Study On The Secondary Metabolites Of Endophytic Fungi CH1307 From Cephalotaxus Hainanensis Li Based On The Solid State Culture

At present,it is a hot spot that researches are focused on looking for biological active secondary metabolites existing in endophytic fungi from medicinal plants.Harringtonine alkali substances with significant anti-leukemia effect is existed in its tissues of Cephalotaxus hainanensis Li,so it becomes an alternative way to obtain such anticancer drugs with similar activity from secondary metabolites of endophytic fungi which screened from such plants.Our laboratory long engaged in screening endophytic fungi from Cephalotaxus hainanensis Li plant,one endophytic fungus CH1307 contain high anti-leukemia cells activity of producting homoharringtonine was isolated,which is identified as Alternaria tenuissima.In our previous studies,the conditions in liquid shake fermentation were optimized to increase HHT production,and some progress had been made.However,it was found that the colony morphology and the production of HHT by the strain were unstable.Therefore,The text based on further separation and purification of this strain,we designed a series of experiments to explore the effect of synthetic levels of secondary metabolites by different fermentation method.In addition,the specific components of secondary metabolites were identified and evaluated.The main findings were as follows:(1)Strain purification and identification of fungi CH1307Through traditional morphological identification methods to further isolated for fungi CH1307 which kept in China General Microbiological Culture Collection Center(CGMCC)were,and found the colony of various shapes.Thus,fungi CH1307 were divided into five morphological types in terms of the colony diameter,hight,shape,the color of the front,aerial hyphae form,pigment production or not and named as CH1307a,CH1307b,CH1307c,CH1307d and CH1307e.At the same time,microscopic observation showed that five morphological types had similar morphological and CH1307c had spores.Molecular biology methods were used for the analysis of sequence alignment and verifying phylogenetic relationships with ITSrDNA sequences of five morphological types.The results showed that the ITSrDNA sequences of five morphological types were same and all belonged to the genus Alternaria.(2)Exploring the metabolites of five colony morphology types of CH1307As the unstability in colonial morphology of CH1307,we still couldn't identified which species the strain belongs to.In view of the instability production and the possibility of variations,we explored the metabolites of five colony morphology types.The crude extracts of secondary metabolites of five colony morphology types of CH1307 were measured by the use of chemical experiments,HPLC analysis,anti-microbial and anti-tumor cytotoxic activity test.The results indicated that the species of compounds of five morphology types were all very rich,covering seven compound classifications.From the peaks of liquid chromatogram,the five morphology types of CH1307 had abundant peaks within a range of 0-45min.The liquid chromatogram of CHI 307c was with the most complex and highest peaks,that of CH1307d and CH1307e were the second,and the overall peak of CH1307a,CH1307b is relatively low.Besides,antibacterial activity showed that secondary metabolites of five colony morphology types had obvious inhibitory activity to seven strains tested(Escherichia coli,Staphylococcu aureus,Bacillus subtilis,Streptococcus agalactiae,Rhizoctonia solani,Sclerotinia sclerotiorum,Fusarium oxysporum f.sp.Cubense).The crude extracts of secondary metabolites from five morphology types of CH1307 did not show inhibition activity to cells K562,while showed better inhibition to cells NB4 and HL60 with IC50 values between 28-46 ?g/mL.The reason selecting CH1307c as a objective strain to further study was that it was a sole one that could trace to produce their spores and have most species and the highest levels of the synthesized compounds.(3)Ferments on promoting synthesis of secondary metabolites of CH1307cAs the productivity and the yield of secondary metabolites by the strain is unstable in liquid fermentation.Effect of different fermentation methods(rice solid-state,wheat bran solid-state,potato liquid-standing and potato dynamic)on promoting synthesis of secondary metabolites of CH1307c by OSMAC strategy was explored.From the findings,different fermentation methods have a significant impact on the categories of the compounds and the output of the product.The yield of crude extractions by rice solid-state fermentation and wheat bran solid-state respectively increased 45.9 times and 28 times than potato dynamic.In addition,the production of potato dynamic is 9.1 as many as potato static.Experimental results showed the species of rice solid-state fermentation was the richest on low-lying land,the second on wheat bran solid-state,and potato dynamic,and the least on potato static.Meanwhile,four fermentation methods had obvious inhibitory activity to seven strains tested(Escherichia coli,Staphylococcu aureus,Bacillus subtilis,Streptococcus agalactiae,Rhizoctonia solan,Sclerotinia sclerotiorum,Fusarium oxysporum f.sp.Cubense).The crude extracts of secondary metabolites from five morphology types of CH1307 did not show inhibition activity to cells K562,while showed better inhibition to cells NB4 and HL60 with IC50 values between 30-40 ?g/mL.Therefore,the rice solid-state fermentation was the optimal choice for this strain.(4)Study on structure identification and activity evaluation of CHI 307cSolid-state fermentation method was carried out as large-scale fermentation of CH1307c.Fermentation production was extracted with ethyl acetate,concentrated,and then obtained a crude extraction of 200.4g.First,silica gel column chromatography,thin layer chromatography,semi-preparative liquid chromatography and recrystallization analysis methods were used for isolation and purification.Second,the structures of those isolated compounds were analyzed by NMR.And there were 8 compounds,as follows:Pestalotiopin A(1),?-Sitosterol(2),Alternariol 5-O-sulfate(3),Alternariol monomethyl ether(4),Scopoletin(5),Cerevisterol(6),Oblongolide V(7),and 3-Methoxyphenoxy(8)?Third,the antitumor activity of the 8 compounds against cells K562 and NB4 were measured.The results showed that the 8 compounds did not have inhibition activity to cell K562 and NB4.Currently,we still not separated to obtain the target product of HHT or HT,also did not get other compounds with leukemia cell activity,but the method established currently having a reference and guidance to the target complete,While 23 fractions have been obtained to lay the foundation for following research.