【作者】 任冬梅；

【导师】 娄红祥；

【作者基本信息】 山东大学 ， 天然药物化学， 2007， 博士

【摘要】 对传统药用植物的化学成分进行系统的研究，是发现具有生物活性化合物的重要途径之一。唇形科(Labiatae)青兰属(Dracocephalum)植物约60种，主要分布于亚洲温带，多在高山及半干旱地区，少数延至中欧及北欧，1种分布于北美。我国约有32种7变种，分布于东北、华北、西北及西南。该属植物在民间被广泛应用，尤其是新疆维吾尔自治区多种该属植物被用作气管炎和心血管疾病的治疗药物。我们选择了两种对心肌缺血具有保护作用的青兰属植物岩青兰(Dracocephalum rupestre Hance)和香青兰(Drcocephalum moldavica L)，对其进行了系统的化学成分研究，并对得到的部分黄酮类化合物进行了肿瘤细胞增殖抑制作用、心肌细胞氧化损伤保护作用和自由基清除活性研究，采用RP-HPLC法测定了岩青兰中具有心肌细胞保护作用的主要黄酮类化合物的含量。岩青兰(Dracocephalum rupestre Hance)为多年生草本，分布于我国辽宁、内蒙、河北、山西、青海等地，以地上全草入药，可清热消炎，凉血止血。用于治疗外感风热，头痛咳嗽，黄疸性肝炎等症。通过硅胶柱色谱、Sephadex LH-20柱色谱、手性高效液相柱色谱(Chiral-HPLC)等方法，从岩青兰地上全草乙醇提取物中分离得到38个化合物，通过波谱学方法，全部化合物鉴定为：间羟基苯甲酸(1)，3-甲氧基-4-羟基苯甲酸(2)，3，5-二甲氧基-4-羟基苯甲酸(3)，5，7-二羟基色原酮(4)，5，7-二羟基色原酮-7-0-葡萄糖苷(5)，白桦脂醇(6)，白桦脂酸(7)，乌索酸(8)，柚皮素(9)，柚皮素-7-O—葡萄糖苷(10)，芹菜素(11)，芹菜素—7—O—葡萄糖苷(12)，北美圣草素(13)，北美圣草素—7—O—葡萄糖苷(14)，木犀草素(15)，木犀草素—7—O—葡萄糖苷(16)，2S，6S-dracorupesin(17)，2R，6S-dracorupesin(18)，2R，6R-dracorupesin(19)，2S，6R-dracorupesin(20)，2R，5″S-dracocephin A(dracocephin A1，21)，2S，5″S-dracocephin A(dracocephin A2，22)，2S，5″R-dracocephin A(dracocephin A3，23)，2R，5″R-dracocephin A(dracocephin A4，24)，2S，5″S-dracocephin B(dracocephin B1，25)，2R，5″S-dracocephin B (dracocephin B2，26)，2R，5″R-dracocephin B(dracocephin B3，27)，2S，5″R-dracocephin B(dracocephin B4，28)，2R，5″S-dracocephin C(dracocephin C1，29)，2S，5″S-dracocephin C(dracocephin C2，30)，2S，5″R-dracocephin C(dracocephin C3，31)，2R，5″R-dracocephin C(dracocephin C4，32)，2S，5″S-dracocephin D(dracocephin D1，33)，2R，5″S-dracocephin D(dracocephin D2，34)，2R，5″R-dracocephin D(dracocephin D3，35)，2S，5″R-dracocephin D(dracocephin D4，36)，β-谷甾醇(37)，胡萝卜苷(38)。其中17-36为新化合物，化合物17—20为具有苯并吡喃结构的两对对映异构体，采用HPLC—CD联用技术在线测定了17—20的CD谱，利用激子手性法则对得到的CD谱进行解释并推定了4个立体异构体的构型。21—24、25—28、29—32、33—36分别为四组连有吡咯烷酮环的黄酮生物碱类化合物，通过手性HPLC—CD联用技术证明每一组化合物均为具有相同平面结构的两对对映异构体。采用Chiralpak AS—H手性柱制备得到了21—36共16个立体异构体，通过X—单晶衍射和CD谱测定完全确定了化合物21—24的立体结构，并依据不同构型立体异构体CD谱差减得到的CD曲线推导出了位于含氮杂环上的手性碳对于CD谱的贡献。进一步根据推得的吡咯烷酮环上手性碳的CD规律以及二氢黄酮C—2的CD规律，正确无误的推定了化合物25—36的立体构型。香青兰(Drcocephalum moldavica L)为维吾尔族和蒙古族习用药材，分布于黑龙江、吉林、辽宁、内蒙古、甘肃、山西、青海、新疆等省区，新疆南部有栽培，一年生草本，用于补脑安神，镇咳止喘，强心利尿。通过MCI柱色谱、硅胶柱色谱、Sephadex LH—20柱色谱从其地上全草乙醇提取物中分离得到9个黄酮类成分，波谱学方法鉴定结构为：木犀草素(15)，栀子素乙(39)，玄参黄酮(40)，鼠尾草素(41)，异鼠李素(42)，山柰酚(43)，金合欢素(44)，agastachoside(45)，日本椴苷(46)；2个三萜类成分，鉴定为：乌索酸(8)和白桦脂酸—28—乙酯(47)。对上述两种植物分离得到的部分黄酮类化合物通过DPPH·捕获试验评价了自由基捕获能力，采用MTT法对肿瘤细胞K562增殖抑制作用和心肌细胞抗氧化损伤作用进行了评价。结果表明，B环具有3′，4′邻二羟基取代的黄酮类成分具有较好的自由基清除活性；多甲氧基取代的黄酮类成分具有显著的K562细胞增殖抑制活性；二氢黄酮类成分对于阿霉素诱导的心肌细胞H9C2损伤有明显保护作用。采用RP—HPLC法，测定了岩青兰中三种主要黄酮类化合物柚皮素—7—O—β—D一葡萄糖苷、北美圣草素—7—O—β—D—葡萄糖苷和木犀草素—7—O—β—D—葡萄糖苷的含量，并对植物不同部位和不同采收季节药材中三种黄酮苷的含量进行了比较，结果表明，三种黄酮苷类成分在植物叶中含量最高，地下部分几乎不含，且三种黄酮苷的含量随季节变化明显。HPLC含量测定方法的建立为药材的合理采收和质量评价提供了依据。更多还原

