【作者】 张春阳；

【导师】 娄红祥；

【作者基本信息】 山东大学 ， 天然药物化学， 2022， 博士

【摘要】 苔藓属于孢子植物,在孢子体成熟之后即释放孢子,世代交替繁殖。苔藓在分类学上界于藻类和蕨类之间,包括苔纲(6000种),藓纲(14000种)和角苔纲(300种)三大类群。苔类植物中的油体——富含醚类、萜类和芳香类化合物的膜结合型细胞器,从微观角度解释了苔类植物的化学及其生物活性多样性的原因。自70年代以来苔类植物的化学生物学研究取得了长足的发展,主要有联苄和萜类两大特征化合物。此外,结合方式多样,骨架特殊的二聚体也是苔纲植物的特色。除了骨架新颖,苔类植物中的化合物具有广泛的生物学活性,比如植物生长调节、抗氧化、抗炎、抗真菌和细胞毒等。本文对采自中国的四种苔类植物,包括浙江九龙山的尖瓣萹萼苔(Radula apiculata Sande Lac.ex Stephani)、湖南张家界的多形带叶苔[Pallavicinia ambigua(Mitt.)Stephani]、四川雅安的裸萼小萼苔(Mylia nuda Inoue&Yang.)和海南三亚的指叶苔[Lepidozia reptans(L.)Dumort]的化学成分及其生物活性进行了系统的阐述。采用柱层析和半制备液相,基于核磁共振、质谱、圆二色谱、红外、紫外、单晶衍射、核磁计算以及含时密度泛函理论ECD计算等技术手段,从这四种苔类植物中分离鉴定了 87个化合物,其中53个首次报导,18个为新骨架。这些化合物大部分都是异戊烯化联苄和开环的萜类化合物及其二聚体,包括异戊烯联苄衍生物及其二聚体、7,8-开环的半日花烷型二萜及其二聚体、1,10-开环香橙烷型倍半萜及其与苯醚类化合物形成的二聚体以及9,10-开环环阿屯烷型三萜。对所有化合物进行生物活性研究,发现异戊烯化联苄和分子中含有α,β-不饱和酮结构的萜类化合物具有较好的细胞毒活性。对采自浙江丽水九龙山国家自然保护区的尖瓣萹萼苔继续研究,获得了 12个异戊烯联苄的衍生物,七个为新化合物,包括四个异戊烯联苄的二聚体[(±)-radulapins L-O(1-4)]。在硕士研究基础上,对从尖瓣萹萼苔中纯化的26个异戊烯联苄进行细胞毒活性筛查,发现二聚体均有显著的细胞毒作用,进一步研究发现,其通过升高ROS水平,降低线粒体膜电势诱导PC-3细胞凋亡。对九个异戊烯联苄二聚体生源推测,发现这类特殊结构的二聚体是Diels-Alder环加成以及自由基耦合作用的结果。对采自张家界的多形带叶苔化学成分的深入研究,发现25个化合物,17个为新化合物,包括十个高度氧化的半日花烷化合物[pallaviambins I-R(21-29)]和四个7,8-开环的半日花烷二聚体[pallaviambins E-H(17-20)],基于结构观察,推测这些二聚体是酶催化下两个半日花烷通过Diels-Alder环加成形成。除此之外,在这个苔中还获得两个Δ13(14)互为顺反异构,具有一个四环[5.2.1.02,5.04,10]十烷的新骨架的微量成分[pallaviambins A(13)和B(14)]。对其前体,化合物33进行光照实验表明,化合物13和14由化合物33经光催化,分子内两个双键在自由基作用下发生[2+2]环加成。在MTT细胞毒活性筛查中,发现一些氧化度高,分子中具有α,β-不饱和酮结构片段的分子具有不同程度的细胞毒作用。以往报道证明具有α,β-不饱和酮结构类化合物的抗肿瘤机制与活性氧水平有关。诱导ROS变化在多种治疗手段中杀灭癌细胞的一种有效机制。药物诱导的“氧化还原重置”则会导致化疗耐药,打破“氧化还原重置”将提供一种克服耐药的新方法。因此,将代表性化合物33进行进一步的探索,发现33对PC-3具有选择性抑制作用,并且抑制肿瘤增殖发生在G2/M期。后续的活性研究发现其能升高ROS水平,破坏线粒体功能诱导PC-3细胞凋亡。从采自四川雅安的裸萼小萼苔中分离鉴定了 32个化合物,其中18个为新化合物,包括四个苯醚类化合物[mylnudethers A-D(38-41)],五个骨架罕见的香橙烷类倍半萜以及八个苯醚与香橙烷型倍半萜形成的二聚体[mylnudones A-H(42-49)]。分析结构,发现八个二聚体中,七个由Diels-Alder环加成途径形成,一个为自由基耦合产物。除此之外还得到了 14个已知化合物。将这些化合物进行抗真菌活性研究,除了 68号的剪叶苔烷型倍半萜对双敲菌株DSY654表现一定的抗真菌毒力,其他的化合物活性表现较弱。从海南三亚的指叶苔中分离纯化并鉴定了 18个化合物,其中11个新化合物,包括九个首次在苔藓植物中发现的9,10-开环的环阿屯烷型的三萜类化合物[lepidozins A-I(70-78)]。在细胞毒活性筛查中发现,化合物70、75和76对系列肿瘤细胞株展示了显著的细胞毒抑制作用。其中化合物76结构中存在一个α,β-不饱和羰基,显示了更强的细胞毒活性。随后研究表明,其通过升高ROS水平介导的线粒体损伤诱导PC-3细胞凋亡。本课题在前人研究的基础上,获得了一些结构新颖,骨架特殊的化合物,尤其是二聚体居多,大部分二聚体都是在植物体氧化酶作用下发生Diels-Alder环加成和自由基耦合的产物。随着分离和鉴定的技术发展,更多微量成分被发现,极大地丰富了天然产物化合物库。对这些化合物进行生物学活性筛查,也表现出令人满意的生物学活性,为新药开发提供了若干个潜在的苗头小分子化合物。苔类植物的化学成分的研究结果表现出一定的种属特异性,这对于苔类植物的化学分类也有一定的参考价值。更多还原

【Abstract】 Bryophytes as sporophytes,which release spores and reproduce alternately from generation to generation after the sporophyte matures,are placed taxonomically between algae and pteridophyte.There are approximately 23,000 species in the world,and divided into mosses(14,000 species),liverworts(6,000 species),and homworts(300 species).The secondary metabolites,isolated from liverworts,are more novel in species and structures than other two bryophytes,which may be morphologically related to the appealing and characteristic cell organelles,possessing a cellular oil