Study On The Endophytic Penicillium Janthinellum From Panax Notoginseng And Its Secondary Metabolites

The long-time co-existence of endophytes and host plant make their relationship complex.Meanwhile,endophytes can produce massive secondary metabolites with lots of activities,such as antitumor,antimicrobe and antivirus.Therefore,endophyte as a resource repository for natural medicines is worth exploring.In this work,an investigation was carried out on an endophyte Penicillium janthinellum(No.SYPF 7899)obtained from the root of three-year-old Panax notoginseng.A total of 32 compounds were isolated from the solid fermentation culture of SYPF 7899 through various chromatographic techniques,such as silica gel column chromatography(CC),ODS CC,Sephadex LH-20 CC and preparative high performance liquid chromatography(p-HPLC).On the basis of physico-chemical property,spectroscopic analyses(UV,IR,MS,1H-NMR,13C-NMR and 2D-NMR),calculated electronic circular dichroism(ECD)and chemical derivatization analysis,the structures of the compounds were identified as brefeldin El(1),brefeldin E2(2),brefeldin E3(3),brefeldin E4(4),brefeldin E5(5),brefeldin A(6),brefeldin A 7-O-acetate(7),alternariol-5-O-methyl ether(8),3'-hydroxyalternariol-5-O-methyl ether(9),mangrovamides A(10),brasiliamide J-a(11),brasiliamide J-b(12),peniciolidone(13),dehydroaustinol(14),dehydroaustin(15),7-hydroxydehydroaustin(16),1,2-dihydro-acetoxydehydroaustin(17),austinol(18),austin(19),5R-isoaustinone(20),11?-acetoxyisoaustinone(21),4-toluene-1,3-dicarbamic acid dimethyl ester(22),glicoricone(23),striatisporolide A-11-formylation(24),4-hydroxy-benzaldehyde(25),orcinol(26),penicillic acid-a(27),furanone glucoside(28),penicillic acid-b(29),5-hydroxy-3-isopropyl-4-methoxyfuranone(30),(21S,3R)-2,3-epoxyhexanoic acid(31),methyl 2-(2-acetyl-3-hydroxy-5-methoxyphenyl)acetate(32).Among them,eight compounds(1-5,11-13)are new compounds.All the isolated compounds were screened for bioactivites,including antimicrobial,antineoplastic and antiviral activities,some of them showed better results.Of which compound 6 and 7 had a signifi cant inhibitory effects on the test strains of Bacillus cereus,Bacillus subtilis,Escherichia coli,Klebsiella pneumonia,Pseudomonas aeruginosa and Staphylococcus aureus.The minimum inhibitory concentrations(MICs)of compound 6 were 20,25,22,35,36 and 28 p,g/mL and compound 7 were 21,28,22,38,39 and 31?g/mL,respectively.Compound 11 and 12 exhibited antibacterial activities against gram-positive strains B.cereus,B.subtillis and S.aureus,the MICs of compound 11 were 25,18 and 18 ?g/mL,and compound 12 were 21,15 and 18 ?g/mL,respectively.Electron microscopic observation showed that B.subtilis grew longer and S.aureus swollen without division after treated with compound 11 and 12.Further,molecular docking studies revealed that the antibacterial mechanism of compound 11 and 12 may combine with filamentous temperature-sensitive protein Z(FtsZ),resulting in the inhibition of cell division.Meanwhile,compound 6 and 7 showed cytotoxicity to the four tumor cell lines,including HepG2,Huh7,KB and MDA-MB-231 by MTT method.The half inhibitory doses(ID50)of compound 6 were 0.024,0.028,0.0625 and 0.064?M and compound 7 were 0.024,0.035,0.06 and 0.064 ?M,respectively,similar to the positive control drug L-deoxycytidine(LODDC).The antitumor actions of compound 6 and 7 was to inhibite the S phase of HepG2.In the antiviral test,compound 6 and 7 showed better antiviral activity against Hepatitis C virus(HCV)with ID50 0.022 and 0.013?M.Endophyte P.janthinellum and its metabolites displayed antagonism against root-rot pathogens of Panax notoginseng,such as Alternaria brassicicola,Fusarium solani and Cylindrocarpon destructans.Compound 6 and 7 exhibited the strongest inhibitory activities(MIC<30?g/mL).Biocontrol agent prepared with P.janthinellum was applied in the field and protected P.notoginseng,as evidenced by the reduced disease incidence,higher plant height,higher stem weight,higher root weight,higher activities of defence enzymes,including peroxidase(POD),superoxide dismutase(SOD)and catalase(CAT),higher saponin accumulation and no damage to the plant root.Furthermore,P.janthinellum was proven to successfully colonize the rhizosphere soil of P.notoginseng for a long time by egfp-labelling experiments.The probiotic effects of P.janthinellum on P.notoginseng are closely related to the colonization,reproductive capability,plant protection and promotion the biosynthesis of pharmaceutical substances,indicating its potential as a biocontrol agent.