【作者】 曹新伟；

【导师】 肖培根； 陈士林； 陈四保；

【作者基本信息】 中国协和医科大学 ， 生药学， 2008， 博士

【摘要】 贝母为一种常用中药,具有十分悠久的药用历史,始载于《神农本草经》,列为中品:“气味辛、平,无毒,主伤寒烦热。淋沥邪气,疝症,喉痹,乳难。”百合科Liliaceae中的贝母属Fritillaria植物是药用贝母的主要来源,此属全世界约有130种,主要分布于北半球的温带地区,尤以地中海北岸、伊朗、土耳其等地区分布的种类最为丰富。贝母种类繁多,在民间多种贝母属植物的鳞茎均以贝母入药,《中国药典》2005年版一部收载有川贝母、平贝母、浙贝母、湖北贝母、伊贝母等.川贝母（Fritillariacirrhosa D.Don）和梭砂贝母（Fritillaria delavayi Franch.）作为川贝母的主要基源植物,以其干燥鳞茎入药,能清热润肺,化痰止咳。用于肺热燥咳,干咳少痰,阴虚劳嗽,咯痰带血。为了进一步寻找新的天然产物成分或者活性成分,阐明川贝母药效作用的物质基础,为贝母属药用植物资源的充分利用和合理开发提供依据,建立科学的质量评价方法,我们对川贝母的两种基源植物:川贝母（F.cirrhosa D.Don）和梭砂贝母（F.delavayi Franch.）的化学成分进行了系统研究,并建立了生物碱和核苷两类成分的含量测定方法,对贝母属植物的质量评价方法作了初步的探讨,为贝母属药用植物的质量控制和品种鉴别提供科学依据。采用多种现代色谱技术进行分离纯化,从川贝母的甲醇提取物中分离得到31个化合物,综合运用理化性质和各种光谱技术(包括IR、UV、ESI-MS、HRESI-MS、¹H NMR、¹³C NMR、DEPT、¹H-¹H COSY、HMQC、HMBC、NOESY和X-ray等),鉴定了27个化合物的结构。其中生物碱类化合物15个,分别为:垂茄次碱（demissidine,QB-3）,川贝酮碱（chuanbeinone,QB-9）,梭砂贝母酮碱（delavinone,QB-10）,贝母乙素（verticinone,QB-11）,西贝素（imperialine,QB-12）,蒲贝素B（puqienine B,QB-16）,异梭砂贝母碱（isodelavine,QB-23）,isoforticine（QB-24）,petilidine（QB-25）,贝母甲素（verticine,QB-26）,贝母辛（peimisine,QB-27）,川贝碱甲（cirrhosinine A,QB-35）,川贝碱乙（cirrhosinine B,QB-46）,垂茄次碱苷（demissidine-3-O-β-D-glucopyranosyl（1→4）glucopyranoside,QB-65）,solanidine3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）]-β-D-glucopyranoside（QB-75）;有机酸类化合物2个:E-肉桂酸（E-cinnamic acid,QB-P-3）,单棕榈酸甘油酯（2-monopalmitin,QB-P-5）;核苷类化合物8个:尿嘧啶（uracil,QB-N-2）,胸嘧啶（thymine,QB-N-3）,胞苷（cytidine,QB-N-6）,肌苷（inosine,QB-N-7）,尿苷（uridine,QB-N-8）,鸟苷（guanosine,QB-N-10）,胸苷（thymidine,QB-N-11）,腺苷（adenosine,QB-N-12）;甾醇及其苷类化合物2个:β-谷甾醇（β-sitosterol,QB-P-1）,胡萝卜苷（daucosterol,QB-P-6）。其中,QB-23,QB-24,QB-35,QB-46和QB-65等5个化合物为新化合物,QB-P-3,QB-P-5,QB-N-2,QB-N-3,QB-N-6,QB-N-7,QB-N-8和QB-N-10等8个化合物为首次从贝母属中分离得到,QB-9,QB-10,QB-12,QB-16,QB-25,QB-75,QB-P-1和QB-P-6等8个化合物为首次从川贝母中分离得到。从梭砂贝母鳞茎中共分离得到33个化合物,通过化合物的理化性质和各种波谱技术鉴定了29个化合物。其中生物碱类化合物12个,分别为:川贝酮碱（chuanbeinone,LBM-11）,梭砂贝母酮碱（delavinone,LBM-12）,梭砂贝母啶碱（delavidine,LBM-14）,去氢鄂贝定碱（ebeiedinone,LBM-15）,贝母乙素（verticinone,LBM-16）,西贝素（imperialine,LBM-17）,异梭砂贝母碱（isodelavine,LBM-31）,梭砂贝母碱（delavine,LBM-41）,鄂贝定碱（ebeiedine,LBM-42）,异贝母甲素（isoverticine,LBM-46）,贝母甲素（verticine,LBM-48）,贝母辛（peimisine,LBM-50）;有机酸类化合物8个:E-3,4,5-三甲氧基肉桂酸（E-3,4,5-trimethoxycinnamic acid,BM-2）,E-对-甲氧基肉桂酸（E-p-methoxycinnamic acid,BM-3）,E-肉桂酸（E-cinnamic acid,BM-4）,E-对-羟基肉桂酸（E-p-hydroxycinnamic acid,BM-7）,E-对-羟基肉桂酸甲酯（E-p-hydroxycinnamic acid methyl ester,BM-8）,阿魏酸（ferulic acid,BM-11）,咖啡酸（caffeic acid,BM-12）,1-O-feruloylglycerol（BM-15）;核苷类化合物6个:尿嘧啶（uracil,LB-1）,胸嘧啶（thymine,LB-2）,尿苷（uridine,LB-5）,鸟苷（guanosine,LB-7）,胸苷（thymidine,LB-8）,腺苷（adenosine,LB-9）;甾醇及其苷类化合物2个:β-谷甾醇（β-sitosterol,BM-1）,胡萝卜苷（daucosterol,BM-17）;萜类化合物1个:苍术内酯Ⅲ（atractylenolideⅢ,BM-5）。其中LBM-14为新化合物,BM-2、BM-3、BM-5、BM-7、BM-8、BM-11、BM-12和BM-15等8个化合物为首次从贝母属中分离得到,LBM-15、LBM-16、LBM-31、LBM-42、LBM-46、LBM-48、LBM-50、BM-1、BM-4、BM-17、LB-1、LB-2、LB-5、LB-7、LB-8和LB-9等16个化合物为首次从梭砂贝母中分离得到。