretion with a special odar,produced in a glandular sac beneath the skin of the abdomen of the male Moschus deer,which has the function of inducing resuscitation,promoting blood circulation to remove meridian obstruction and reducing swelling and relieving pain.It is the main treatment of calenture,coma,stagnation of qi,and sudden syncope,etc..Modern studies have shown that the pharmacological activity of musk is closely related to its rich chemical types.However,the current research on its chemical composition mainly focuses on the none-polar chemical components,and lacks systematic and comprehensive analysis.In order to systemactively clarify the chemical composition of musk and establish an effective quality control system,the chemical components of musk are analyzed in a qualitative and quantitative way in this paper,and the following results are obtained:1.By using dichloromethane-methanol-water(4:2:2),the musk was extracted and the none-polar extraction and the polar extraction were obtained respectively.A wide range of qualitative and quantitative analysis was carried out on the none-polar extraction of musk by NMR,GC-MS,and UC-MS.The results showed that the main difference between cultural musk and wild musk were fatty acids and glycerides.The content of muscone in cultural musk is lower than that of wild musk.The content of steroids in cultured musk is higher than that of wild musk.The muscone content of the cultural musk is less than 2%,while it’s more than 2%in wild musk.By introducing chiral shift reagent and UFE-chiral-achiral-UFC-MS system,artificially adding muscone which dissolved in oil to increase the congtent of muscone in wild musk was proved.2.NMR and RPLC-HILIC-MS were used to analyze the chemical constituents in the polar extraction of musk.22 compounds were preliminarily identified through 1H-NMR、13C-NMR and 2D-NMR combined with HMDB database.The map of each batch samples was transformed into matrix,and then analyzing in the multivariate statistical analysis software.It was found that there were significant differences in the profile of polar chemical composition between cultural and wild musk,mainly were amino acids and organic acid.By means of extensively collecting standard products and establishing LC-MS analysis method,20 compounds were found by comparing the retention time and ion pairs in polar extraction of musk.A total of 32 chemical constituents,including amino acids and organic acids,were identified by two qualitative analysis results.3.RPLC-HILIC-MS was used to quantitatively analyze 22 chemical constituents which were carried out across large,medium and small polarity ranges in musk.The results showed that the extracts of musk methanol contained both polar and none-polar chemical components.The contents of amino acids,organic acids and muscone in wild musk were higher than that of cultural musk.Alanine,tyrosine,leucine/isoleucine,valine,threonine and proline were the main amino acids in wild musk,while leucine/isoleucine,arginine,valine,glycine and threonine were for cultural musk.The content of steroids in cultural musk is higher than that of wild musk.The content of muscone in wild musk is 2.0～4.6%,and less than 2%in cultural musk.This method can be used for the overall quality control of musk.4.The content of protein in cultural and wild musk was determined by BC A method,and there was no significant difference between them.The content of protein accounted for 3～5%of the total weight of the musk.The map of SDS PAGE showed that both the wild musk and the cultured musk have a molecular weight of about 70 kDa,50 kDa and 25 kDa.Besides,multiple bandings between 4～25 kDa was observed in cultural musk..A total of 94 proteins in musk were annotated by Swissprot database,including 74 commmon proteins,mainly keratin,and 20 differential proteins.19 were specific to the wild musk,mostly myosin;The Serpin A3-4 is unique to cultural musk.By Sephadex G-50 and G-100 chromatogram column,the keratin of 48.7 kDa was separated from the musk water extract.In conclusion,the chemical composition of small and medium molecules of musk is basically clarified in this project,and the protein and peptide components are preliminarily revealed;To establish a comprehensive quality analysis method for natural musk(including wild musk and cultural musk);There are some differences in the chemical composition of wild musk and cultural musk.Triglycerides and muscone are artificially added in most wild musk.These results will provide the most direct information on the basis of the chemical and pharmacodynamic basis of musk.