【作者】 李艳平；

【导师】 李蓉涛；

【作者基本信息】 昆明理工大学 ， 环境生物学， 2013， 博士

【摘要】 论文共由六章组成。第一章综述了杜鹃花科(Ericaceae)活性成分的研究进展。第二章详细论述了杜鹃花科马醉木属(Pieris)植物马醉木(Pieris japonica)中木藜芦烷二萜成分的分离和鉴定及生物活性研究。第三章报道了大戟科黑面神属(Breynia)植物黑面神(Breynia fruticosa)的化学成分及生物活性研究。第四章介绍了对蕨类七指蕨属(Helminthostachys)植物七指蕨(Helminthostachys zeylanica)的化学成分研究。第五章报道了对淫羊藿异戊烯基黄酮抗骨质疏松活性研究的结果。第六章对论文所做工作进行了总结和讨论。从上述三种药用植物中共分离鉴定了60个化合物,其中18个为新化合物。已鉴定的化合物类型涉及二萜、三萜、木脂素、黄酮和酚性成分等。新化合物主要为二萜类成分,其中2个为结构新颖的18位含氯的3,4-开环的多酰化木藜芦烷二萜,此外还有2个6,10环氧的木藜芦烷二萜以及1个4,7环氧的木藜芦烷二萜；对分离得到的木藜芦烷二萜类化合物进行了昆虫拒食活性筛选,对黄酮及酚性成分做了抗氧化活性筛选。一、马醉木(Pieris japonica)系杜鹃花科马醉木属有毒植物,家畜误食茎和叶会引起中毒反应。国内外很多学者很早就对马醉木属植物进行过研究。迄今已经从中分到了三萜,甾体和二萜等类型的化合物,主要以木藜芦烷二萜为主。我们对采自云南寻甸的马醉木的花进行了系统的化学成分研究,从乙酸乙酯部分中分离鉴定了34个化合物。其中14个为新化合物,分别被命名为：neopierisoid A (1), neopierisoid B (2), neopierisoid C (3), neopierisoid D (4), neopierisoid E (5), neopierisoid F (6), neopierisoid G (7), neopierisoid H (8), neopierisoid I (9), neopierisoid J (10), neopierisoid K (11), neopierisoid L (12) neopierisoid M (13)和benzyl2-hydroxy-4-O-β-D-glucopyranosyl-benzoate (14),20个己知化合物分别被鉴定为：pierisformotoxin G (15), secorhodomollolide A-B (16-17), pierisformotoxins E-F (18-19), secorhodomollolides E-H (20-23), secorhodomollolides C-D (24-25), pierisoids A-B (26-27), pierisformotoxins A-B (28-29), pierisformosins B, C (30,31),(2S,3R)-ent-catechin (32), quercetin (33),和1-phenylbutane-2,3-diol (34)。本实验分离得到的木藜芦烷型二萜多为3,4-裂环的多酰化木藜芦烷二萜,其中包括16个3,4-裂环且C-3与C-5成五元环内酯的木藜芦烷二萜,9个3,4-裂环且C-3与C-10成五元环内酯的多酰化木藜芦烷二萜,3个5/7/6/5环骨架的木藜芦烷型二萜以及2个3,4-开环未环合的木藜芦烷型二萜。值得一提的是,在3,4-裂环的多酰化木藜芦烷二萜中有两个含氯的3,4裂环且C-3与C-5成五元环内酯的木藜芦烷二萜,具有一定的昆虫拒食活性,这是首次从杜鹃花科中分离得到的含氯的二萜。而其良好的拒食活性说明杜鹃花科有毒植物的木藜芦毒素成分可能与该植物的化学防御功能有一定的关系；此外,我们对分离得到的黄酮和酚性成分进行了抗氧化活性的筛选,发现了多酚羟基的酚性成分具有较强的抗氧化活性。二、黑面神(Breynia fruticosa)为大戟科(Euphorbiaceae)黑面神属(Breynia)植物。作为中国传统的民间草药,黑面神具有清湿热,化淤滞的功效。主治腹痛吐泻,疗毒,疮疖,湿疹,皮炎,跌打肿痛等症。此外,还可作为蛇药治疗毒蛇咬伤。我们对采自云南西双版纳的黑面神全株进行了系统的化学成分研究,从其甲醇提取物的乙酸乙酯部分和正丁醇部分中分离鉴定了20个化合物,主要为酚类成分。其中2个为新的木脂素化合物,分别被命名为：(7S,8R,7’S)-9,7’,9’-trihydroxy-3,4-methylenedioxy-3’-methoxy[7-O-4’,8-5’]neolignan (1),(7S,8R,7’S)-9,9’-dihydroxy-3,4-methylenedioxy-3’,7’-dimethoxy[7-O-4’,8-5’] neolignan (2),18个为已知化合物,分别被鉴定为：9,9’-dihydroxy-3,4-methylenedioxy-3’-methoxy [7-O-4’,8-5’] neolignan (3)、(-)-machicendiol (4)、松脂素(5)、4”-苯酚基-6-O-甲基丙酰基-β-D-吡喃葡萄糖苷(6)、hydroquinone O-[6-(3-hydroxyisobutanoyl)]-β-galactopyranoside (7) β-D-glucopyranoside,4-hydroxyphenyl,6-(2-methylbutanoate)(8)、arbutin6’-butyrate(9)、熊果苷(10)、pyrocatechol-O-β-D-glucopyranoside (11)、α/β-D-glucopyranose,6-(4-hydroxybenzoate)(12)、α/β-D-glucopyranose,6-(3-phenyl-2-propenoate)(13)、熊果酸(14)、apocynol A (15)、柚皮素(16)、反式肉桂酸(17)、肉桂酸(18)、对二苯酚(19)和3,4-二羟基肉桂醛(20)。此外,我们通过ORD、CD和ECD的方法,首次确定了2个新木脂素(7S,8R,7’S)-9,7’,9’-trihydroxy-3,4-methylenedioxy-3’-methoxy[7-O-4’,8-5’]neolignan (1)和(7S,8R,7’S)-9,9’-dihydroxy-3,4-methylenedioxy-3’,7’-dimethoxy[7-O-4’,8-5’] neolignan(2)的绝对构型,以及两个未确定构型的已知化合物9,9’-dihydroxy-3,4-methylenedioxy-3’-methoxy [7-0-4’,8-5’] neolignan (3)、(-)-machicendiol(4)的绝对构型。