Chemical Constituents Of Three Traditional Chinese Medicines With Their Biological Activities

In this paper,the chemical composition and bioactivity of three representative medicinal plants with abundant natural resources,definite medicinal effects and potential druggability were studied.The plants were Malus hupehensis(Pamp.)Rehd.,Sapindus mukorossi Gaertn.and Pueraria lobata(Willd.)Ohwi.The extracts were fractionated using macroporous adsorption resin chromatography,Polyamide nylon 66 resin,silica gel chromatography,sephadex LH-20,as well as reversed-phase high performance liquid chromatography(RP HPLC).As a result,104 compounds were isolated,including 12 novel compounds.The structures of these compounds were confirmed by mass spectrometry(MS),1D and 2D nuclear magnetic resonance(NMR)techniques,X-ray single crystal diffraction,infrared(IR)spectrum,ultraviolet(UV)spectrum,hydrolysis reaction,etc.The isolated compounds were evaluated for biological activity such as antioxidant activity,antifungal activity,hSGLT2 inhibitory activity.The results showed that some of the compounds had significant biological activity.Chapter 1 described the research on the chemical constituents of the leaves of M,hupehensis(Pamp.)Rehd.,a raw material for tea production,collected from the Tea Planting Base owned by Linyi Shansong Biological Product Co.,Ltd.A total of 22 phenolic compounds were isolated and characterized,the compound 5-O-?-D-glucopyranoside-4-chromanone(1)was discovered for the first time.DPPH radical scavenging activities of these compunds were evaluated.Most of the compounds showed DPPH radical scavenging activities.Among the dihydrochlcones,3'-hydroxyphloretin(7)showed a better antioxidant activity than edaravone which was used as a positive control.Dihydroflavones exhibited moderate activities,and 5,7,3',5',-tetrahydroxy-dihydroflavone(11)showed significant activities better than edaravone.All the flavones displayed significant antioxidant activities except compound 16.Among these flavones,quercetin-3-O-a-L-rhamnoside(18)showed potent activities,which possessed comparable antioxidant ability with vitamin E.Besides,chrysin(14)and quercitrin(17)displayed good antioxidant activities.Besides,HPLC profiles for the extract from the leaves of M.Hupehensis or the plant's fruit wine were analyzed and the differences in the chemical constituents between them were compared briefly,and the material base for health care of the fruit wine was clarified.Chapter 2 discussed the study on Sapidus Mukorossi Gaertn,a plant that has been utilized in modern cosmetics and cleaning products for its known role in germicidal effect.Chemical constituents in the pulp of this plant were investigated in a systematic manner:the isolated compounds were evaluated for their antifungal activities,the mechnism of antifungal was explored.20 compounds were obtained from the water extraction of the pulp of S.mukorossi,mainly of which were oleanane-type,hederagenin-type or dammarane-type triterpene saponins.Compound 23 to Compound 27 were newly discovered,that is,oleanolic acid 3-O-K?-L-arabinofuranosyl-(l?3)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinpyranosde(23),hederagenin 3-0-5'"-O-acetyl-a-L-arabinofuranosyl-(1?3)-a-L-hamnopyranosyl-(1?2)-a-L-arabinopyranoside(24),23-0-acetyl-hederagenin-3-O-?-D-xylopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranoside(25),gypsogenin 3-O-a-L-arabinopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1->2)-a-L-arabinopyranoside(26)and betulinic acid 3-O-?-D-xylopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranoside(27).Antifungal activities of these compounds were evaluate against Candida albicans and Trichophyton rubrum.The results indicated that oleanolic acid 3-O-?-D-xylopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranoside(30),oleanolic acid 3-O-a-L-arabinopyranoside-(1?3)-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranoside(31)and a-hederin(34)showed good antifungal activity against Candida albicans with MIC80 of 16,8 and 16 ?g/mL,respectively.They also exhibited good antifunal activity against T.rubrum with MIC80 of 8,8 and 16 ?g/mL,respectively,In addition,study on the mechanisim of their antifungal activities were initialed.Chapter three described the studies on the chemical constituents of the root of Pueraria lobata(Leguminosae)which used for the production of granules and injections,together with the Human-SGLT2 inhibitory activity evaluation.Hence,62 compounds were isolated,including 6 new compounds:pulobatones A(43)?pulobatones B(44)?8-C-?-glucofuranosyl-7,3',-trihydroxyisoflavone(45)?8-C-a-glucofuranosyl-7,3',4'-trihydroxyisoflavone(46)?8-C-?-glucofuranosyl-7,4'-dihydroxy-3'-methoxy isoflavone(47)?8-C-a-glucofuranosyl-7,4'-dihydroxy-3'-methoxyisoflavone(48).Many types of monomeric compounds were identified,including isoflavones,isoflavone glucosides,puerarin,lignans and alkaloids.The study can help us to understand the medicinal material basis and its efficacy.In addition,we performed impurity spectral analysis of trace impurities in Puerarin Injection,and identified the trace impurities in Puerarin Injection by using the monomer compounds isolated from Pueraria lobata which help us to understand the mechanism in the optimization and upgrading process to the next generation.Finally,we evaluated the hemolytic properties of puerarin and Puerarin Injection,and found that the impurities are not the main cause of hemolysis.The innovation of this dissertation is:1.A new class of dihydrochromanone compounds(1)was isolated from the leaves of Malus hupehensis for the first time,the compounds were evaluated for their DPPH free radical scavenging activity and positive control drug edaravin and VE.2.Based on the compounds extracted from the leaves of M.Hupehensis,the ingredients in the M.Hupehensis wine were identified.3.Five new triterpene saponins(23-27)were isolated from the pulp of Sapindus mukorossi Gaertn for the first time.They were oleanolic acid,ivy saponin and lupinane,respectively.4.The compounds with antifungal activity were selected and the time-sterilization curve of Candida albicans was tested.The active site of 75%ethanol fraction was subjected to in vivo evaluation,and its activity on the mouse model of vaginitis caused by Candida albicans was comparable to that of amphotericin B.5.By testing cell membrane permeability,change of intracellular ROS,and under the transmission electron microscope observation of Candida albicans treated by tested compounds,it was speculated that the compounds derived from Sapindus mukorossi Gaertn play antifungal effect by destroying the cell membranes of fungal.6.Six new isoflavones and isoflavone glucoside compounds(43-48)were isolated from Pueraria lobata for the first time.Based on the crystals structure of the known compound neopuerarin B(49),the 13C NMR spectrums of neopuerarin B and Neopuerarin A(50),the stereo configurations of the new compound(45-48)had been charicterized.7.The impurities of Puerarin injection were studied,six trace impurities were separated and their hemolytic properties were evaluated,it is found that the Puerarin and impurities are not the main cause of hemolysis.