【作者】 刘涛；

【导师】 穆青；

【作者基本信息】 复旦大学 ， 药物化学， 2013， 博士

【摘要】 伞形科(Apiaceae)植物化学成分丰富,主要有香豆素类、精油、黄酮类、酚类、生物碱等。伞形科植物具有多种应用价值,可作为药材、蔬菜、香料、农药等。现已发现其多种化学成分具有很强的药理活性,其中抗菌活性成分备受关注。多伞阿魏(Ferula ferulaeoides)是伞形科阿魏属植物。该属药用植物作为传统中药被用于治疗神经障碍、炎症、消化紊乱、风湿症和关节炎等多种疾病。现代药理研究表明,阿魏具有抗病毒、抗炎、杀虫、抗癌和抗菌等多种药理活性。目前文献共报道多伞阿魏化学成分约55个多为倍半萜及其衍生物。论文第一部分,以抗耐药金黄色葡萄球菌作用为主要活性指导,研究多伞阿魏化学成分,寻找具有抗耐药金黄色葡萄球菌或者协同抗菌作用的化合物,并对活性化合物构效关系和抗耐药菌机制(外泵抑制作用)进行研究。同时,对多伞阿魏中分离到的天然产物进行了抗肿瘤,抗病毒和杀虫活性评价。首先,采用GC-MS联用技术对多伞阿魏二氯甲烷部位化学成分进行分析测定,鉴定了其中34个化合物。主要成分为萜类化合物,含量最高的成分为愈创木烷型倍半萜guaiol (37.01%)。采用色谱分离技术从该部位分离纯化得到40个化合物,通过波谱解析等手段鉴定了34个化合物的结构(苯乙酮衍生物16个,香豆素13个,倍半萜5个)：其中12个为新化合物(9-10,12-16,24-28),另有10个化合物(1-4,24,29-31,33-34)为首次从该植物中发现。结合文献报导,对化合物生物演化途径进行了推导。通过结构修饰得到了天然产物衍生物19个(F系列)。本文对多伞阿魏中天然产物抗耐药金黄色葡萄球菌活性进行了重点研究。研究中引入和部分使用了一种抗菌物质快速活性筛选方法—TLC-生物自显影法,并利用此方法从多伞阿魏中分离得到两个具有抗耐药金黄色葡萄球菌活性的化合物(5和11)。采用微量肉汤稀释法对多伞阿魏中天然产物最低抑菌浓度(MIC)进行了测定,研究发现倍半萜类化合物没有抗耐药金黄色葡萄球菌活性。香豆素类化合物中仅有7-羟基呋喃香豆素(17,18,21,22,27和28)对含有TetK外泵蛋白的四环素类抗生素耐药菌株XU212表现出独特的抑制活性,最低抑菌浓度达到2 μg/mL。苯乙酮类化合物(5-13)表现出普遍的抗耐药金黄色葡萄球菌活性,活性最强的化合物10对实验菌株XU212和EMRSA-16两种实验菌株最低抑菌浓度为0.5 μg/mL。对活性化合物抗耐药金黄色葡萄球菌的外排泵抑制作用机制研究,发现了两个新天然产物(9和12)能够抑制细菌外泵作用,两个化合物存在时,细菌细胞内溴化乙锭的荧光吸收在实验时间内下降速度减慢,而且此时的荧光吸收会保持在一个相对较高的水平。为了探讨苯乙酮类化合物的构效关系,我们对活性天然产物进行了结构修饰,得到天然产物衍生物19个。通过与分离所得到的16个天然苯乙酮类化合物活性比较,发现化合物的二羟基苯基,环上羟基,倍半萜侧链及其双键,酮基及其α位对化合物抗耐药菌活性具有明显影响。论文对分离到的不具有直接抑菌活性的倍半萜和香豆素类化合物进行了协同抗菌实验筛选,结果显示香豆素类化合物(17,18,21,22,25)与抗生素联用可以明显改变细菌对抗生素的耐药性,降低抗生素最低抑菌浓度,表现出协同抗菌活性。其中,化合物17和22与相应浓度抗生素配合使用可以使其最低抑菌浓度由64 μg/mL降低至1 μg/mL,即抗生素用量减少为原来的1/64。对于多伞阿魏中分离到的天然产物,论文还进行了抗肿瘤,抗病毒及杀虫活性研究。化合物的抗肿瘤结果显示,倍半萜和苯乙酮类化合物对人类肝癌细胞株(SMMC-7721)和人类胃癌细胞株(SGC-7901)没有抑制作用。香豆素类化合物对两种肿瘤细胞表现出一定抑制活性,IC50为22.5-69.0μM。化合物的抗病毒活性的结果显示,香豆素类化合物(17-19)在不显示毒性条件下对乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)表现出抑制活性。其中,化合物17和18对乙肝病毒DNA同样起到一定的抑制作用。本研究对所得化合物的杀虫活性的检测发现,倍半萜类化合物(31)对蚜虫、粘虫、小菜蛾及家蝇显示出较好的杀灭活性。论文第二部分,对芹菜籽抗植物病原菌活性及组分进行了研究。芹菜籽是伞形科植物芹菜的种子,其功能性成分具有良好的抗氧化,抗肿瘤,抑菌及杀虫等生物活性。研究发现芹菜籽石油醚部位(QCZ-P)对三种测试菌株大豆丝核病菌(Rhizoctonia solani),大豆黑斑病菌(Alternaria fasciculate)和棉花枯萎病菌(Fusarium oxysporium)具有最好的抑制活性。在100 gg/mL浓度下对三种植物病原菌的抑制率分别为48.1%,36.8%和52.1%。GC-MS分析测试鉴定了其中58个化合物,主要为萜类化合物。活性指导下对QCZ-P的分离得到一个活性组分QCZ-P-4,其对三个测试菌株抑制率分别达到64.6%,54.7%和88.4%。进一步采用GC-MS对该组分进行分析测试,鉴定了其中5个化合物。芹菜籽无毒,原料易得,降解途径无污染等特点,是天然植物源农药的理想来源。芹菜籽抗耐药金黄色葡萄球菌活性研究表明其对耐药金黄色葡萄球菌在低浓度下没有抑制作用。更多还原

