Ustilaginoidins From Rice False Smut Balls Along With Their Preparation,Detection And Biological Activities

Rice false smut disease emerged in the rice panicles was caused by the pathogenic fungus Ustilaginoidea virens Takahashi(teleomorph:Villosiclava virens Tanaka and Tanaka).This pathogen produced toxic mycotoxins that had negative effects on the growth of rice as well as the safety of humans and animals.Thus it caused low yields of rice and poor quality of rice and its products and has become one of the most destructive diseases in rice-growing areas in the world.A key characteristic of rice false smut was the formation of rice false smut balls(FSBs)with red or black color in the spikelets,and there were two types of secondary metabolites isolated from FSBs,namely ustiloxins and ustilaginoidins.Ustilaginoidins are a series of mycotoxins that possess the bis-naphtho-y-pyrone skeleton structure.Ustiloxins belong to cyclopeptides.There were little known about the compositions of ustilaginoidins in the FSBs and their biological activities.This study aimed at isolation and identification of ustilaginoidins from FSBs and evaluates their phytotoxicity and cytotoxicity,then employed high-speed counter-current chromatography(HSCCC)and macroporous resins for purification and enrichment of the main ustilaginoidins from rice FSBs.The main results were as follows.(1)Silica gel and Sephadex LH-20 chromatography as well as HSCCC were employed for isolation and purification of ustilaginoidins from the crude EtOAc extract of FSBs,and 25 pure compounds were obtained.Their structures were elucidated on the basis of UV,IR,HRESIMS,1H and 13C NMR,HSQC,HMBC and CD spectra analysis.They were all bis-naphtho-y-pyrone compounds.Among them,ustilaginoidins A,B,C,D,E,F,G,H,I and J were the known compounds previously isolated from rice FSBs,ustilaginoidins K,L,M,N,P,E1 and isochaetochromin B2 were the known compounds firstly isolated from rice FSBs,and compounds of B1-2,B4,B4-2,B3-1,B5-1,B5-2,Cl,C4 were new ustilaginoidins isolated for first time.They all possess an R-configurated chiral axis at C-9,9’.New compounds B4 and C4 have the special isopropyl alcohol moiety,and B3-1 has the special formic ether moiety.(2)In the biological activity tests,ustilaginoidins B,I and C4,C1 exhibited moderate inhibitory effects toward the radicle and germ growth of rice seeds.When at the concentration of 400 μg/mL,their inhibition ratios on the radicle growth of rice variety Lijiang were between 60.40%and 68.09%.Ustilaginoidin I showed cytotoxic activity on the human carcinoma cell line HCT116 with IC50 value of 4.06 μM,and ustilaginoidin J showed cytotoxicity against the cell line BGC823 with IC50 value of 4.98μM.(3)High-speed counter-current chromatography(HSCCC)was employed for purification of seven main ustilaginoidins from FSBs.Three independent two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water 6.5:3.5:5.0:5.0,4.0:5.0:5.0:6.0 and 3.0:5.0:4.0:6.7(v/v)were selected and successfully separated ustilaginoidins A,G,B,H,I,C,J from each other.A total of 6.2 mg of ustilaginoidin A,5.1 mg of ustilaginoidin G,3.9 mg of ustilaginoidin B,1.2 mg of ustilaginoidin H,5.7 mg of ustilaginoidin I,3.5 mg of ustilaginoidin C,and 6.1 mg of ustilaginoidin J with purities all above 80%were yielded from total 62 mg of samples in three HSCCC runs.This method reduced the loss of compounds,showed higher separation efficiency and less labour and separate time,thus provided an alternative efficient,simple and fast strategy for the purification of ustilaginoidins from FSBs.(4)As the contents of ustilaginoidins were very low in the crude extract,macroporous resins were employed for the enrichment of the target compounds.Resin HP2MGL showed both the highest adsorption ratios and desorption ratios for the ustilaginoidins among 20 commercial macroporous resins.The optimum enrichment process for adsorption of ustilaginoidins on resin HP2MGL was pH of the initial sample solution at 6.00,flow rate at 3 BV/h,processing volume at 24 BV,and temperature at 25 ℃.The optimum process for desorption was ethanol concentration at 100%,pH of 6.00,flow rate at 3 BV/h,and eluent volume at 6 BV.After treated with HP2MGL,the contents of representative ustilaginoidins A,B,C,G,I and J in the final product increased to 29.90%,5.27%,4.21%,4.43%,4.90%and 3.05%,respectively from 4.21%,0.65%,0.37%,0.38%,0.39%and 0.28%in the untreated extract.The high recoveries ranged from 86.71%to 91.73%were obtained.Another resin DM130 was also studied for the adsorption and desorption of the target ustilaginoidins,the recoveries of the ustilaginoidins were not high as those treated with resin HP2MGL,and resin DM130 was considered to be unsuitable for the enrichment of the ustilaginoidins.(5)The isolated 25 ustilaginoidins were analyzed for their finger-print spectrum using HPLC,and retention times(RT),UV spectrum and correspond molecular ions[M-H]-were confirmed and the peaks in the mixed standard and crude extract were also identified from each other.The developed method showed good resolution for the 25 compounds and high stabilities in the intra-or inter-day analysis and could be an alternative method for the fast recognition of ustilaginoidins in FSBs.(6)The outer chlamydospore part were separated from the middle or inner parts of rice FSBs,and the chlamydospores were soaked with organic solvents and then extracted with petroleum ether.The petroleum ether crude extract were chromatographed on silica gel column,and a white needle crystal was obtained.It was identified as ergosta-5,7,22-trien-3-ol based on the 1H NMR.In conclusion,this study dealt with the isolation and structural identification of ustilaginoidin mycotoxins from rice FSBs,and eight new ustilaginoidins were reported for the first time.This study also optimized the purification and enrichment process of ustilaginoidins by means of HSCCC and macroporous resins,and provided a basis for the reasonable utilization of the ustilaginoidins.Also this study established the foundation for the future studies such as the biological activity and action mode of the ustilaginoidis,clarification of the metabolic pathways of the ustilaginoidins,the physiological and ecological roles of the mycotoxins during the disease development,as well as establishment of standard system and finger-print spectrum of ustilaginoidins.