Study On Chemical Constituents And Anti-cancer Activities Of Secondary Metabolites Produced By 3 Types Of Medicinal-edible Fungi

Cancer is one of the most serious and widespread disease possessing great threat to human life and health.Under the rapid development of anti-tumor drug discovery,many significant breakthrough have been made during last several decades.Among all the anti-cancer drugs,medicinal-edible fungi possess several advantages,such as abundant resources,the renewability of the secondary metabolites and the diversity of species.What’s more,the modern pharmacological studies have demonstrated that the secondary metabolites of certain types of medicinal-edible fungi possess excellent bioactivities,including anti-cancer,immunoregulation,and antioxidant activity.In our primary screening process,the crude extracts of solid cultures of Pleurotus ostreatus,Hypsizygus marmoreus,and Lepista sordida exhibited excellent antioxidant and anti-cancer activity.However,the current focus was mainly on the studies of the fruiting body,cultivation,fermentation processes,polysaccharides,and the nutritional value assessment.Less attention has been paid to its solid fermentation product.High-speed counter current chromatography（HSCCC）is a new type of support-free liquid-liquid partition chromatography.The key of HSCCC is selecting a suitable solvent system.The combination of HSCCC and the solid fermentation technology provide a reliable scientific approach for the rapid isolation of active compounds and the exploration of its efficient industrial production.The dissertation consists of three sections,and studied the chemical and active constituents of secondary metabolites derived from Pleurotus ostreatus,Hypsizygus marmoreus,and Lepista sordida,and introduces the separation and purification of active constituents by HSCCC.1.The chemical constituents and bioactivity of Pleurotus ostreatus,Hypsizygus marmoreus,Lepista sordida have been systemically studied throughvariousmethodsofchromatographictechniqueand biotechnology,including Gas Chromatography-Mass Spectrometry（GC-MS）,Nuclear Magnetic Resonance（NMR）,Near Infrared（IR）,and UV-vis Spectrophotometry（UV）.55 compounds are identified,including1 new compound,and 50 compounds that are isolated for the first time.Besides,another 39 volatile components were identified by GC-MS.Among all the compounds,naphthalene-γ-pyranone（Asperpyrone F）,phenolic compound（hydroxytyrosol）and benzaldehyde compounds have been screened out,which display strong cytotoxicity towards HepG2,A549 and SW480,respectively.And the antioxidant activity of phenolic compound（hydroxytyrosol）and benzaldehyde compounds are superior than the positive control group of vitamin C.This chapter provides a better understanding of the chemical constituents of the secondary metabolites of Pleurotus ostreatus,Hypsizygus marmoreus,and Lepista sordida,which provides a solid theoretical and material basis for the its in-depth anti-cancer and antioxidant activity research and development.This is indicative of the fact that the medicinal-edible fungi,which can produce secondary metabolites with novel structure and various activities under solid culture conditions is an important source of active natural products.1.1 A total of 24 compounds were isolated and identified from the ethyl acetate extract of Pleurotus ostreatus,mainly consisted of dinaphthalene-γ-pyrones,steroids,and fatty acid compounds.They are asperpyrone F（1）,aurasperone A（2）,fonsecinones B（3）,aurasperone E（4）,fonsecinones A（5）,asperpyrones C（6）,asperpyrones B（7）,flavasperone（8）,5,8-Dihydroxy-10-methoxy-2-methyl-4H-naphth-o-[1,2-b]pyran-4-onen（9）,pyrophen（10）,isopyrophen（11）,7-hydroxy-4-meth-oxy-5-methylcoumarin（12）,5-Hydroxymethyl furaldehyde（13）,hydroxytyrosol（14）,p-Hydroxy-benzeneethanol（15）,2-ethyl-1,4-benzenediol（16）,stearic acid（17）,oleinic acid（18）,glyceryl monooleate（19）,9,12-octadecadienoic acid-2,3-dihydroxypropyl ester（20）,ergosterol（21）,brassicasterol（22）,campesterol（23）,and3β-hydroxy-5α,8α-epidioxyergo-sta-6,22-diene（24）.Thereinto,compound 1 is a new natural product,compounds 2-11,13-19,21,23were isolated from this genus for the first time.26 of volatile components were obtained by GC-MS analysis,which are mainly acid,aldehyde,furan,and ester compounds.Compounds 1-7,14-16 were selected for screening the cytotoxicity against human