在化学成分研究的基础上,本文采用HPLC-ELSD法建立了同时测定贝母类药材中8个生物碱含量的方法,8个生物碱类成分分别为平贝碱甲（pingbeimineA）、贝母辛（peimisine）、贝母甲素（verticine）、贝母乙素（verticinone）、异梭砂贝母碱（isodelavine）、垂茄次碱苷（demissidine-3-O-β-D-glucopyranosyl（1→4）glucopyranoside）、梭砂贝母酮碱（delavinone）和川贝酮碱（chuanbeinone）;利用新建立的HPLC-ELSD分析方法对13种贝母60批次样品中的8个生物碱进行了含量测定。建立了13种贝母药材的HPLC色谱模式,并根据贝母药材中生物碱的分布特点,对贝母属植物的化学分类学进行了探讨。同时,首次建立了用HPLC-DAD法同时测定贝母类药材中9个核苷类成分含量的方法,9个核苷类成分分别为尿嘧啶（uracil）,胞苷（cytidine）,鸟嘌呤（guanine）,尿苷（uridine）,胸嘧啶（thymine）,肌苷（inosine）,鸟苷（guanosine）,胸苷（thymidine）,腺苷（adenosine）。60批次贝母样品的含量测定结果表明,核苷类成分普遍存在于各种贝母药材中,尿苷（Uridine）、鸟苷（Guanosine）和腺苷（Adenosine）为贝母类药材中主要的核苷类成分,由于核苷类化合物具有多种生物活性,因此核苷类可能为贝母类药材生物碱以外的活性成分。贝母中生物碱和核苷类成分定量分析方法的建立,对贝母类药材的质量控制、品种鉴定具有重要的参考价值,为贝母属植物的化学分类学提供新的科学依据,同时为贝母类药材的临床合理用药提供参考。本文还通过查阅大量文献,归纳总结,对贝母属植物的植物资源、传统疗效、化学成分、药理活性及质量控制研究的最新进展进行了全面综述,对有效开发、合理利用本属药用植物资源具有重要的参考价值。更多还原

【Abstract】 The genus Fritillaria belongs to family Liliaceae,which includes about 130 species worldwide and occurs mainly in the temperate regions of the northern hemisphere.Most species are distributed in Cyprus,Iran and Turkey.The bulbs of many Fritillaria species （’Beimu’ in Chinese） have been used as antitussive,anti-asthmatic and expectorant agents in traditional Chinese medicine for more than 2000 years.Fritillaria cirrhosa D. Don and Fritillaria delavayi Franch.are recorded as two of plant sources of ’Chuanbeimu’ in China Pharmacopoeia（2005 Edition）,which documented to treat cough, bronchitis and hemoptysis.In order to search for biologically active constituents,as well as develop a method for the quality control of Fritillaria raw materials,the bulbs of F. cirrhosa D.Don and F.delavayi Franch.were subjected to systematically phytochemical studies.The analytical methods based on HPLC-ELSD and HPLC-DAD technologies were also established.Thirty-one compounds were isolated from the methanol extracts of the bulbs of F. cirrhosa by various column chromatographies.On the basis of physicochemical and spectral analysis(including IR,UV,HRESI-MS,ESI-MS,¹H NMR,¹³C NMR,DEPT, ¹H-¹H COSY,HMQC,HMBC,NOESY and X-ray),structures of twenty-seven compounds were determined,as demissidine（QB-3）,chuanbeinone（QB-9）,delavinone （QB-10）,verticinone（QB-11）,imperialine（QB-12）,puqienine B（QB-16）,isodelavine （QB-23）,isoforticine（QB-24）,petilidine（QB-25）,verticine（QB-26）,peimisine （QB-27）,cirrhosinine A（QB-35）,cirrhosinine B（QB-46）,demissidine-3-O-β-D-gluco-pyranosyl （1→4） glucopyranoside（QB-65）,solanidine 3-O-α-L-rhamnopyranosyl- （1→2）-[β-D-glucopyranosyl-（1→4）]-β-D-glucopyranoside（QB-75）,E-cinnamic acid （QB-P-3）,2-monopalmitin（QB-P-5）,uracil（QB-N-2）,thymine（QB-N-3）,cytidine （QB-N-6）,inosine（QB-N-7）,uridine（QB-N-8）,guanosine（QB-N-10）,thymidine （QB-N-11）,adenosine（QB-N-12）,β-sitosterol（QB-P-1）,daucosterol（QB-P-6）.Among