对黑面神中分离得到的木脂素类化合物做了细胞毒活性筛选,发现从黑面神中分离得到的5个木脂素类化合物在浓度为50μM时并未表现出明显的细胞毒性,说明这类成分可能不是有毒药用植物中的毒性成分。三、七指蕨(Helminthostachys zeylanica (L.) Hook.),俗名入地蜈蚣,属于七指蕨科七指蕨属多年生陆生蕨类植物,民间常用来治疗痨热咳嗽,跌打肿痛,咽痛,痈疮,毒蛇咬伤等症。近年来,台湾学者从七指蕨中分离得到结构类型丰富的异戊烯基黄酮类化合物,这些化合物也表现出了免疫调节,抗肿瘤,以及抗氧等活性。我们对采自云南西双版纳的七指蕨全株的化学成分进行了研究,从乙酸乙酯部分中分离鉴定了6个化合物。其中1个为新的异戊烯基黄酮,命名为neougonin A (1),5个已知的异戊烯基黄酮,分别为：4"a,5",6",7",8",8"a-hexahydro-5,3’,4’-trihydroxy-5",5",8"a-trimethyl-4H-chromeno[2",3":7,8]flavone(2),4"a,5",6",7",8",8"a-hexahydro-5,3’,4’-trihydroxy-5",5",8"a-trimethyl-4H-chromeno[2",3":7,8]flavone (3), ugonin J (4), ugonin E (5), ugonin N (6)。四、在小檗科淫羊藿属植物中,异戊烯基黄酮化合物是其中的特征性成分,这类黄酮除了传统的抗微生物、杀虫和类雌激素等作用外,还有降血压、抗血凝、抗病毒、抗骨质疏松等活性。我们对前期从淫羊藿中分离得到的异戊烯基黄酮化合物进行了抗骨质疏松活性的筛选,此外,通过活性追踪方式,确定了淫羊藿中的活性段位为乙酸乙酯萃取相经氯仿丙酮划段的7：3部分,并从该活性段位得到了两个异戊烯基黄酮化合物-脱水淫羊藿素和2”-羟基-3”-烯-脱水淫羊藿素。对这两个异戊烯基黄酮进行抗骨质疏松活性初步研究,发现了它们具有促进小鼠前成骨细胞增殖,分化和矿化的作用。更多还原

【Abstract】 The thesis consists of six chapters. Chapter one presents a review on the bioactive chemical constituents of plants in family Ericaceae. The second chapter elaborates the studies on the grayanane diterpenoids from Pieris japonica. Chapters three and four deal with the isolation and determination of the chemical constituents of two plants--Breynia fruticosa and Helminthostachys zeylanica. In the fifth chapter, bioactivities of the prenylated flavonoids isolated from the Epimedium brevicornum Maxim. are reported. And the last chapter is the conclusion of the whole thesis.Sixty compounds, including eighteen new ones, were isolated from the above three specicies, and these compounds contain diterpenoids, triterpenoids, lignans, and flavonoids. The structures of the compounds were elucidated on the basis of extensive spectroscopic analysis. The new compounds are mainly diterpenoids, among them, two unique chlorinated multi-acylated3,4-seco grayanane diterpenoids, two novel6,10epoxy3,4-seco grayanane diterpenoids and a4,7epoxy3,4-seco grayanane diterpenoids were obtained. The absolute configuration of one of the chlorinated diterpenoids neopierisoid A was ditermined by X-ray diffraction. The antifeedant effect of some of the obtained diterpenoids and the antioxidant activity of flavonoids and phenolic compounds in Pieris japonica were evaluated.Pieris japonica, a well-known poisonous plant belonging to the family Ericaceae, has a rich distribution in hill and valley regions. It has been reported that livestock fall into a coma after accidental ingested of the plant. Previous chemical investigations on this plant resulted in the isolation of triterpenoids, diterpenoids, flavonoids and phenolic glycosides, while grayanane diterpenoids are the main constitutes in P. japonica. Our