body.Great progress has been made in the chemical and biological study of liverworts since the 1970s,and it has been found that there are two kinds of characteristic compounds of bibenzyl and terpenoids in liverworts.In addition,the dimers with various binding modes and special skeleton are also the characteristic of liverworts.Apart from the novel skeleton,the compounds from liverworts show a wide range of biological activities,such as cytotoxic,antifungal,anti-inflammatory,antioxidant,plant growth regulation,anti-alzheimer’s disease and others.In this research,four Chinese liverworts,including Radula apiculata Sande Lac.ex Stephani,Pallavicinia ambigua(Mitt.)Stephani,Mylia nuda Inoue&Yang,and Lepidozia reptans(L.)Dumort,were phytochemically investigated.Using column chromatography and semi-prepared liquid phase,based on nuclear magnetic resonance,mass spectrometry,circular dichroism,infrared,ultraviolet,single crystal diffraction,nuclear magnetic calculation and time-dependent density functional theory ECD calculation,a total of 87 compounds,including 53 new ones,were isolated and identified from these four liverworts.Most of these compounds are bibenzyls,terpenes and their dimers,of which prenylated bibenzyls and their dimers,7,8-seco-labdane and their dimeric compounds,1,10-seco-aromadendrane and their dimers with phenylether,as well as 9,10-seco-cycloartanes.The biological activity of the isolated compounds was studied,and it was found that prenylated bibenzyls and the terpenoids which contain α,β-unsaturated ketones showed better cytotoxic activity.In the doctoral stage,continuing the master’s work,we carried on the inverstigation of the remaining components of the Radula apiculata that collected from Jiulongshan National Nature Reserve in Lishui,Zhejiang Province,and obtained 12 derivatives of isopentenyl bibenzyl and seven new compounds(1-7),including four dimers of isopentene bibenzyl[(±)-radulapins L-O(1-4)].Combined with 14 compounds obtained during the master’s degree,we screened the cytotoxic activity of 26 isopentenyl bibenzyl that purified from Radula apiculata.It was found that dimer showed significant cytotoxic effect.Further studies found that it increased the level of ROS and decreased the mitochondrial membrane potential to induce apoptosis of PC-3 cells.The biogenic sources of nine isopentene dibenzyl dimers were speculated.It was found that these dimers with special structure were the result of Diels-Alder cycloaddition and free radical coupling.Based on the investigation of the chemical constituents of Pallavicinia ambigua,which were collected from Zhangjiajie,25 compounds were obtained,of which 17 were new compounds,including 10 highly oxidized labdanes[pallaviambins I-R(21-29)]and four dimeric compounds of 7,8-seco-labdanes[pallaviambins E-H(17-20)].These dimers were formed by Diels-Alder reaction under the action of oxidase.In addition,two cis-trans