| Selaginella pulvinata,a traditional Chinese medicine,was proved to have a variety of physiological activities,such as anti-tumor,anti-diabetics,anti-bacterial,anti-viral and so on.S.pulvinata was included in The People’s Republic of China Pharmacopoeia of 2015 version.The biflavones were found to be as major physiological activity components in Selaginella genus.Amentoflavone,a main biflavone in S.tamariscina,shows a varity of activities such as hypoglycemic activity,anti-oxidation activity and anti-inflammatory activity.The extraction process and the enrichment process of amentoflavone from S.pulvinata were optimized,the chemical composition of the extract was qualitatively identified by LC-MSn analysis,the hypoglycemic activity and anti-oxidation activity of the extract were evaluated by in vitro activity models,and a HPLC fingerprint for selaginella genus was established to evaluate the differences between different varieties of Selaginella.The main contents were as follows:(1)The response surface was designed with ethanol concentration,ethanol times,extraction time and extraction times.The optimal extraction conditions were as follows: ethanol concentration: 71%,extraction time: 60 min,extraction frequency: 3 times,amount of ethanol consumption: 12 times,the extract rate of amentoflavone could reach17.28 mg/g.The purification process of the crude extract were optimized by macroporous resin or secondary precipitation.The optimal parameters of macroporous resin enrichment method are as follows: extract concentration of 100 mg/mL,the sample flow rate of 1.0 mL/min,80%ethanol elution,elution velocity of 1.0 mL/min,dosage of solvent elution volume 4 BV column,in this process the content of amentoflavone in S.pulvinata extracts could increase from 13.79% to 52.46%;The optimal parameters of secondary precipitation process were as follows: ethanol concentration 40%,water temperature 15 ℃,stirring speed 600 rpm,speed of adding water 3 mL/min.The content of amentoflavone could increase up to 47.92%.As a result,the macroporous resin process is more suitable for S.pulvinata extract to enrich the content of amentoflavone.(2)The chemical composition of the extract was qualitatively identified by LC-MSn experiment.As a result,27 compounds of S.pulvinata extract were identified,including of 13 flavonoid compounds,10 acetylene phenolic compounds and 4 other types of compounds,it could effectively clarify the efficacy of S.pulvinata extract and providereference value.(3)The α-glycosidase inhibitory activity and anti-oxidation activity of S.pulvinata extract were evaluated.The results showed that the extract exhibits α-glycosidase inhibitory with IC50 value of 0.1978 mg/mL,the IC50 value of positive control was 2.6996 mg/mL.The experiment of DPPH show that the S.pulvinata extract had weak antioxidant activity with the IC50 value of 184.8 μg/mL and it was a kind of slow antioxidants(the IC50 value of VC was 12.84 μg/mL).(4)The HPLC fingerprint of 14 species plants of the Selaginella genus was established(including 7 characteristic peak).The similarity of the 14 species were more than 0.8,through cluster analysis and principal component analysis could quickly divide the samples into 4 classes.The peak of amentoflavone is a key component in the classification.The fingerprint method combined with the stoichiometry could provide a scientific basis for the quality evaluation of Selaginellae herba.In this dissertation,we have made a systematic study on the extract of S.pulvinata,which provided scientific basis for industrial production,pharmacological material basis,drug development research and so on. |
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