Study On The Bioactive Constituents Of Dryopteris Crassirhizoma And Its Endophytic Fungi

The rhizomes of Dryopteris crassirhizoma（“mian ma guan zhong”in Chinese）are one of the main ingredients of two well known traditional Chinese medicines（TMCs）“kang gan ke li（KGKL）”and“lian hua qing wen（LHQW）”in Chinese)which were widely used to treat cold and influenza.The latter TMC（LHQW）,a patented Chinese medicine product which was previously applied for the treatment of many public health emergencies in China such as SARS,H1N1 and H3N2 influenza has recently been approved by National Medical Products Administration（NMPA）in treating fever,cough,or fatigue caused by COVID-19.123 Compounds were isolated by silica gel,TLC,Sephadex LH-20 gel,MCI,MPLC,semi-preparative HPLC,including 45 new compounds,were isolated from“mian ma guan zhong”and its endophytic.Their structures were assigned by the 1D and 2D NMR,ESIMS,HRESIMS,ECD,OP,IR,UV/Vis,and X-Ray spectroscopic analyses.Structural types of the isolates involved acylphloroglucinol meroterpenoids,tocopherol derivatives,ergosterols,aromatic compounds,alkaloids,ect.Seventy-eight compounds,including thirty novel meroterpenoids（3～10,13,14,19～34,39,40,41,and 42）with an unprecedented skeleton consisting of dimeric or trimeric acylphloroglucinols and sesquiterpene were isolated from the rhizomes of D.crassirhizoma.Additionally,nine previously undescribed compounds including six tocopherol derivatives（43～48）and three acylphloroglucinol derivatives（49～51）were isolated from the rhizomes of D.crassirhizoma.The stereochemistries of eight reported meroterpenoids with undefined chiral centers were reassigned.Two intriguing methods by analyzing a)the regularity of chemical shift variation of protons and carbons around the stereogenic centers,and b)pyridine-induced deshielding effect of hydroxy groups,to discriminate relative configurations of flexible long-chain alcohol with chiral centers separated by three or seven covalent bonds,were successfully applied.A non-enzymatic biosynthesis of 1～24 was assumed based on a rare single-crystal cluster formed with two diastereomeric enantiomer pairs（±1/±2）and chiral HPLC analyses.The novel structures（25～34）including absolute configurations were established by comprehensive spectroscopic analyses and quantum chemical electronic circular dichroism（ECD）calculations.Their biosynthetic pathway was assumed.The biomimetic synthesis of dimeric acylphloroglucinol-nerolidol meroterpenoid was achieved using the proposed biosynthetic pathway.The absolute configurations of compounds（43～48）were established by NMR spectroscopic analyses and calculated ECD spectra.Compounds 43 and 44 are the first tocopheroid derivatives possessing unique 2,5-dimethylcyclopent-4-ene-1,3-dione carbon skeleton,and compounds 45～48were new 5a-norcyclopentenones having a spirofused bicyclic carbon skeleton.New compounds:（±）-dryoptols/（±）-3’’-epi-dryoptols A-O（1～24,35～38,41,and 42）,dryoptins/11’’-epi-dryoptins A～F（25～34,39,and 40）,crassirhizomic acid A～E（43～48）,crassirhizomic acid F（49）,crassirhizomiate acid F methyl ester（50）,and crassirhizomic acid G（51）.Meroterpenoids 13 and 14 showed obvious inhibitory effects on NO production in LPS-induced RAW264.7,and suppressed the expression of i NOS,COX-2,IL-1β,and IL-18.Their anti-inflammatory activity was closely related to the inhibition of the formation and function of inflammasomes.Additionally,the known 54 showed antiviral efficacy against the influenza viruse A/Puerto Rico/8/1934（H1N1）.The trimeric acylphloroglucinol meroterpenoids 31/32 showed significant antifungal activity against standard Candida albicans with MIC₅₀ value of1.61μg/m L[fluconazole（FLC）:3.41μg/m L].When combined with FLC,compound45 showed significant antifungal activity against standard C.albicans with MIC₅₀ value of 1.19μg/m L（FLC:3.41μg/m L）,compounds 59 and 61 exhibited strong antifungal activities against FLC-resistant C.albicans with MIC₅₀ values of 8.39,and 7.16μg/m L（FLC:>100μg/m L）,respectively.Furthermore,the anti-plant pathogenic fungi and bacterial activities have been evaluated in vitro,compounds 26,29/30,43,47,50,56,58,and 61 exhibited inhibitory effects against several pathogenic fungi and bacteria,with MIC values of 6.25～50μg/m L.Meanwhile,endophytic fungi of medicinal plants are natural resources for obtaining the active chemical components.Screening lead compounds from secondary metabolites of endophytic fungi is one of the important means of drug discovery.There are no reports on endophytic fungi and their active secondary metabolites of D.crassirhizoma.a series of endophytes were isolated from the root of D.crassirhizoma.According to the antimicrobial activity and chemical screening result,three bioactive strains Trichoderma afroharzianum（HP-3）,Alternaria alstroemeriae（HP-7）,and Penicillium reticulisporum（HP-13）were selected for scale-up fermentation,and the Et OAc extract of their broth were then investigated,leading to the isolation of 45compounds including two new compounds 2-hydroxyphenethyl 2-phenylacetate and2,4-dihydroxy-3-farnesyl-5-methoxy benzoic acid.In addition,selected compounds were tested for their activity against plant pathogenic fungi and bacteria.