【Abstract】 Systematical study on the chemical constituents of traditional Chinese herbs is one of important routes to discover bioactive compounds. There are about 60 species belong to the genus Dracocephalum, family Labiatae. These plants distribute in alpine and semidry regions mainly in temperate Asia, with a few in Europe, one in North America. There are about 32 species, 7 variations in China. Several species belong to the genus Dracocephalum are used as folk medicine for the treatment of tracheitis and cardiovascular disease in Xinjiang autonomous region. Dracocephalum rupestre Hance and Drcocephalum moldavica L can be applied for the protection of cardiac ischemia. This dissertation mainly focused on the chemical studies of these two herbal plants. Bioactivities including antitumor, protecting cardiac cells and the free radical scavenging were investigated for some flavonoids. Determinations of the dominating flavonoids in Dracocephalum rupestre were achieved by HPLC.Dracocephalum rupestre Hance is a perennial herb distributed in northern China, i.e. Liaoning, Neimeng, Shanxi, Qinghai Province. Its aerial parts were commonly employed in relieving headache, soothing sore throat, subsiding cough, and preventing icterohepatitis in traditional Chinese medicine. By Silica gel chromarography, Sephadex LH-20 chromatography and Chiral HPLC, thirty-eight compounds were isolated from the ethanol extract of this plant. Based on spectroscopy methods, all the compounds were elucidated as: m-hydroxybenzoic acid (1), 3-methoxy-4-hydroxybenzoic acid (2), 3,5-dimethoxy-4-hydroxybenzoic acid (3), 5,7-dihydroxychromone (4), 5,7-dihydroxychromone-7-O-glucoside (5), betulin (6), betulinic acid (7), ursolic acid (8), naringenin (9), naringenin-7-O-glucoside (10), apigenin (11), apigenin-7-O-glucoside (12), eriodityol (13), eriodityol-7-O-glucoside (14), luteolin (15), luteolin-7-O-glucoside (16), 25,65-dracorupesin (17), 2R,6S-dracorupesin (18), 2R,6S-dracorupesin (19), 2S,6R-dracorupesin (20), 2R,5"S-dracocephin A (dracocephin A1, 21), 2S,5"S-dracocephin A (dracocephin A2, 22), 2S,5"S-dracocephin A (dracocephin A3, 23), 2R,5"R-dracocephin A (dracocephin A4, 24), 2S,5"S-dracocephin B (dracocephin B1, 25), 2R,5"S-dracocephin B (dracocephin B2, 26), 2R,5"R-dracocephin B (dracocephin B3, 27), 2S,5"R-dracocephin B (isodracocephin B4, 28), 2R,5"S- dracocephin C (dracocephin C1, 29), 2S,5"S-dracocephin C (dracocephin C2, 30), 2S,5"R- dracocephin C (dracocephin C3, 31), 2R,5"R-dracocephin C (dracocephin C4, 32), 2S’,5"S-dracocephin D (dracocephin D1, 33), 2R,5"S-dracocephin D (dracocephin D2, 34), 2R,5"R-dracocephin D (dracocephin D3,35), 2S,5"R-dracocephin D (dracocephin D4, 36), sitosterol (37), daucosterol (38). 17—36 were novel compounds. 