【Abstract】 Plants of Apiaceae are rich in chemicals including coumarins, essential oils, flavonoids, phenols, alkaloids. They have a variety of applications such as medicine, vegetables, spices, chemicals, etc. Various chemical constituents with strong pharmacological activities have been found and the antibacterial active ingredients are drawing our attention. Plant Ferula ferulaeoides belongs to the genus Ferula (Apiaceae). As a traditional herb, several species of Ferula have a reputation in the treatment of various diseases such as neurological disorders, inflammations, digestive disorders, rheumatism and arthritis. In pharmacological research, Ferula were found to possess biological effects including anti-virus, anti-inflammatory, cytotoxicity, insecticide and antibacterial activities. So far, there have been about 55 compounds isolated from this herb, most of which are sesquiterpenes and their derivatives.In the first section, chemical compositions of F. ferulaeoides were studied by the direction of antibacterial activity against drug-resistant S. aureus. The inhibit and sinergestic effect of the natural compounds against drug-resistant S. aureus, structure-activity relationship and mechanism of active compounds were studied. Biological activities of the compounds including anti-tumor, anti-virus and insecticidal activity were also evaluated.In phytochemical research, dichloromethane fraction of F. ferulaeoides was analysed by GC-MS and thirty-four compounds were identified. The main component of the fraction was guaiol (37.01%). Forty compounds have been isolated from this fraction of F. ferulaeoides. Thirty-four compounds were identified. Twelve of them are new compounds (9-10,12-16, 24-28), another known ten (1-4,24,29-31,33-34) are first reported from this plant. Biosynthetic pathways leading to these compounds were proposed. Fifteen derivatives were obtained according to structural modifications of the natural products.In pharmaceutical research, attentions have been focus on activity of natural products from F. ferulaeoides against drug-resistant S. aureus. A rapid screening method, TLC-bioautography, was introduced and improved. Two compounds (5 and 11) with activity against drug-resistant S. aureus were obtained from F. ferulaeoides using this method. The minimal inhibitory concentrations (MICs) for natural compounds were determined using the standard broth micro-dilution method. Results showed that sesquiterpenoids and most of the coumarins had no antibacterial activity. Only individual coumarins (17,18,21,22,27 and 28) showed unique inhibitory activity against tetracycline resistant strains Xu212 which possessed the TetK tetracycline efflux protein with MIC values at 2 μg/mL. Compounds of acetophenone (5-13) derivatives showed widespread activity against S. aureus. Compound (10) showed the best activity against strain XU212 and EMRSA-16 with MIC value at 0.5 μg/mL。 The mechanism of active compounds against drug-resistant S. aureus was studied and two new natural compounds (9 and 12) were found to possess effect of efflux pump inhibition. In the presence of compounds 9 and 12, the fluorescent absorption of intracellular EtBr decreased slowly within the experimental duration, and the absorption was at a higher level than that in the absence of compounds 9 and 12.Sixteen derivatives were obtained by structural modifications of the natural products. Structure-active relationships study showed that 2’,4’-dihydroxy-phenyl, the length of side chain, hydroxyl of benzene ring, the double bonds, ketone group and its alpha bits played important roles in their activity against drug-resistant S. aureus.Synergistic effects of compounds that had no inhibit activity against drug-resistant S. aureus were studied. Five coumarins (17,18,21,22 and 25) were screened to have synergistic effect. The combination of antibiotics with these compounds can greatly reverse bacteria resistance to antibiotics. Compound 17 and 22 can improve effect of the antibiotics whose MIC can be changed from 64 μg/mL to 1 μg/mL.The anti-tumor, antiviral and insecticidal activities of natural products from F. ferulaeoides were studied. Compounds of phenyl ethyl ketone and terpene had no inhibition on SMMC-7721 and SGC-7901 while coumarins showed certain inhibitory activity with IC50 from 22.5 to 69.0 μM. Compounds of coumarin (17-19) showed inhibitory activity to both HBsAg and HBeAg under the condition of non-toxic. Compounds 17 and 18 also showed inhibitory activity to the DNA of HBV. Sesquiterpenoids compound (31) showed good killing activity against common plant insects.In the second section, the activity against phytopathogenic fungi of celery seed and its chemical compositions were studied. Celery seed belonging to Apiaceae is the seed of celery. Its functional components were reported to have good antioxidant, anticancer, antibacterial, insecticidal bioactivity and so on. In pharmaceutical research, the low polarity of celery seed, QCZ-P, showed the best inhibitory activity to test strains Rhizoctonia solani, Alternaria fasciculate and Fusarium oxysporium. Bioactive directed separation offered a more active fraction QCZ-P-4, with the best activity of 64.6%,54.7% and 88.4% against R. solani, A. fasciculate and F. oxysporium, respectively. QCZ-P-4 was shown to contain 5 compounds by GC-MS. This indicated that it might be developed to be a natural non-toxicity pesticide. The activity against drug-resistant S. aureus of QCZ-P and QCZ-P-4 showed they had no inhibition activity in low concentrations.更多还原