them,there are five new compounds,including QB-23,QB-24,QB-35,QB-46 and QB-65;eight compounds,QB-P-3,QB-P-5,QB-N-2,QB-N-3,QB-N-6,QB-N-7, QB-N-8 and QB-N-10 were isolated from Fritillaria genus for the first time;QB-9, QB-10,QB-12,QB-16,QB-25,QB-75,QB-P-1 and QB-P-6 were isolated from this plant for the first time.Thirty-three compounds were isolated from the bulbs of F.delavayi,and twenty-nine of them were identified as chuanbeinone（LBM-11）,delavinone（LBM-12）, delavidine（LBM-14）,ebeiedinone（LBM-15）,verticinone（LBM-16）,imperialine （LBM-17）,isodelavine（LBM-31）,delavine（LBM-41）,ebeiedine（LBM-42）, isoverticine（LBM-46）,verticine（LBM-48）,peimisine（LBM-50）, E-3,4,5-trimethoxycinnamie acid（BM-2）,E-p-methoxycinnamie acid（BM-3）, E-cinnamic acid（BM-4）,E-p-hydroxycinnamic acid（BM-7）,E-p-hydroxycinnamic acid methyl ester（BM-8）,ferulie acid（BM-11）,caffeic acid（BM-12）,1-O-feruloylglycerol （BM-15）,uracil（LB-1）,thymine（LB-2）,uridine（LB-5）,guanosine（LB-7）,thymidine （LB-8）,adenosine（LB-9）,β-sitosterol（BM-1）,daucosterol（BM-17）,atractylenolideⅢ（BM-5）.Among them,LBM-14 is a new compound;eight compounds,BM-2,BM-3, BM-5,BM-7,BM-8,BM-11,BM-12 and BM-15 were obtained from this genus for the first time,and sixteen compounds LBM-15,LBM-16,LBM-31,LBM-42,LBM-46, LBM-48,LBM-50,BM-1,BM-4,BM-17,LB-1,LB-2,LB-5,LB-7,LB-8 and LB-9 were isolated from this plant for the first time.A facile method based on high performance liquid chromatography coupled with evaporative light scattering detector（HPLC-ELSD） was developed for the simultaneous quantitative determination of eight alkaloids including pingbeimine A,peimisine, verticine,verticinone,isodelavine,demissidine-3-O-β-D-glucopyranosyl（1→4）-glucopyranoside in 60 batches of samples from 13 Fritillaria species.The chromatographic patterns of 13 Fritillaria species were also established,and the chemotaxonomic characteristics were proposed.Furthermore,a simple method based on high performance liquid chromatography coupled with diode-array detector（HPLC-DAD） was developed for the simultaneous determination of nine nucleosides and bases, including uracil,cytidine,guanine,uridine,thymine,inosine,guanosine,thymidine, adenosine in the same above 60 batches of samples.The results showed that uridine, guanosine and adenosine are the major nucleosides in Fritillaria bulbs,and may be the main bioactive constituents other than alkaloids.To conclude,The newly developed HPLC-ELSD and HPLC-DAD methods for simultaneous determination of eight alkaloids and nine nucleosides could be used as powerful tools for quality control of Fritillaria bulbs.In addition,advance in the research of chemical constituents,pharmacological activities and quality control of medicinal plants from Fritillaria genus were reviewed with 223 references.更多还原