17-20 were characterized as two diastereomeric pairs of enantiomers with an unprecedented flavanone fused benzopyran skeleton. Using HPLC-CD, the on-line CD spectra of 17-20 were obtained and the exciton chirality method was used for the determination of the absolute configuration of them. Compounds 21—24、25—28、29—32、33—36 were all conjugates of flavanone with pyrrolidin-2-one and each group were two diastereomeric pairs of enantiomers with the same planar structure. The single stereoisomers of 21-36 were prepared by chiral HPLC using Chiralpak AS-H column.The stereochemical features of 21-24 were elucidated on the basis of measurements of NMR, CD and by combined with X-ray analysis. Investigating the CD behavior of the stereoisomers, the chiroptical contributions of the two stereocenters were found to behave additively. By substraction of their respective contributions, the CD contributions of the chiral carbon in the pyrrolidin-2-one ring were discussed. Based on the deduced CD rules, the stereostructures of compounds 25-36 were assigned unambiguously.Drcocephalum moldavica L, habitually used herb by Weiwuer and Meng nationality, mainly distributed in Heilongjiang, Jilin, Liaoning, Neimenggu, Gansu, Shanxi, Qinghai and Xinjiang in China. It was a annual herb and was cultivated in southern Xinjiang autonomous region. The aerial parts of this herb were employed in soothing the nerves, relieveing a cough and protecting the heart. By MCI gel chromarography, Silica gel chromatography and Sephadex LH-20 chromatography, thirteen compounds were isolated from the ethanol extract of this plant. Based on spectroscopy methods, all the compounds were elucidated as: luteolin (15), gardenin B (39) , scrophulein (40) , salvigenin (41) , isorhamnetin (42) , kaempferol (43) , acacetin (44) , agastachoside (45) , tilianin (46) ,ursolicacid (8) and betulinic acid—28—ethyl ester (47) 。For some flavonoids isolated from the two herbs, DPPH·scavenging test was applied to study the free radical scavenging activity. The cytotoxity to K562 cells (antitumor effect) and protection to cardiac cells was also studied by MTT. 3’,4’-dihydroxyl substituted flavonoids possessed preferable DPPH- scavenging ability; flavonoids with multi-methoxyl substitution showed potent anticancer effect; flavanones showed significant protective effects on doxorubicin-mediated cytotoxicity in H9C2 cells.Using RP-HPLC method, the contents of naringenin-7-O-β-D-glucoside, eriodityol-7-O-β-D-glucoside and luteolin-7-O-β-D-glucoside in D. rupestre were determined. The flavonoids contents in different parts of D. rupestre and in the aerial parts collected in different seasons were compared. It indicated that the content of the three flavonoid glucosides was maximum in the leaves, and it was different in different seasons. The HPLC method was suitable for the quality evaluation of the herbs.更多还原