meticulous investigation on the chemical constituents, especially grayanane diterpenoids, from the flowers of P. japonica led to the isolation of34compounds, including13new grayanane diterpenoids and a new phenolic glycoside:neopierisoid A (1), neopierisoid B (2), neopierisoid C (3), neopierisoid D (4), neopierisoid E (5), neopierisoid F (6), neopierisoid G (7), neopierisoid H (8), neopierisoid I (9), neopierisoid J (10), neopierisoid K (11), neopierisoid L (12), neopierisoid M (13), and benzyl 2-hydroxy-4-O-β-D-glucopyranosyl-benzoate (14). The known compounds were identified as:pierisformotoxin G (15), secorhodomollolides A-B (16-17), pierisformotoxins E-F (18-19), secorhodomollolides E-H (20-23), secorhodomollolides C-D (24-25), pierisoids A-B (26-27), pierisformotoxins A-B (28-29), pierisformosins B, C (30,31),(2S,3R)-ent-catechin (32), quercetin (33),1-phenylbutane-2,3-diol (34). Most of the isolated diterpenoids are3,4-seco grayanane diterpenoids, including sixteen3,4-seco grayanane diterpenoids with the lactone group located between C-3and C-5, nine compounds with the lactone group located between C-3and C-10and two3,4-seco grayanane diterpenoids without acylated substitute. Other grayanane diterpenoids (three ones) have normal5/7/6/6skeleton. It is meaningful that among the31grayanane diterpenoids, we firstly reported two unusual defensive chlorinated diterpenoids bearing5,7,6,5ring system and12chiral center from the whole family Ericaceae. The antifeedant effect of some of the obtained diterpenoids compounds in Pieris japonica were evaluated. The results showed the unusual highly acylated chlorinated3,4-seco-grayanane diterpenoids, neopierisoids A and B, had antifeedant activity against pieris brassicae indicating the toxic properties of P. japonica might serve as a defensive role in the plant. The antioxidant activity of flavonoids and phenolic compounds in Pieris japonica were also evaluated.Breynia fruticosa (L.) Hook. f. is widely distributed in southern China and used as a Chinese folk medicine for the treatment of chronic bronchitis and inflinflammation. In addition, B. fruticosa had been used as an important ingredient of the plant medicine in the treatment of snakebite. Our further investigation of this plant collected from Xishuangbanna of Yunnan Province led to the isolation of20compounds, including2new ones:(7S,8R,7’S)-9,7’,9’-trihydroxy-3,4-methylenedioxy-3’-methoxy[7-O-4’,8-5’]neolignan (1),(7S,8R,7’S)-9,9’-dihydroxy-3,4-methylenedioxy-3’,7’-dimethoxy [7-0-4’,8-5’] neolignan (2). The18known compounds were identified as:9,9’-dihydroxy-3,4-methylenedioxy-3’-methoxy [7-0-4’,8-5’] neolignan (3),(-)-machicendiol (4), pinoresinol (5),4"-phenol group-6-O-methyl-propionyl-β-D-pyranglucoseglycoside (6), hydroquinone