Studies On The Chemical Constituents Of The Cultured Armillaria Mellea

Armillaria mellea(Tricholomataceae),known as zhen-mo and mihuanxun in China,is an edible and medicinal mushroom which has a strong symbiotic relationship with a slow growing and expensive traditional Chinese medicine ’Gastrodia elata‘’.The sporophores of A.mellea showed the similar clinical use as G.elata which have been used for treating megrim,headache,neurasthenia,insomnia,acroanesthesia,hypertension,epilepsy and nyctalopia in China.Studies showed that the biological activities and clinical applications of A.mellea submerged culture products are also similar to those of G.elata.The main chemical constituents of the A.mellea mycelia are protoilludane sesquiterpenoid,purine and organic acid.Protoilludane sesquiterpenoid is a kind of characteristic constituent of A.mellea which has been found to possess various biological effects such as anti-tumor,antibacterial and so on.To take full development and utilization of the medicinal value of A.mellea,it is necessary to clarify the basis of the efficacy substances.Therefore,on the basis of references about the protoilludane sesquiterpenoid or the chemical compositions of A.mellea,we systematically studied the chemical components of the A.mellea mycelia,which will provide theoretical basis for further development of the A.mellea mycelia.In this study,36 compounds were isolated by using various column chromatography,such as VLC,Silica gel,Sephadex LH-20,ODS,as well as preparative TLC,preparative HPLC and recrystallization.On the basis of spectroscopy technology as well as the physical and chemical property analysis,the structures of the 34 compounds were identified as follows:10a,13a-dihydroxyarmillaridin(MHJ 1*),4’-methylmelledonal(MHJ 2),armill.aritin(MHJ 3),mellolide B(MHJ 4),5’-methoxyarmilane(MHJ 5*),5’-methoxy-6’-chloroarmillane(MHJ 6),armillarilin(MHJ 7),armillarinin(MHJ 8),armillarigin(MHJ 9),melleolide(MHJ 10),armillaripin(MHJ 11),10-dehydroxymelleoloede(MHJ 12),5’-demethoxyarmillaribin(MHJ 13*),armillaridine(MHJ 14*),(4R,5S,7R,95)-3’,5-epoxy-4-dehydroxyarmillaridiene(MHJ 15*),(4R,5S,7R,9S,13R)-3’,5-epoxy-4-dehydroxyarmillarin(MHJ 16*),6’-chloroarmillaridine(MHJ 17*),(4R,5R,7R,9S,13R)-3’,5-epoxy-4-dehydroxyarmillarin(MHJ 18*),armillarin(MHJ 19),armillaridin(MHJ 20),armillaribin(MHJ 23),1-dehydroxyarmily everninate(MHJ 24*),(4R,5R,7R,9R,13’S)-3’,5-epoxy-4-dehydroxyarmillarin(MHJ 25*),armillarivin(MHJ 26),4-dehydroxyarmillarin(MHJ 27*),4-dehydroxyarmillaridin(MHJ 28*),5’-methoxyarmillasin(MHJ 29*),5’-methoxy-4-dehydroxyarmillasin(MHJ 30*),Daidzein(MHJ 31),5’-methoxy-8-hydroxymelledonal(MHJ 32*),genistein(MHJ 33),10β,13α-dihydroxymelleolide(MHJ 34*),armillarizin(MHJ 35),armillaricin(MHJ 36).Compound MHJ 1,5,13-18,24-25,27-30,32 and 34 were new compound.Based on the studies on the chemical components,we conducted the biological activities screening of the compounds which content are higher and the structure are typical among the compounds obtained from A.mellea.The experimental results showed that armillarigin,melleolide,armillaridin and armillaribin had no protective effects on a noxic/hypoglycemic trauma of cells in cerebral cortex in primary cultures rats,but they showed inhibition effects on the growth of A549 cell and Bel7402 cell.The growth inhibition of the tumor cell of HCT-8 was lower.The anti-ureaplasma urealyticum effects of the petroleum ether extract and ethyl acetate extract were higher than melleolide,armillaribin and orsellinic acid.Orsellinic acid showed significant antiangiogenic activity in zabrafish embryos,whereas melleolide and armillaribin showed signs of toxicity.The chemical compositions and activities of polysaccharides from wild sporophores and submerged cultured products(mycelia,fermentation mixture and fermentation broth)were compared in this paper.Among the four samples,CFBP showed the highest yield(24.38%±3.34%)and lowest carbohydrate content(52.73%± 3.41%).SP showed a moderate yield(16.89%± 0.71%)and the highest carbohydrate content(68.48%± 0.14%).The yield of CFMP was the lowest(13.77%±1.03%).The carbohydrate contents of CMP and CFBP were moderate(57.68%±0.20%,60.35%± 0.16%).All of the polysaccharides were heteropolysaccharides that were composed of D-xylose,D-glucose and D-galactose in different molar ratios without protein.Glucose was the dominant monosaccharide in SP(55.83%),CMP(46.49%)and CFMP(44.20%),and galactose was the dominant monosaccharide in CFBP(43.96%).The contents of xylose in the polysaccharides of cultured products(19.10%in CMP,20.30%in CFMP and 31.71%in CFBP)were much higher than that of SP(9.8%).SP and CFBP have different NMR spectra with CMP and CFMP,which may be caused by the different linkage types,the different branch degree.CMP had excellent antioxidant activity that could match SP.CFMP showed moderate antioxidant activity.These results indicated that the carbohydrate content and the corresponding glucose percentage had a positive influence on the activity,whereas the influence of the corresponding xylose and galactose percentages were negative.In addition,UPLC-ESI-Q TOF has been used to study the fragmentation regularity of protoilludane sesquiterpenoid and the ethyl acetate extracts from mycelia,fermentation mixture and fermentation broth of A.mellea from different batchs and manufacturers.The compounds presented in the.extracts have been identified based on a detailed analysis of the retain time,UV,MW and fragmentation behaviour of standard compounds and the data in the previous reported papers.According to the results,we can conclude that(1)46 compounds had been identified in the extracts of cultured products of A.mellea.13 of them had never been reported from A.mellea.(2)The structure of 6 new protoilludane sesquiterpenes had been deduced according to the fragmentation regularity and structure characteristic of protoilludane sesquiterpene.(3)More compounds had been found in the extraxt of mycelia.The compounds in the extract of mixture powders were similar to that of mycelia.The compounds in the extract of fermentation were fewer.(4)The chemical compositions of the extracts from different manufacturers and batches were significantly different.So it is necessary to formulate the specification and standardization of the fermentation method of A.mellea quickly.