Chemical Investigation On Huperzia Serrata And Studies On The Metabolites And Biotransformation Process Of Its Fungal Endophytes

Fungal endophytes refer to fungi that live within a plant for at least part of its life without causing apparent disease.During the process of co-evolution,metabolic interactions may be established between fungal endophytes and the hosts.Neither the function nor the underlying mechanism of fungal endophytes in the metabolic interaction was disclosed,though,fungal endophytes have been proved to contribute to the hosts'resistance to insect,disease and drought,as well as to the formation of active constituents in the hosts.Also,such interactions may regulate the metabolism of the endophytes and endow them with the ability to produce metabolites with unique chemical diversities and distinct bioactivities.With the aim of discovering new lead compounds with novel structures and potent bioactivities,a study was carried out to investigate the bioactive compounds derived from the traditional Chinese medicinal plant Huperzia serrata and its fungal endophytes.Studies include the isolation and screening of fungal endophytes,purification and elucidation of the alkaloids constituents of Huperzia serrata and the metabolites of the target strains,the biotransformation of huperzine A by the fungal endophytes,as well as the preliminary evaluation for the bioactivities of the pure compounds.49 fungal endophytes were isolated from the inner tissues of Huperzia serrata collected in Xishuangbanna Tropical Plant Garden,Yunnan Province and Panan County,Zhejiang province.In the preliminary screening,one strain with the ability to transform huperzine A and another three strains producing abundant metabolites were chosen for further studies based on the TLC analyses.By means of spectroscopic analyses,103 pure compounds including 31 new ones were obtained.Chemical study on the constituents of the plant Huperzia serrata furnished eighteen alkaloids(1-18)including four new ones(1-4),among which 1-3 were alkaloids with dihydroxy-substituted lycopodine skeleton,and 4 was a phlegmarine-type lycopodium alkaloid possessing pyridine and N-methoxy-substituted piperidine partial structures.Thirty two compounds(19-50),including two new ones tryhistatin(19)and(2S)-2,3-dihydro-7-hydroxy-6,8-dimethyl-2-[(E)-prop-1-enyl]-Chrom an-4-one(23),were identified from the strain Penicillium chrysogenum HS-3.Complete NMR data assignment was accomplished for 16-hydroxyroquefortine C(20)and cyclo(D-Pro-L-Trp)(36)for the first time.And perlolyrine(37),a constituent of traditional Chinese medicinal plant Ligusticum chuanxiong,Codonopsis pilosula and Panax Ginseng,was obtained for the first time from an endophyte.Eleven compounds(51-61)were obtained from the metabolites of Peyronellaea glomerata XW-12.Among them,compound 56,a known one without any available chemical information,was obtained from a natural source for the first time,as well as its complete NMR data assignment.Nine compounds(62-70)with unusual C25 steroid skeleton were separated and elucidated from the strain Penicillium chrysogenum P1X.Three of them,11-hydroxyneocyclocitrinol(62),norcyclocitrinol A(63)and pesudocyclocitrinol A(64)were new and norcyclocitrinol A(63)represented the first example of C25 steroid with a C4 side chain.Twenty eight compounds(71-91)including eighteen new ones were isolated from the strain Ceriporia lacerata C13A.Fourteen(71-84)of the new isolates belong to tremulane sesquiterpenes.In particular,ceriponol B(72)possessed an unprecedented 12-nortremulane skeleton,while ceriponol K(81)and ceriponol P(84)contained an unusual 7-oxa-bicyclo[4.2.1]nonane and an unusual 5,6-seco moiety,respectively.Compounds 87-88 were new lanostane triterpeniods while Ceriponone A-B(85a and 85b)and 8-methoxyceriponone A-B(86a and 86b)were furan derivatives obtained as two pairs of insepratable diastereoisomers.The biotransformation of Huperzine A with the strain Ceriporia lacerata C13A was also studied,leading to the isolation and elucidation of 8 compounds.Among them,93-95,tremulane sesquiterpenes characteristic of Ceriporia lacerata C13A were isolated again.The other five compounds 99-103 were originated from huperzine A.Among them,99-102 were novel "sesquiterpene-alkaloid" hybrids featuring an unprecedented moiety in which tremulane sesquiterpenes were connected to the primary amino group of huperzine A via the hemiacetal methine.To the best of our knowledge,it was the first case of biotransformation in which microorganism attached its own secondary metabolites to the exogenous substrate.All the new compounds and some of the known compounds were evaluated for their in vitro acetylcholinesterase inhibitory(AchEI)and cytotoxic activities.Both ompounds 24 and 25 showed potent cytotoxic activity against Hela cell lines(IC50 = 1.6 and 7.4?M,respectively)and compounds 24,25,76 and 81 exhibited weak cytotoxic activity against HepG2 cell lines(IC50 = 27.2,44.4,32.3 and 35.8 ?M,respectively).In the AchEI assay,activities of compounds 99-102 decreased drastically while compared to huperzine A,but the structural resamblence with ZT-1 rendered them the potential of being developed as prodrugs.In summary,103 pure compounds including 31 new ones were obtained through this work.In the process of exploring the biotransformation of host-originated substance by fungal endophytes,a thoroughly new mode of transformation was discovered,in which endophytes attach its own secondary metabolites to the exogenous substrates via covalent bonds to form completely new compounds.The results indicated that fungal endophytes could establish direct chemical interactions between fungal metabolites and plant constituents,which represented a new form of metabolic interations between them.Rational and efficacious implementation of this new mode will offer a new way of acquiring lead compounds and will also provide a new breakthrough point for probing the interactions between fungal endophytes and their hosts.