O-[6-(3-hydroxyisobutanoyl)]-β-galactopyranoside (7),β-D-glucopyranoside,4-hydroxyphenyl,6-(2-methylbutanoate)(8), arbutin6’-butyrate (9), arbutin (10), pyrocatechol-O-β-D-glucopyranoside (11), α/β-D-glucopyranose,6-(4-hydroxybenzoate)(12), α/β-D-glucopyranose,6-(3-phenyl-2-propenoate)(13), ursolic acid (14), apocynol A (15), naringenin (16), trans-cinnamic acid (17), cinnamic acid (18), diphenol (19),3,4-dihydroxy-cinnamic aldehyde (20). By the using of calculations of OR spectrum, together with OR and calculated ECD spectra method, we established the absolute configurations of C-7’ hydroxy group substituted neolignans for the first time. The neolignans were tested for in vitro cytotoxicity against A459,150HCT116, MCF-7and U87-MG human cancer cell lines using the MTT method. However, none of them showed obvious cytotoxicities with values of IC50>50μM, which suggested that these compounds were not the poisonous constituents in the plant.In Chinese traditional medicine, Helminthostachys zeylanica (L.) Hook., known as "Ding-Di-U-Gon", have been used for centuries in the treatment of inflammation and various hepatic disorders. The plant has been shown to exhibit anti-inflammatory, antiphlogistic, antipyretic, and hepatoprotective activities. Flavonoids with prenyl or geranyl groups have been reported to have specific bioactivities, such as cytotoxic activity, inhibitory activity and strong antioxidant effects. The flavonoids, ugonins A-L, a have been identified from H. zeylanica.The present study on the ethanolic extract of the dried whole plant of H. zeylanica collected from Xishuangbanna of Yunnan Province resulted in the isolation and characterization of a new prenylated flavonoid, neougonin A, and five known compounds:4"a,5",6",7",8",8"a-hexahydro-5,3’,4’-trihydroxy-5",5",8"a-trimethyl-4H-chromeno[2",3":7,8]flavone(2),4"a,5",6",7",8",8"a-hexahydro-5,3’,4’-trihydroxy-5",5",8"a-trimethyl-4H-chromeno[2",3":7,8]flavone (3), ugonin J (4), ugonin E (5), ugonin N (6).Epimedium brevicornu Maxim. is one of the most frequently used Chinese herbs that has been used for thousands of years in formulas prescribed for the nourishment of bone and gonadal functions. Prenyl-flavonoids were the main and characteristic components of the plant E. brevicornu and showed anti-microbial, insecticidal as well as lowering blood pressure, anti-bloodcoagulation, anti-viral, anti-osteoporosis activity. Two prenyl-flavonoids, anhydroicaritin and2"-hydroxy-3"-en-anhydro-icaritin, were isolated from the main effective fraction (CHCl3-Me2CO,7:3part) and proved, for the first time, to have the ability to promote proliferation and differentiation of murine osteoblastic MC3T3-E1cells, which suggested that both compounds have the potential to be candidates for osteogenic compounds for bone regenerative medicine.更多还原