Extraction,Purification,Structural Identification And Bioactivities Of Effective Constituents From Two Species Of Pteris

Chinese herbal medicine has become a major source of lead compounds for the treatment of various serious diseases,and has made great contributions to human health.As an important resource of Chinese herbal medicine,the genus Pteris is well worth developing.There are about 66 Pteris species in China,and approximately 30 Pteris species are used as traditional medicinal plants with the functions of heat-clearing,detoxification,draining dampness,treating stranguria,decreasing swelling,alleviating pain,hemostasis and treating dysentery.Modern studies have shown that Pteris species contain flavonoids and terpenoids,and have anti-tumor,anti-inflammatory,antioxidant,anti-bacterial/viral and antidiabetic effects.The development and utilization of Pteris species have become a research hotspot.Flavonoids are the main active components of the genus Pteris.In recent years,the research and application of flavonoids have drawn many attentions.Because the extraction yield and purity of flavonoids have important influences on biological activity.Hence,the optimization of the extraction and purification process of flavonoids with the goal of obtaining high yield and high purity is an urgent problem to deal with.In addition,there are many studies on the chemical constituents and pharmacological activities of Pteris multifida,Pteris vittata,Pteris semipinnata and Pteris cretica,while there are relatively few studies on the other Pteris species,which leads to uncertainty about the active ingredients and questionable effectiveness,thereby limiting their development and utilization.Hence,to promote the scientific development and utilization of the abundant wild medicinal plant resources of the genus Pteris in China,two medicinal plants with great development potential but little research are selected as experimental materials,namely Pteris ensiformis Burm.and Pteris esquirolii Christ.The main research methods and conclusions are as follows:1)Optimization of extraction and purification process of total flavonoids from Pteris ensiformis Burm.（Pe B–TP）was performed.First,heating reflux extraction parameters（extraction solvent,time,temperature and liquid-solid ratio）of Pe B–TP were optimized based on single-factor experiment combined with orthogonal experiment.Under optimal conditions（70%ethanol,120 min,70℃and 20 m L/g）,the Pe B–TP yield was 35.73±0.52 mg/g.Ultrasonic-assisted extraction parameters（extraction solvent,time,temperature and liquid-solid ratio）of Pe B–TP were optimized based on single-factor experiment combined with response surface experimental design.Under optimal conditions（67%ethanol,38 min,66℃and 20 m L/g）,the Pe B–TP yield was 32.47±0.40 mg/g.It could be seen that Pe B–TP yield extracted by heating reflux extraction method was higher than that of ultrasonic-assisted extraction method.Then,NKA-II resin was selected as the best resin through investigating the adsorption/desorption performances of 10 kinds of macroporous resin for Pe B–TP.The results of the adsorption kinetics,adsorption isotherms and adsorption thermodynamics studies showed that the adsorption of Pe B–TP on NKA-II resin was fitted best to the pseudo-second-order kinetic model and Langmuir model,and this adsorption was a spontaneous,exothermic,physical adsorption process with entropy reduction.Based on the dynamic adsorption/desorption experiments,the optimal conditions for the purification of Pe B–TP by NKA-II resin column chromatography（1 BV=19.5 m L）were determined as Pe B–TP concentration 1.84 mg/m L,feed rate 2 BV/h,feed volume 5 BV,eluent 50%ethanol,elution rate 2 BV/h and eluent dosage 7 BV.The results of scale-up purification experiment（1BV=720 m L）showed that the purity of Pe B–TP was increased to 3.16-fold of that before