A549,MG63,SW480 and HepG2 cell lines.The result shows that compounds 1-7 displayed good cytotoxicity towards HepG2 at 10μg/ml.And the inhibition rate of compound 1 is 59.3%.Compounds 14-16 have significant cytotoxicity on A549.The inhibition rate of 14 is 64.9%.The DPPH radical scavenging activity evaluation of these compounds were conducted at the concentration of 10μg/ml.Compounds 1-7,14-16 all exhibited good antioxidant activities.Especially,the antioxidant activity of compound 15was comparable to the positive control group vitamin C.1.2 A total of 29 compounds were isolated and identified from the ethyl acetate extract of Hypsizygus marmoreus,mainly including benzaldehydes,terpenoids,steroids,and fatty acid compounds.They are echinuline（1）,citreorosein（2）,1,8-dihydroxyanthraquinone（3）,1-dihydroxyanthraquinone（4）,anthrarufin（6CI）（5）,alizarina（6）,alatinone（7）,rheochrysidin（8）,5-hydroxy-2-methylanthraquinone（9）,4,6-dimethoxyphthalide（10）,4,7-dimethoxyphthalide（11）,2-（3,8-epoxy-7,9,11-heptadienyl）-6-hydroxy-5-methyl-16-butenyl)benzal dehyde（12）,2-（3,8-epoxy-7,9-hep-tadienyl）-6-hydroxy-5-（17-methyl-16-butenyl）benzaldehyde（13）,dihydroauroglaucin（14）,2-（E-3-heptneyl）-3,6-dihydroxy-（3-methyl-2-butenyl）benzaldehyde（15）;isotetrahydro-auroglaucin（16）,iso-dihydroauroglauci（17）,flavoglaucin（18）,chaetopyranin（19）,6’-Oxo-chaetopyranin（20）,javanicin（21）,（2α）-A’-Neogammacerane-2,22-diol（22）,β-sitosterol（23）;stigmasterol（24）,ergosta-4,6,8（14）,22-tetraen-3-one 24-ethyl homolog（25）,episterin（26）,octadecanoic acid（27）,oleinic acid（28）,and glyceryl monooleate（29）.Thereinto,compounds 1-29 were obtained from this genus for the first time.While 13 of volatile components were identified by GC-MS analysis,which are mainly fatty acid compounds.Compounds 12-20 were selected for their anti-cancer activity study,with human A549,MG63,Sw480,and HepG2 as model cancer cell lines.It was demonstrated that benzaldehyde compounds displayed greater cytotoxicity towards Sw480cell line,while 19 has an IC₅₀0 of 13μg/mL.The DPPH radical scavenging activity study exhibited that 12-20 possess good antioxidant activity.The IC₅₀0 of 13-15 and 17 were 12.5,10.6,and 3.7μg/mL,respectively,displaying a better performance over the positive control group vitamin C.1.3 Compound 1-hydroxy-6-methyl-9,10-anthraquinone was isolated from the ethyl acetate extract of Lepista sordida for the first time,which demonstrated cytotoxicity towards all A549,MG63,SW480 and HepG2human cancer cell lines.While it possess greater cytotoxicity against A549,with IC₅₀0 of 48.2μg/mL.2 HSCCC method was employed for active compound separation and purification of Pleurotus ostreatus,Hypsizygus marmoreus,Lepista sordida crude extract.The key is the suitable solvent system.The solvent system was screened based on the average polarity p?and K values of crude sample according to the‘like dissolve like’principle.Whether the polarity of solvent system matches that of target compounds is the key in solvent systems screening.In the 5^stt section of chapter 1 and 2,the corresponding solvent system was optimized based on this principle.The rapid separation of active compound derived from the solid fermentation product of Pleurotus ostreatus and Hypsizygus marmoreus was achieved by utilizing HSCCC.5 types of naphthalene-γ-pyranone,and 6 types of benzaldehyde derivates were obtained.Our study proved the feasibility of optimization of the solvent system based on the average polarity p?and K values of crude samples.It provided new insights for HSCCC solvent system optimization,expanded the solvent system selection method for HSCCC,and expended the usage range of UV absorption method in solvent screening.More importantly,this method can be applied for the separation of various compounds with similar structures or isomers.The thin layer chromatography（TLC）-based GUESS method is a rapid and reliable method for HSCCC solvent system screening through the HEMWat meter.It was based on the connection between the Rf value of target compound and its corresponding K value in the solvent analyzed by TLC.In chapter 3,one anthraquinone compound was isolated from the crude sample of solid cultures of the fungus Lepista sordid by HSCCC based on the optimized solvent system.These results suggest that HSCCCisapowerfulmethodfortheseparationof naphthalene-γ-pyrones,benzaldehyde derivatives,and anthraquinone