Study On The Main Chemical Compounds Of Fritillaria Unibracteata Var.Wabuensis And The Metabolites Of Its Partial Endophytic Fungi

Fritillaria unibracteata Hsiao et K.C.Hsia var.wabuensis(S.Y.Tang et S.C.Yue)Z.D.Liu,S.Wang et S.C.Chen(FUW)is one kind of medicinal plant belongs to the Liliaceae family Fritillaria.And FUW was recorded in the 2010 edition of China Pharmacopoeia as sources of bulbus Fritillariae cirrhosae(FC)(Pharmacopoeia of the People's Republic of China).Due to long-term mining and excavation,wild FC resources are decreasing.And FUW is currently hard to find in the wild area as well.In recent years,the cultivation and domestication of FUW has achieved a key breakthrough,which makes FUW the most suitable variety for cultivation among the plants of bulbus FC and the cultivation area of FUW is expanding.The bulbus of FUW will play an important role in meeting the market demand for bulbus FC in terms of a larger in size,easy to cultivate,and having more positive therapeutic effects.In addition,endophytic fungi invade or live inside of the tissues of plants at least for a part of their life cycle,without causing any noticeable symptoms of infection or disease.More and more research proves that many bioactive constituents derived from plants can also be obtained from their endophytic fungi,and the other bioactive compounds have been isolated from endophytic fungi as well.Due to the difficulty in obtaining the resources of FUW in the past,the basic research on the metabolites of FUW is still quite lagging,and the study on endophytic fungi isolated from FUW has not been reported.In order to improve the quality control system of the bulbus of FUW,extensively and in-depth exploit the chemical composition of the endophytic fungi,and expand its development potential,the study the main chemical compounds of FUW and the metabolites of its partial endophytic fungi have been carried out.The main findings are as follows:1)A high-performance liquid chromatography coupled with evaporative light scattering detection(HPLC-ELSD)method was developed for the determination of sipeimine,peimisine and peiminine contents in bulbs of FUW.The optimal conditions of separation and detection were achieved on a Tigerkin C₁₈ analytical column(4.6×150 mm,5?m)with an isocratic mobile phase system consisting of acetonitrile-water(70:30)containing 0.1%diethylamine at the flow-rate of 1.0 m L/min,and the temperature for the detector drift tube was set at 75°C,and the pressurized air flow-rate was at 2.5 L/min.And the calibration curves for sipeimine,peimisine and peiminine were linear over the range of 0.192?3.84?g,0.191?3.82?g and0.178?3.55?g,respectively.The RSD of test on precision,stability and repeatability were all less than 3%.Moreover,the recoveries were 97.75%,100.47%and 99.40%with less RSD than 3%,respectively.In addition,the contents of three alkaloids in BFW were 0.0835%,0.0271%and 0.0174%with RSD 1.3%,2.0%and 2.6%,respectively.2)A sensitive and reliable HPLC-diode-array detection method was developed to simultaneously determine 6 nucleosides(cytidine,uridine,thymidine,adenosine,guanosine and inosine)and 4 nucleobases(adenine,cytosine,uracil and thymine).Complete separation of all the analytes was achieved on a Zorbax 300(?)Extend C18 column with a gradient of methanol(A)-ultrapure water(B)at a flow rate of 1 m L/min in less than 30 min.The diode-array detector wavelength was set at 260 nm for the UV detection of nucleosides and nucleobases.The optimized method provided good linearity,satisfactory precision,good repeatability and good recovery.In addition,the developed method was successfully applied to simultaneous determination of ten nucleosides and nucleobases from FUW,and their content changes of various cultivation time(1?7 years)were further analyzed for the first time.And the results showed that there are obvious differences between the contents of 10kinds of nucleosides and nucleobases in different growth years of FUW.The contents of uridine,guanosine and adenosine were higher,which were the main bioactive components in the bulb of FUW.The contents of thymidine and deoxycytidine were in medium level.3)GC-MS was used to detect the ether extract from the dry and fresh samples of FUW by soxhlet extraction method(SEM)and ultrasonication extraction(UE),respectively.The relative contents of chemical composition from FUW were determined by area normalization.The antioxidant capacity was evaluated by DPPH and ABTS⁺radical scavenging assay.The results showed that there was higher extraction efficiency by UE than SEM.69 and 58 kinds of components from the fresh samples by SEM and UE were determined but 73 and 57 kinds of components from dry samples by SEM and UE were determined,respectively.Moreover,the percentages of those components were 62.16%,53.70%,65.18%and 58.76%in the total number of components as well as 80.01%,68.91%,68.62%and 74.89%in the total content,respectively.The main components were n-Hexadecanoic acid,Linoleic acid ethyl ester,(Z,Z)-9,12-Octadecadienoic acid and 1,2-Benzenedicarboxylic acid,mono(2-ethylhexyl)ester in all samples.The ether extracts from fresh and dry samples by SEM and UE all showed antioxidant activity.4)Rich polysaccharides were directly observed in the bulbs of FUW using the periodic acid-Schiff(PAS)method and microscope.An acidic water-soluble heteropolysaccharide(FWPS1-1)was isolated from FUW through extraction with water,ethanol precipitation,decoloration,deproteinization,dialysis and separation using a DE-52 anion-exchange column and a Sepharose G-150 gel filtration column.FWPS1-1(Mw:?7.44 k Da)has many branches and long side chains,holds the triple-helix conformation,was composed of mannose,galacturonic acid,galactose,xylose and arabinose with a molar ratio of2.62:5.59:10.00:0.76:9.38,and features side chains that may be composed of arabinose,mannose,galactose and galacturonic acid,while the backbone may be composed of xylose,arabinose and galactose.In addition,the backbone of FWPS1-1 mainly consists of?