Chemical Components Analysis and Microbial Fermentation of Seven Traditional Chinese Medicines from Yunnan

Author:Dong Jian Wei

Supervisor:ding zhong tao


Degree Year:2017





Microbial fermentation is an important processing method for traditional Chinese medicines(TCMs),which frequently brings about various biotransformation,changes of the contents of effective components,improvenment of bioactivities,and decrease of toxicities.Study on fermented medicine is meaningful for further development of TCMs.This paper aims at the chemical components analysis and microorganism fermentation of seven TCMs from Yunnan Province.On the basis of chemical research,the chemical constituents and bioactivities of fermented TCMs were compared with original materials.And the microorganism fermentation processing methods for above TCMs were proposed.Chapter I reviewed the progress in microbial fermentation of TCMs in recent years,mainly aiming at "bidirectional relationship" between TCMs and microorganisms,including changes of TCMs by microorganisms and influences on microbial metabolisms by TCMs.Chapters II-VI discrible the studies on chemical constituents and microbial fermentation of seven TCMs from Yunnan province..1.Stephania epigaea a medicinal liana belonging to the genus Stephania Lour.(Menispermaceae).In this chapter,fourteen alkaloids,including two new compounds,epiganines A and B(1 and 3),were isolated and identified from S.epigaea.Compound 1 is a first reported proaporphine alkaloid bearing an N-isopropanenitrile.Compounds 3-6 and 13 showed significant acetylcholinesterase(AChE)inhibitory activities(IC50<10 μM).Eleven alkaloids were isolated and identified from Stephania intermedia which is also a liana of Stephania.Compound 21 possessed significant AChE inhibitory activity with an IC50 value of 4.52 ± 0.33 μM.On the basis of chemical constituents studies,the fermentation of S.epigaea by 16 fungi was carried out.The results revealed that aporphine alkaloids in S.epigaea could be regio-and stereo-selectively transformed to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana 82H2 fermentation.Meanwhile,four new 4R-hydroxyaporphine alkaloids,(4R,6aR)-4-hydroxydicentrine(26),(4R,6aR)-4-hydroxyromeline(28),(4R,6aR)-4-hydroxyphanostenine(29),and(4R,6aS)-N-methyl-4-hydroxylaurotetanine(30)were obtained.New 4R-hydroxyaporphine alkaloids exbibited same grade bioactivities as original alkaloids and possessed a considerably improved water solubility(several/several dozens folds as original aporphine alkaloids).2.Illigera aromatica S.Z.Huang et S.L.Mo is an endemic medicinal liana belonging to the genus of Illigera BI.(Hermandiaceae).Twenty-three compounds,including nine new compounds,dimericilligerates A-D(1-4),illigerates A-D(9-11,5)and illigeranine(12),were isolated and identified from I.aromatica.Compounds 1-4 are novel menthane-type dimers with a new skeleton of C(7)-C(7,)bond;and compound 5 is a novel drimenol-type sesquiterpene derivative with a skeleton of seven-membered B ring.Compounds 1-4 showed moderate inhibitory effects against NO production in LPS-stimulated RAW264.7 macrophages and cytotoxic activities.Compound 9 possessed significant inhibitory effect against NO production.Compounds 9 and 11 had moderate butylcholinesterase(BuChE)inhibitory effects.Aporphine alkaliods 12-14 possessed moderate AChE and BuChE inhibitory activities.The chemical constituents of C.rogersoniana 82H2 fermented I.aromatica were also investigated in this chapter.Nine compounds,including two novel menthane-type dimers,dimericilligerates E and F(26 and 27),two new monoteroenes,illigerates E and F(28 and 29),and one new aporphine alkaloid,(4R,6a S)-4-hydroxyactinodaphnine(34),were isolated and identified.Compounds 26 and 27 possessed significant NO production inhibitory effects and cytotoxic activities.C.rogersoniana fermentation could facilitate hydrolysis of phenylpropionic acid esters to free menthane-type dimers which possessed more significant cytotoxic activities.Aporphine alkaloid(S)-actinodaphnine was also transformed to(4R,6aS)-4-hydroxyactinodaphnine(34)which exhibited stronger AChE inhibitory effect than raw material.3.Ophioglossum thermale Kom,"Yizhijian" in TCM,belongs to the genus Ophioglossum the family Ophioglossaceae.Ten chemical constituents,including two new peroxy fatty acids,thermalic acids A and B(1 and 2),were isolated and identified from O.thermale.Both 1 and 2 showed strong antibacterial activities against Staphylococcus aureus,Bacillus subtilis,and Escherichia coli.Twenty-three fungi fermentation of O.thermal implied that Talaromyces purpurogenus M18-11 could improve the total phenolic,total flavonoid contents,and antioxidant activity of plant material.4.Bletilla striata(Thunb.ex A.Murray)Rchb.f.and Bletilla formosana(Hayata)Schltr.which are the plant of the genus Bletilla the family Orchidaceae,were fermented by several fungi.Total phenilic contents,antioxidant and antibacterial activities of B.striata were improved after fermentation by Fusarium avenaceum YIM3065 and Fusarium oxysporum YIM 3068.These changes might be ascribed to deglycosylatation of some of the phenolic glycosides and saccharides,and metabolism of bibenzyl compounds during fermentation.The total phenolic content and antioxidant activity of B.formosana were increased after fermentaion by Helminthosporium maydis YIM3076.Curvularin(1)isolated from H.maydis fermented B.formosana was the main bioactive compound leading the improvement of total phenolic and antioxidant activity.5.Gastrodia elata Bl.,a valuable and expensive TCM,belongs to the genus Gastrodia the family Orchidaceae which is same as B.striata.In this chapter,Fusarium sambucinum B10.2 was screened from 20 fungi to ferment Gastrodia elata for improving antimicrobial activity.A new tetracyclic triterpene sulfate with strong antimicrobial activity,sambacide(1),was isolated and identified from F.sambucinum fermented G.elata,whose content was 37.49 ± 056 mg/g.Cultivation of F.sambucinum using different substrates revealed 1 could also be produced on potato,whose contents(17.27 ± 0.52 mg/g)was about half of on G.elata.However,when using rice,rice bran,wheat bran,and cornmeal as culture medium,1 only could be produced in very small amounts,indicating the presence of precursor or inductor for the production of 1 in G.elata and potato.In this paper,seventy-five compounds including twenty-three new compounds were isolated and identified;and a regio-and stereo-selective biotranformation was found.Additionally,this paper presents the method for processing S.epigaea,I.aromatica,O.thermale,B.striata,B.formosana,and G elata by microbial fermentation,which is meaningful for further development of TCMs.