| Melaleuca alternifolia,a member of Myrtaceae Melaleuca L.,is native to New South Wales of Australia and some districts of New Zealand,and was successfully introduced to China in the 1990s,and has formed a certain scale of planting in Guangzhou,Hainan,Fujian,Yunnan,and etc.The resource of Melaleuca alternifolia is also rich in Guangxi,production bases had been set up in many districts like Qingzhou,Yongning,Yulin,and etc.At present,the research of Myrtaceae Melaleucal has been mainly focused on plant cultivation and essential oil(tea tree oil),while the non-volatile phytochemistry of Myrtaceae Melaleucal has few related reports.In this thesis,the leaves and bark of Myrtaceae Melaleucal were study systematically,twenty one compounds were separated and indentified,pharmacological activity research for the crude extration were conducted.Meanwhile,gallc acid,which was separated from this plant,was selected as origination material,sulfonamides as the synthon,a series of gallic acid derivatives were synthetized and their effects on cartilage cell proliferation in vitro were studied,the details are as following:1.The constituents in leaves and barks of Melaleuca alternifolia were study systematically,the extracts were divided into petroleum ether portion,chloroform portion and ethyl acetate portion,then were column chromatographed through silica,Sephadex LH-20,and et al.repeatedly and recrystallized,21 compounds were separated and identified as n-nonacosanol,n-dotriacontanol,n-gheddic acid,?-sitosterol,n-tricontyl-4-cinnamate,triacontyl-3,4,-dihydroxy-cinnamate,3-(4-hydroxy-3-methoxy-pheny l)-acry li c acid triacontyl ester,cholesterol,betulinic acid,betulin,urs-12(13)-en-3-on-28-oic acid,3?-acetoxyurs-11-en-28,13-olide,3?-O-acetyl-11?,12a-epoxy-oleanane-28,1 3 ?-olide,ursolic acid,3?-acetoxyurs-12-en-28-oic acid,ursolic acid 3?-O-caffeate,quercetin,kaempferol,gallic acid,ethyl gallate,and protocatechuic acid.2.Pharmacology and acute toxicity research of alcohol extract and aqueous extract of Melaleuca alternifolia were conducted.Several assays like test of auricle tumefaction induced by croton oil in mice,double broth dilution,hot plate test,allergic test on the skin of mice were adopted,results suggest that both alcohol extract and aqueous extract could prevent the ear swelling,relieve the pain caused by heat stimuli,inhibit passive skin permeability in mice.In addition,aqueous extract could increase itch-threshold of cavy caused by histamine phosphate.For bacteriostatic action,alcohol extract has the effect of growth inhibition on staphylococcus aureus,epidermis staphylococcus and Candida albicans,besides those,aqueous extract has the effect on Escherichia coli.The LD50 of alcohol extract was 50.90 g/kg,while the maximum administration dosage of aqueous extract was 225.62g/kg.3.Gallic acid was selected as origination material,sulfonamides as the synthon,13 gallic acid derivatives were synthesized.1H-NMR,13C-NMR and MS methods were applied to confirm the structures.4.Articular chondrocytes of rabbit were cultured in vitro and treated with different concentrations of gallic acid derivatives.MTT assay was applied to detect the proliferation of rabbit chondrocytes which treated with different concentrations of gallic acid derivatives.The property changes of cartilage cells were observed after different staining methods,i.e.eosin(HE)staining,safranin O-fast green staining,calcein/PI staining,Hoechst 33258 staining,and ?/?collagen immunization staining.Biological changes of the chondrocytes were observed by determination of glycosaminoglycan,collagen ?,collagen ?,collagen X and Sox9 gene expression by RT-PCR.The results showed that derivative 1,7 to 11 could effectively promote chondrocyte growth in vitro.5.AutoDock Vina 1.1.2 was adopted for docking calculations,six derivatives(1,7 to 11)which showed positive effect in vitro present strong affinity toward ADAMTS-5. |
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