title: Microbial biotransformation of gentiopicroside by the endophytic fungus Penicillium crustosum 2T01Y01.
abstract: Endophytic fungi are symbiotic with plants and possess multienzyme systems showing promising metabolite potency with region selectivity and stereoselectivity. The aim of this study was to use these special microorganisms as an in vitro model to mimic the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, compound 1). The fungi isolated from a medicinal plant, Dendrobium candidum Wall. ex Lindl., were screened for their biotransformation abilities with GPS as the substrate, and one strain with high converting potency was identified as Penicillium crustosum 2T01Y01 on the basis of the sequence of the internal transcribed spacer of the ribosomal DNA region. Upon the optimized incubation of P. crustosum 2T01Y01 with the substrate, seven deglycosylated metabolites were detected by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS). Preparative-scale biotransformation with whole cells of the endophytic fungus resulted in the production of five metabolites, including three novel ones, 5α-(hydroxymethyl)-6β-methyl-3,4,5,6-tetrahydropyrano[3,4-c]pyran-1(8H)-one (compound 2), (Z)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 3), and (E)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 4), along with two known ones, 5α-(hydroxymethyl)-6β-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 5) and 5α-(hydroxymethyl)-6α-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 6), aided by nuclear magnetic resonance and high-resolution mass spectral analyses. The other two metabolites were tentatively identified by online UPLC/Q-TOF MS as 5-hydroxymethyl-5,6-dihydroisochromen-1-one (compound 7) and 5-hydroxymethyl-3,4,5,6-tetrahydroisochromen-1-one (compound 8), and compound 8 is a new metabolite. To test the metabolic mechanism, the β-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with ρ-nitrophenyl-β-d-glucopyranoside as a probe substrate, and the pathway of GPS biotransformation by strain 2T01Y01 is proposed. In addition, the hepatoprotective activities of GPS and metabolite compounds 2, 5, and 6 against human hepatocyte line HL-7702 injury induced by hydrogen peroxide were evaluated.
authors.affiliation: MOE Key Laboratory for Standardization of Chinese Medicines and SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Wen-Liang
authors.initials: WL
authors.lastname: Zeng
authors.forename: Wan-Kui
authors.initials: WK
authors.lastname: Li
authors.forename: Han
authors.initials: H
authors.lastname: Han
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Tao
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Zheng-Tao
authors.initials: ZT
authors.lastname: Wang
authors.forename: Kai-Xian
authors.initials: KX
authors.lastname: Chen
mesh_terms: D001711:Biotransformation; D002853:Chromatography, Liquid; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D031667:Dendrobium / Q000382:microbiology; D060026:Endophytes / Q000378:metabolism; D057888:Iridoid Glucosides / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010407:Penicillium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D057888:Iridoid Glucosides; C012997:gentiopicroside