Study On Seperation Methods And Antimicrobial Activities Of TL1-1 Produced By The Endophytic Fungi D.Eschscholzii Of Tenodora Aridifolia

5-Hydroxy-2-methylchrommanone(TL1-1),one kind of phenols,derived from the endophytic fungi Daldinia eschscholzii,which inhabited in the mantis gut(Tenodora aridifolia).According to the reports,TL1-1 exhibited significant cytotoxitc activity against the human leukemia cell line(HL-60).Moreover,TL1-1 could inhibit the growth of Microbotryum violaceum,a common form of fungal pathogenic pathogen.However,the restriction of fermentation inhibited the further study of the bioactivities of TL1-1.Besides,many conventional and modern methods for the separation and purification of TL1-1 from Daldinia eschscholzii have been developed including preparative HPLC,TLC.However,these methods are not suitable for large-scale separation,which are severely restricted by the absence of systematicness and economy.As a result,this paper studied two methods for separating TL1-1,including silica gel column chromatography as well as macroporous resin,based on the success of 500 L fermentation of Daldinia eschscholzii.In addition,we added the antibacterial experiment of TL1-1.The study applied silica gel column chromatography to separate TL1-1 from the broth of Daldinia eschscholzii.The mass ratio of loading amount and si gel cc was 1:20 and the petroleum ether and ethyl acetate were used as eluent(100:1,75:1,50:1,20:1,10:1,5:1),the volume of each rate was 2 BV,and the flow rate was 1BV/h.When the elution conditions(petroleum ether:ethyl acetate)was 20:1,TL1-1 was enriched,and the purity and recovery were 59.75%and 68.82%,respectively.However,there are lots shortcomes about si gel cc,such as the low recovery and purity,the mass use of organic solvent and so on,which are not satisfied with the requirement of easy and economic production.In this paper,a systematic separation process for the large-scale production of TL1-1,based on a macroporous resin,was seriously developed.Based on batch experiments,XAD-16 resin was selected from ten types of resin because of its superior adsorption and desorption performance.Adsorption equilibrium data for this resin fitted well to pseudo-first order kinetics,Weber-Morris model and the Freundlich model;these data were elucidated from kinetic experiments and adsorption isotherms.Dynamical adsorption and desorption experiments optimized the conditions of loading amount(fermentation:si gel cc = 1:20),pH 4.0 and loading flow rate(2 BV/h)for TL1-1 on XAD-16 resin.The eluent was followed as gradient concentration:deionized water(4 BV),40%ethanol solution(4 BV),95%ethanol solution(4 BV),and the elution flow rate was controlled to 2 BV/h.In the elution volume range of 1.5-3.5 BV of 95%ethanol elution,TL1-1 was obtained.And the purity and recovery of TL1-1 were 84.64%and 75.06%,respectively.Based on the optimal conditions described above,the separation of TL1-1 was scaled-up.The results revealed that the purity(80.33%)and recovery yield(72.02%)of TL1-1 after scale-up,which was similar to that obtained using the small column.Hence,the XAD-16 resin was shown to be an efficient and promising tool for the large-scale separation of TL1-1 because of its high adsorption and recovery characteristics.In this paper,the structure of TL1-1 obtained by XAD-16 resin separation was confirmed.The initial qualitative analysis was carried out by HPLC,the retention time of the obtained compound was consistent with that of the standard TL1-1.The molecular weight of TL1-1 was determined by LC-MS,and the molecular weight of TL1-1 was 178,which was consistent with the reported results.The structure of TL1-1 was confirmed by 1H NMR,and the data of hydrogen were consistent with those reported in the literature.The isolated TL1-1 was also tested for its biological activities against four plant pathogens.While TL1-1 showed greate activity against Ralstonia solanacearum.This paper mainly introduced two methods for the separation of TL1-1 from Daldinia eschscholzii.Compared with silica gel column chromatography,macroporous resin was more convenient,safe and economical.Hence,the XAD-16 resin was shown to be an efficient and promising tool for the separation of nature products.