Structural Identification And Potential Antitumor Activity Study Of Chemical Constituents Of Symbiotic Fungi From Harmonia Axyridis

Natural products,which are special substances found in animals,plants and microorganisms,are important sources of drug discovery.At present,the probability of separating new active natural products from traditional separation sources is continuously decreasing,and the exploration of organisms in unusual niches has attracted the attention of scholars.The discovery of new biologically active natural products from the secondary metabolites of insect intestinal bacteria is one of the important research areas for the development of symbiotic bacteria resources.Insect symbionts have become the most potential group for discovering new compounds and new biologically active substances,which is a research focus for the development of new drugs and new pesticides.In this study,the symbiotic fungi of Harmonia axyridis?Coleopteran:Coccinellidae?were separated.The symbiotic fungi were subjected to the fermentation culture for the separation and identification of secondary metabolites,and the cytotoxic activities of some compounds were evaluated.1.The symbiotic fungi of Harmonia axyridis were identified by morphology combined with the similarity and phylogenetic status of the reference sequences in GenBank.Four fungi were identified,namely Fusarium equise,Setosphaeria rostrate,Alternaria alternata and Exserohilum mcginnisii.2.Four symbiotic fungi were solid-fermented by rice,and then the secondary metabolites were separated by silica gel column chromatography,Sephadex LH-20 gel chromatography,ODS reversed phase column chromatography,preparative silica gel thin layer chromatography,high performance liquid chromatography and recrystallization.Structural analysis was carried out by means of nuclear magnetic resonance,infrared spectroscopy,ultravoilet spectroscopy,high resolution mass spectrometry,ECD calculation and single crystal diffraction.A total of 63 compounds were isolated and identified in the secondary metabolites of four symbiotic fungi,including nine new compounds?compounds 1,5,6,16,17,21,39,61 and 62?,and one new natural product?compound 41?.3.MTT assay was used to test the anti-tumor cytotoxic activity of some isolated compounds?compounds 1,2,21,22,27,and 30?,which illustrated that compound 2performed obvious inhibitory activity to four types of human tumor cells including human breast cancer cells?MCF-7?,human gastric cancer cells?MGC-803?,human cervical cancer cells?Hela?and human hepatoma cells?Huh-7?.IC₅₀0 values were 21.00,2.37,46.53 and13.10?g/mL,respectively(IC₅₀0 values for control cisplatin were 2.05,3.47,5.13 and 8.27?g/mL).Compound 2 had a stronger inhibitory effect on MGC-803 cells than that of cisplatin,which has the value for further research and development.4.The results of migration assay showed that the mobility rate of MGC-803 cells was significantly lower than that of the cells in the control group at 48 h when the concentrations of compound 2 were 3 and 6?g/mL?P<0.01?;When the concentrations of compound 2 were12.5,25 and 50?g/mL,the mobility rate of MCF-7 cells was significantly lower than that of the cells in the control group?P<0.01?;With the concentrations of compound 2 at 10 and 20?g/mL for 24 h,the mobility rate of Huh-7 cells was significantly reduced compared with that of the cells in the control group?P<0.05?.The results of the shuttle test showed that when the concentrations of compound 2 were 1.5,3 and 6?g/mL at 24 h,the number of transmembrane cells of MGC-803 was significantly lower than that of the control group?P<0.01?;At 48 h,when the concentrations of compound 2 were 3 and 6?g/mL,the number of transmembrane cells of MGC-803 was significantly lower than that of the control group?P<0.01?;When the concentrations of compound 2 were 6.25,12.5 and 25?g/mL,the number of transmembrane cells of MCF-73 was significantly lower than that of the control group?P<0.01?at 48 h.5.DAPI staining results showed that the concentrations of compound 2 were 1.5,3 and 6?g/mL at 24 h,the apoptotic bodies were presented in MGC-803 cells.At 24 h,the apoptotic bodies appeared in MCF-7 cells when the concentrations of compound 2 were 12.5,25 and 50?g/mL,respectively.6.Apoptosis and cycle test results showed that the apoptosis rates of MGC-803 cells were 24.15%and 42.47%when the concentrations of compound 2 were 3 and 6?g/mL;When the concentrations of compound 2 were 12.5,25 and 50?g/mL,the apoptosis rates of MCF-7 cells were 43.14%,52.99%and 53.76%,respectively.When the concentration of compound 2 was 1.5?g/ml,the cycle arrest of MGC-803 cells was in G1 phase;At the concentrations of 3 and 6?g/mL,the cycle arrest of MGC-803 cells was in S phase?P<0.05?.At the concentrations of 12.5,25 and 50?g/mL,the cycle arrest of MCF-7 cells was in S phase.Western blot results showed that compound 2 could induce MGC-803 cells apoptosis through mitochondrial Bcl-2 family which triggered endogenous apoptotic pathway?up-regulation of Bax and down-regulation of Bcl-2?.7.RT-CA assay showed that compound 2 performed anti-mitotic effect on MGC-803cells with the concentration of 1.5?g/mL,had DNA damaging effect to MGC-803 cells with the concentrations of 3 and 6?g/mL,presented DNA damaging effect on MCF-7 cells with the concentrations of 12.5,25 and 50?g/mL,and had the cytoskeletal inhibitory effect on Huh-7 cells when the drug concentrations were 2.5,5 and 10?g/mL.In this study,four symbiotic fungi were separated from Harmonia axyridis,and nine new compounds were isolated and identified from the secondary metabolites.Compound 1 is a new natural product of[6/6/5/5]tetracyclic system polyketide carbon skeleton with 8 chiral centers,and compound 62 is a new compound with enol structure.Compound 2 could induce MGC-803 cells apoptosis by up-regulating Bax protein expression and down-regulating Bcl-2protein expression,thus inhibiting cell proliferation.Meanwhile,Compound 2 showed anti-mitosis effect,DNA damaging effect,and cytoskeletal inhibition effect.Therefore,compound 2 has the potential to be developed as anti-tumor drugs.