【作者】 刘鑫；

【导师】 于淼；

【作者基本信息】 哈尔滨商业大学 ， 中药学， 2024， 硕士

【摘要】 元宝草(Hypericum sampsonii Hance)为金丝桃属(Hypicum Linn)中的传统中草药,内含丰富的化学成分,主要有多环多异戊烯基间苯三酚衍生物、各类酮属及挥发油等,这些成分同时赋予了元宝草抗癌、抗肿瘤、抗抑郁等多重现代药理作用。近年来,药用植物内生真菌作为一类特殊的天然生物资源,因其次级代谢产物的丰富性和结构新颖性备受关注,而有关元宝草内生真菌的研究尚处于探索的初级阶段,还未有系统性的研究框架来对其进行全面深入的探讨,因此,本论文以元宝草内生真菌次级代谢产物为研究对象,利用分离纯化手段、结构鉴定技术和生物活性评价方法,以期发现具有生物活性的天然产物。研究内容主要包括:内生真菌的分离鉴定、小发酵及活性筛选、目标真菌大发酵及次级代谢产物分离纯化、单体化合物结构鉴定及生物活性评价。从元宝草中分离出11株真菌,ITS序列比对确定各内生真菌种属。随后采用大米、燕麦两种培养基进行小发酵培养,提取物经TLC、HPLC化学分析以及抗菌、抗肿瘤、抗炎活性筛选,基于次级代谢产物丰富度及活性强弱,发现内生真菌Penicillium citrinumS23的大米发酵产物丰富且具有显著的生物活性。因此,采用大米培养基对S23扩大规模发酵,利用正相硅胶、反向ODS、凝胶柱层析以及半制备HPLC等色谱分离方法从乙酸乙酯提取物中分离获得15个单体化合物,其中新结构化合物3个(1*、2*、3*)。利用NMR(1D和2DNMR)和HRESIMS数据鉴定了 15个化合物的结构。其结构类型包括有桔霉素二聚体 5个:Citrinone A(1*)、Citrinone B(2*)、Citrinone C(3*)、1-epi-citrinin H1(4)、Penicitrinol A(5),桔霉素单体化合物 4 个:Stoloniferol B(10)、Sclerotinin B(11)、Decarboxydihydrocitrinin(12)、Hydrogenolysis(13),稀有 C25 甾体类化合物 4 个:Neocyclocitrinol A(6)、24-Oxocyclocitrinol(7)、Neocyclocitrinol B(8)、Neocyclocitrinol C(9),异戊烯基化的醌类化合物1个:Quinone Alcohols(14),喹诺酮亚胺类化合物1个:Quinolonimide(15)。最终,对这些化合物进行生物活性评估。目的在于鉴定Penicillium citrinum S23中的有效活性成分,同时验证了这些成分在经过一系列分离纯化过程后,其生物活性是否保持稳定,和与初步筛选实验的结果是否一致。生物活性研究结果表明,化合物4对SA存在中敏抗菌作用,化合物1*、2*、3*、13和14在MCF-7细胞中表现出强抗肿瘤活性,化合物5、6与14在HepG2细胞中表现出强抗肿瘤活性,化合物1*、2*、3*、4、5,14有显著抗炎作用,但4同时伴随明显Raw 264.7巨噬细胞毒性,与初筛活性结果吻合。更多还原

【Abstract】 Hypericum sampsonii Hance is a traditional Chinese medicine in the genus Hypericum Linn,which is rich in chemical constituents,mainly PPAPs,various ketone genera and volatile oils,etc.These constituents simultaneously endow Hypericum Hance with multiple modern pharmacological effects such as anticancer,antitumour and antidepressant effects.In recent years,endophytic fungi of medicinal plants,as a special class of natural biological resources,have attracted much attention due to the abundance and structural novelty of their secondary metabolites,while the research on endophytic fungi of Hypericum sampsonii Hance is still in the primary stage of exploration,and there is no systematic research framework for a comprehensive and in-depth discussion of them.Thus,the present work was carried out to study the secondary metabolites of endophytic fungi of Hypericum sampsonii Hance,using isolation and purification means,structural identification techniques and bioactivity evaluation methods,with the aim of discovering natural products possessing bioactivity.The study mainly includes:isolation and identification of endophytic fungi,small fermentation and activity screening,large fermentation of target fungi and purification of secondary metabolites,structure identification of single compounds and evaluation of biological activities.Eleven fungal strains were isolated from Hypericum sampsonii Hance,and ITS sequence comparison was performed to determine the genus of each endophytic fungus.Then.two media,rice and oat,were used for small-scale fermentation culture,and the extracts were screened for TLC and HPLC chemical analysis as well as antimicrobial,antitumour,and anti-inflammatory activies,and based on the abundance of secondary metabolites and the strength of their activities,it was found that the endophytic fungus Penicillium citrinum S23 was rich in rice fermentation products and possessed significant bioactivities.Therefore,rice medium was used for the scale-up fermentation of S23,and 1 5 monomeric compounds were isolated from the ethyl acetate extract using chromatographic separation methods such as CC,ODS,GFC,and semiprep HPLC,including three(1*,2*,3*)compounds with new structures.The structures of the 15 compounds were identified using NMR(1D and 2D NMR)and HRESIMS data.The structural types include five citrinin dimers:Citrinone A(1*),Citrinone B(2*),Citrinone C(3*),1-epi-citrinin H1(4),Penicitrinol A(5),and four citrinin monomer compounds:Stoloniferol B(10),Sclerotinin B(11),Decarboxydihydrocitrinin(12),Hydrogenolysis(13),and four rare C25 steroids:Neocyclocitrinol A(6),24-Oxocyclocitrinol(7),Neocyclocitrinol B(8),Neocyclocitrinol C(9),one isopentenylated quinone:Quinone Alcohols(14),and one quinoloneimide:Quinolonimide(15).Lastly,the compounds were evaluated for their biological activity.The aim was to identify the active components in Penicillium citrinum S23,and also to verify whether the biological activity of these components remained stable after a series of isolation and purification processes,and whether it was consistent with the results of the preliminary screening experiments.The results of the bioactivity studies showed that compound 4 has a moderately sensitive antimicrobial effect on SA,compounds 1*,2*,3*,13,and 14 showed strong antitumour activity in MCF-7 cells,compounds 5,6 and 14 showed strong antitumour activity in HepG2 cells with,compounds 1*,2*,3*,4,5,and 14 had a significant anti-inflammatory effect but 4 was accompanied by significant Raw 264.7 macrophage toxic activity,the results are consistent with the results of the initial screening acti vity.更多还原