-type glycosidic bonds,Bioactivity tests in vivo showed that the polysaccharide exhibited weak DPPH radical scavenging activity and low FRAP capacity but high ABTS radical scavenging activities,good Fe(II)-chelating abilities and remarkable DNA damage protective activities.5)The endophytic fungi were isolated from fresh samples collected at different times using tissue block method and homogenization method,and were identified based on morphological methods(for partial strains)and the phylogenetic analysis of internal transcribed spacer(ITS)sequences.Fifty-three fungal endophytes were isolated from the bulbs of FUW.Of them,49 strains were identified and grouped into 17 species,or 15 genera,and priority was conferred to the Fusarium genus.Then by using color reaction analysis,TLC,HPLC-ELSD,it was demonstrated that four strains could produce Fritillaria-alkaloid,namely WBS007,7WBY2,6WBY3 and 6WBY4.In order to verify the results from HPLC-ELSD,the samples of WBS007 and 6WBY3 were selected to analyze further using UPLC-MS/LC-MS.The results were consistent with HPLC-ELSD.WBS007 and 7WBY2could produce peimisine and peiminine,6WBY3 could produce peimisine and imperialine-3?-D-glucoside,and 6WBY4 could produce peimisine.6)In the present study,fermented filtrates of 53 fungal endophytes isolated from the bulbs of FUW displayed antioxidant activities.The DPPH activity,total antioxidant capacities(ABTS),reduction power(FRAP),total phenolic content(TPC),total flavonoid content(TFC)and total saponin content(TSC)were evaluated using petroleum ether,ethyl acetate,n-butyl alcohol and ethanol fractions extracted from five representative fungal cultures.The results showed that the last three fractions showed more potent antioxidant activity than the first fraction.Significant positive correlations were found between the compositions and antioxidant capacities.In addition,multifarious natural antioxidant components were identified from the fungal extracts,including gallic acid,rutin,phlorizin,2,4-di-tert-butylphenol and 2,6-di-tert-butyl hydroquinone.These were determined preliminarily by TLC-bioautography,HPLC and GC-MS analysis.7)The compounds of the strain WBS017 from the rice medium fermentation or from the OSMAC approach were isolated and purified using silica vacuum liquid chromatography(VLC),TLC,sephdax column chromatography,reverse C18 column chromatography and HPLC semi-preparation.And then the structures of compounds were elucidated using ESI-MS/HRESI-MS,UV,¹H-NMR,¹³C-NMR,¹H-¹H COSY,HSQC,HMBC,Mosher'method,etc.and combining with literatures.Finally,seventeen compounds were isolated from this strain,including four new hybrid polyketides compounds,named cladosins L-O.In addition,compound 7 showed a strong cytotoxicity against mouse lymphoma cell line,and novel compounds cladosin L and cladosin O showed a moderate cytotoxicity against mouse lymphoma cell line.And cladosin L showed antibacterial activity against to Staphylococcus aureus.8)Four homogeneous exopolysaccharides(EPSs),namely 6WBY3EPS-3,6WBY3EPS-4,WBS019EPS1-2 and WBS020EPS1-2,were obtained from three endophytic fungal strains.The results of physicochemical characteristics of extracellular polysaccharides indicated 6WBY3EPS-3(17.41×10⁶Da)was composed of mannose,glucose and galactose,was an acidic polysaccharose with abundant galactofuranose,mannofuranose and a few?-D-glucopyranose,had anomeric hydrogen with main?-Glycosidic configuration;6WBY3EPS-4(8.84×10⁵Da)was composed of mannose,rhamnose,glucose and galactose,possessed pyranose ring mainly,and had anomeric hydrogen with main?-Glycosidic configuration;WBS019EPS1-2(2.33×10⁴ Da)mainly composed of mannose,rhamnose,glucose,galactose and arabinose,possessed pyranose ring,and may be a neutral glycoprotein,had anomeric hydrogen with?-and?-Glycosidic configuration.WBS020EPS1-2(3.137×10⁴Da)was comprised of mannose,rhamnose,glucose,galactose and arabinose,had the monosaccharide with a pyranose,which was connected by an?-glycosidic linkage,and contained a small quantity of binding proteins.In addition,6WBY3EPS-3,6WBY3EPS-4 and WBS019EPS1-2 had a moderate in vitro antioxidant activity,and WBS020EPS1-2 had a good in vitro antioxidant activity with the IC₅₀values of 1.93 and 4.52 mg/m L for ABTS free radical scavenging activity and ferrous ion chelation activity,respectively.In summary,the developed methods were simple,rapid,suitable and reliable for the determination of the contents of three alkaloids or nucleosides and nucleobases in FUW,which could provide reference for quality evaluation and effective control of FUW.Low polarity components and polysaccharide components analysis of FUW bulbs further promoted the understanding of the basis of FUW pharmacodynamics.The study on endophytic fungi from FUW not only revealed that there were a good bio-diversity of endophytic fungi in healthy FUW bulbs,but also could produce a variety of natural products with physiological and biochemical activities.The studies on the alkaloids,antioxidant active ingredients,new natural products and polysaccharides of endophytic fungi have laid a good foundation for the development and utilization of endophytic fungi resources.