compound.This method provides a scientific,convenient and efficient way for the rapid isolation of active anti-tumor metabolites of fungi.Which pave the way for its anti-tumor,and antioxidant activity study,as well as the industrial production.2.1 Under the guideline of the average polarity of solvent system p?and the K value of crude sample,the chapter 1 focused on the methodology of separation of known compounds with optimized solvent system.The solvent system composed of PEMW（5:5:5:5,v/v）was screened out.The average polarity of the solvent system p?is 4.92,which is quite close to that of reported naphthalene-γ-pyrone compound 5.04.Meanwhile,the K value of the crude sample is 0.63,which is within the range of“optimum point”.Two groups of naphtho-γ-pyrone isomers（fonsecinone B and asperpyrone F;asperpyrone C,fonsecinone A and aurasperone A）were acquired from the crude of Pleurotus ostreatus through HSCCC,with the purities of 89.0,94.1,95.9,90.9,and 96.2%and recovery rates of 91.3,93.5,90.7,93.4,and 95.8%,respectively.Thus,an efficient and rapid separation procedure was established.It was shown that when the average polarity of the solvent system p?is quite close to that of reported similar compound,the K value of the crude sample is within the range of“optimum point”.This result illustrated the feasibility of optimizing solvent system with the average polarity of solvent system p?and the K value of crude sample as the guideline.While with the average polarity of solvent system p?of the similar compound as the reference,the screen pool of target solvent system could be significantly narrowed,thereby dramatically improving the screening efficiency.2.2 The chapter 2 studied on the methodology of separation of unknown compounds with optimized solvent system according to the guideline of the average polarity of solvent system p?and the K value of crude sample.The optimized solvent system was composed of PEMW（8:3:8:1,v/v）.In addition,the linear relationship between the average polarity of solvent system p?and the K value of crude sample of Hypsizygus marmoreus was found out.The corresponding equation was Y=19.333X²-118.44X+182.14,with R² of 0.997.While Y stands for K value and X stands for the average polarity of solvent system p?.Two groups of analogue benzaldehyde derivatives（iso-dihydroauroglaucin,isoaspergin,isotetrahydro-auroglaucin,andflavoglaucin;6’-Oxo-chaetopyranin and chaetopyranin）were obtained from Hypsizygus marmoreus by HSCCC,with the purities of 93.8,95.6,97.4,95.0,90.7,and 90.1%,and recovery rate of 95.4,94.2,95.7,93.4,95.8,and 92.8%,respectively.Therefore,the rapid separation procedure of Hypsizygus marmoreus was established.The results suggest that when the volume of solvent changed regularly,the average polarity of solvent system p?increased linearly with the K value of crude sample.It applicable to optimize the solvent system for HSCCC separation analogues based on K value of crude sample.Compare with the former method,which is based on the calculation of the average polarity p?and K values,this method is more convenient and rapid.Moreover,this method did not rely on the average polarity p?of a known similar compound,which dramatically widened the application of average polarity p?and K values in solvent system selection,and it can be used for the separation of unknown samples.2.3 The TLC-based GUESS method is a simple and quick method for HSCCC solvent system selection via the HEMWat meter.It has been proven that when the Rf value of target compound lies between 0.29 and0.71（optimum value of 0.5）,the K value will be located within the range of 0.4 to 2.5（optimum value of 1）.In chapter 3,the solvent system n-hexane-ethyl acetate-methanol-water（9:1:9:1,v/v）was screened out based on the TLC-based GUESS method,with petroleum ether as a replacement of n-hexane.The upper solvent was used to expand the target compound with a Rf value close to 0.5.The target compound1-hydroxy-6-methyl-9,10-anthraquinone with the purity of 95.6%and recovery rate of 97%was isolated from the crude of Lepista sordid via a one-step HSCCC method.The results indicate that the TLC-based GUESS method is applicable in the solvent system selection,which could greatly shorten the separation time.This method provide a more reliable and efficient way for the separation of active fungal metabolites with anti-tumor activity.