| Selaginella tamariscina(Beauv.)Spring distributed in China’s northeast,north China,east China,central China and Shaanxi,Sichuan and other places,for the perennial herbs,also known as the immortal grass,nine death reviving grass,etc.Traditional Chinese medicine Selaginella has the effect of promoting blood circulation and menstruation.It is used for dysmenorrhea,dysmenorrhea and injury.Stir-fried charcoal can remove blood stasis and stop bleeding,used for vomiting blood,leakage and so on.The genus Selaginella has been one of the research hotspots in recent years.Modern pharmacological studies have shown that the genus Selaginella has many pharmacological activities,such as anti-tumor,antimicrobial,antiviral,anti-inflammatory,anti-aging and hypoglycemic activities.In addition,the chemical constituents of Selaginella are diverse,including flavonoids,acetylene phenol,phenylpropanoids,alkaloids,etc.In this paper,the 75%ethanol extract of Selaginella was studied to enrich the chemical constituents of Selaginella.At the same time,the content determination and fingerprint study of Selaginella were carried out to provide more theoretical basis for the further study and in-depth development.The extraction method of Selaginella was carried out by means of flash extraction,and use macroporous adsorptive resins,silica gel column chromatography,polyamide column chromatography,ODS column chromatography and pHPLC and other modern chromatography techniques were used to separate and purify the chloroform extraction parts of Selaginella.Combined with physical and chemical properties and spectroscopic methods,the structures of the isolated compounds were identified,and 13 compounds were obtained,respectively selariscinin F(1)、amentoflavone(2),selariscinin D(3),selaginellin M(4)selaginellin A(5),selaginellin U(6),selaginellin T(7),apigenin(8),uncinataflavone A(9),1,6-caprolactam(10),3,5,7-Trimethoxy-4’-hydroxyflavonol(11),3,7-dimethoxy-5,4’-dihydroxyflavonoid(12).Compound 1 was found to be a new compound that had not been reported in literature after retrieval in the Scifinder database.Compounds 10,11,12 were isolated from the genus Selaginella for the first time.A method for simultaneous determination of amentoflavone and selaginellin in Selaginella by HPLC was established.Chromatographic column is ChromCore C18(250×4.6 mm,5μm),Mobile phase system is methanol(A)-0.1%formic acid(B),gradient elution(0-20min 15-60%A,20-40min 60-80%A),velocity of flow 0.8mL/min,column temperature20℃,check wavelength 388nm,sample load 10μL,amentoflavone recovery rates 97.02%(RSD=1.449),selaginellin recory rates 96.86%(RSD=1.661).The contents of amentoflavone and selaginellin in 10 batches of Selaginella from different producing areas were determined as follows 0.40～8.00mg/g and 7.65～153.0μg/g。Fingerprint of 10 batches of Selaginella from different producing areas was studied.Chromatographic column was nanoscale chromatographic column(250×4.6 mm,5 μm),Mobile phase system is methanol(A)-0.1%formic acid(B),gradient elution(0-5 min:10-20%A,5-20 min:20-40%A,20-30 min:40-70%A,30-70 min:70-90%A),velocity of flow 0.8 mL/min,column temperature 20℃,check wavelength 330 nm,sample load10 μL.A total of 8 common peaks were identified in 10 batches of Selaginella from different producing areas.The common peaks were identified by HPLC-Q-TOF-MS,and the peaks 2,3,and 6 were determined to be apigenin-6,8-di-C-glucoside,selaginellin and cypress bisflavonoids.According to the fingerprint study of 10 batches of Selaginella from different producing areas,it was found that there were great differences in Selaginella from different producing areas.Therefore,the quality standard of Selaginella should be comprehensively considered in combination with the influence of differences in geology,environment and climate. |
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