purification,with a recovery yield of 80.25%.2)Bioactivities evaluation of different polar fractions of P.ensiformis,and isolation,identification of effective constituents were performed.Results of bioactivities evaluation of95%ethanol extract of P.ensiformis and its five fractions of different polarities indicated that ethyl acetate fraction（Pe B–Et OAc）had good antioxidant,anti-inflammatory,anti-tumor andα-glucosidase inhibitory activities as a whole.Next,effective components in Pe B–Et OAc were investigated by various chromatographic,spectroscopy techniques.Twenty-one compounds were isolated and identified,among which seventeen compounds were first found from P.ensiformis,including eight flavonoids（quercetin-7-O-β-D-glucopyranoside,quercetin-3-O-β-D-glucopyranoside,apigenin-7-O-β-D-neohesperidoside,luteolin-7-O-β-D-glucopyranoside,apigenin-7-O-β-D-glucopyranoside,hydroxygenkwanin and 3’,5,7-trihydroxy-4’-methoxyflavone）,three pterosins（（2S,3S）-wallichoside,（3R）-pterosin D and（2R,3R）-pterosin L）,three nitrogen-containing compounds（L-pyroglutamic acid,nicotinamide and L-phenylalanine）,one phenolic acid（isoferulic acid）,one furan compound（5-hydroxymethylfurfural）and one coumarin（7-hydroxycoumarin）.Eight compounds（quercetin7-O-β-D-glucopyranoside,hydroxygenkwanin,3’,5,7-trihydroxy-4’-methoxyflavone,L-pyroglutamic acid,nicotinamide,L-phenylalanine,isoferulic acid and 5-hydroxymethylfurfural）were first isolated from the genus Pteris.3)The active components of ethyl acetate fraction of Pteris esquirolii Christ（Pe C–Et OAc）were studied.By various chromatographic,spectroscopy techniques,nineteen compounds were first isolated and identified,including six pterosins（（±）-14-hydroxypterosin B,（2S,3S）-wallichoside,（2S,3S）-pterosin S,（2S,3S）-pterosin C,（2R,3S）-pterosin C and dehydropterosinB）,nineflavonoids（quercetin-7-O-β-D-glucopyranoside,hesperetin-7-O-rutinoside,quercetin-3-O-α-L-rhamnopyranoside,luteolin-7-O-β-D-glucopyranoside,genistin,kaempferol,luteolin,naringenin and hydroxygenkwanin）,one phenylpropanoid glycoside（syringin）and three phenolic acids（chlorogenic acid,vanillic acid and p-hydroxybenzoic acid）.Compound（±）-14-hydroxypterosin B was a new pterosin,eight compounds（quercetin7-O-β-D-glucopyranoside,hesperetin-7-O-rutinoside,quercetin 3-O-α-L-rhamnopyranoside,genistin,naringenin,hydroxygenkwanin,syringin and vanillic acid）were first isolated from the genus Pteris.4)The inhibition ofα-glucosidase and antitumor activity of pterosins and flavonoids were studied.The inhibition of pterosins againstα-glucosidase was evaluated based on molecular docking technology,results showed that（2S,3S）-wallichoside had the strongest binding ability with 3A4A,and the result of verification experiment further showed that the EC₅₀was 0.33 mmol/L.Results of anti-tumor effects of pterosins and flavonoids using CCK-8method showed that hydroxygenkwanin had strong inhibitory effect on the proliferation of HCT-116 cells(IC₅₀=5.88μmol/L).Further research showed that hydroxygenkwanin could reduce the colony formation ratio of HCT-116 cell,increase the intracellular ROS level,induce cell cycle arrest at G0/G1 phase by down-regulating the cyclin-D1 and CDK-4proteins expression,and induce apoptosis by up-regulating the Bax and cleaved caspase-3proteins expression and down-regulating Bcl-2 protein expression.In conclusion,the Pe B-TP obtained by the extraction and purification process established in this study has high purity and recovery yield,and this process has an excellent prospect for industrial application.Meanwhile,this study helps to clarify the pharmacodynamic substance basis of P.ensiformis and P.esquirolii,lays a foundation for the research and development of naturalα-glucosidase inhibitors and anti-colon cancer drugs,also provides scientific reference for the rational development of these two medicinal plant resources of the genus Pteris.