isomers[pallaviambins A(13)and B(14)],with a new skeleton of tetracyclic[5.2.1.02,5.04,10]decane,were also obtained from the p.ambigua.The photoinduced rearrangement of compound 33,indicated that compounds 13 and 14 are formed by[2+2]cycloaddition of compound 33 under photocatalysis.It has been reported that the anti-tumor mechanism of compounds with α,β-unsaturated ketones is related to reactive oxygen species(ROS).ROS induction usually is an effective mechanism to kill cancer cells in treatments.Drug-induced "redox reset" will lead to chemotherapy resistance,breaking the "redox reset" will provide a new way to overcome drug resistance.Compound 33 with two α,β-unsaturated ketone structural fragments was found that showed a selective inhibitory effect on PC-3 after cytotoxic activity screening,and the inhibition of tumor proliferation occurred in G2/M phase.Subsequent activity studies found that it’s resulted in an increase in the level of ROS and apoptosis of PC-3 cells.32 compounds including 18 new ones were isolated and identified from Mylia nuda,which was collected from Ya’an,Sichuan Province,including four phenylethers,five rare sesquiterpenes and eight new skeletons of dimers[mylnudones A-H(42-49)]formed by phenylether and aromadendrane-type sesquiterpenes,of which compounds 42-48 were formed by Diels-Alder reaction while compound 49 via free radical coupling.14 known compounds were also obtained.The antifungal activities of these compounds were studied,except that compound 68 showed certain antifungal toxicity,the other activities were weak.Twelve new compounds including nine 9,10-seco-cycloartanes(70-78),along with six known compounds(81-87),were isolated from the Chinese liverwort Lepidozia reptans.In the cytotoxic activity screening,it was found that compounds 70,75,and 76 showed significant cytotoxic inhibitory effect on a series of cell lines.The representative compound 76 containing α,β-unsaturated ketone structure was further explored,and it was found that this triterpenoid could induce apoptosis of PC-3 cells by increasing ROS-mediated mitochondrial damage.On the basis of previous studies,we have obtained some compounds with novel structure and special skeleton,especially dimers,most of which are the products of Diels-Alder cycloaddition and free radical coupling under the action of plant somatic oxidase.With the development of separation and identification technology,more trace components have been found,which has greatly enriched the library of natural product compounds.The biological activity screening of these compounds also showed satisfactory biological activity,which provided a number of potential small molecular compounds for the development of new drugs.The results of the study on the chemical composition of liverworts show a certain species-genus specificity,which also has a certain reference value for the chemical classification of liverworts.更多还原