药材川贝母的品质研究
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摘要
川贝母来源于百合科Liliaceae贝母属Fritillaria6种植物的干燥鳞茎,收载于2010版《中国药典》,具有清热润肺、化痰止咳、散结消痈之功效。川贝母是贝母类药材中药用价值最高的一个复合群。随着市场需求的快速增长以及过度采挖造成的生境破坏导致川贝母资源日益短缺,在地方省市出现了以次充好、以假乱真的现象,严重影响了川贝母成药产品的质量。本论文从川贝母产地适宜性分析、化学成分定量、真菌毒素检测、有效成分生物合成途径解析等方面对川贝母类群进行了系统地研究,为严格控制川贝母质量稳定,保证临床疗效和用药安全,推进川贝母资源开发奠定了理论基础。
市场上贝母类药材种类繁多,外观形态相似,鉴别难度较大。由于川贝具有很高的开发价值,价格较高,市场上经常出现贝母混售或以其他伪品充当川贝母出售的现象,致使川贝母的质量控制难度加大。为有效监管川贝母质量,保证临床疗效,本文分别对几种川贝母药材的脂溶性成分(主要是生物碱类)和水溶性成分(主要是核苷类成分)进行了检测和分析。采用HPLC-ELSD建立了可同时检测川贝母药材中4种生物碱(贝母辛、西贝素、贝母素甲和贝母素乙)含量的方法,并利用该方法对99批川贝母药材进行了含量测定。分别建立了5种川贝母药材的HPLC-ELSD指纹图谱,对共有峰进行了标定,并通过相似度对药材品质进行了评价。建立了HPLC-DAD方法同时检测川贝母药材10个核苷类成分含量的方法。99批川贝母样品含量测定结果表明尿嘧啶、胞苷、尿苷、次黄嘌呤、肌苷、鸟苷、胸苷、腺苷、腺嘌吟、2-脱氧腺苷10种核苷是川贝母药材共有成分,这对更好地评价川贝母品质具有重要的参考价值。同时以暗紫贝母为例建立了UPLC快速测定川贝母药材中10种核苷成分含量的技术体系,代替了传统的液相法进行核苷测定。川贝母中脂溶性和水溶性成分含量测定方法的建立,对有效区分贝母种类,科学评价川贝母品质,保证临床疗效具有重要的现实意义。
川贝母药材在生长、储存、运输过程中都有可能受到霉菌感染,受到真菌毒素的污染,对临床用药安全造成严重的威胁。本文采用HPLC-MS对黄曲霉毒素(aflatoxins, AF)、赭曲霉毒素A (Ochratoxin A, OTA)、雪腐镰刀菌烯醇(nivalenol, NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)、玉米赤霉烯酮(zearalenone, ZEN)、T-2毒素(T-2toxin)等毒素进行检测。分别建立了利用HPLC-MS/MS同时定性定量测定AFB1和OTA含量和采用HPLC-MS同时检测DON、NIV、ZEN及T-2毒素的方法,严格控制川贝母药材质量,为临床用药安全提供保障。
市场需求的快速增长使川贝母野生资源处于濒危境地,寻找川贝母潜在的生态适宜区进行野生抚育和人工种植是缓解资源紧张行之有效的方法。本文采用《中药材产地适宜性分析地理信息系统》(TCMGIS)对川贝母类群的生态适宜区进行研究,计算出与川贝母生态相似度为95%～100%和90%-95%的区域,科学、准确地分析出川贝母类群潜在的生产适宜区,为川贝母的引种栽培和生产布局合理化进行指导。
川贝母作为传统的化痰止咳药,甾体生物碱是其主要活性成分。但川贝母中生物碱的含量很低,提取分离对自然资源消耗很大,这在一定程度上限制了川贝母进一步的研究与开发。为解决这一难题,很多化学家已经将目光转向天然产物的遗传分析,通过构建cDNA文库来获悉编码生物合成关键酶的编码基因,从而解析生物合成的机制,进而达到通过合成生物学的手段来获得特定单一产物的目的。本文利用高通量测序技术对中药川贝母的基原植物之一——川贝母(ritillaria cirrhosa D.Don)转录组进行研究,获得了45,073条单一序列。发现了许多编码川贝母生物碱生物合成途径关键酶的相关基因,包括编码参与甲羟戊酸(MVA)途径所有酶和可能对下游反应进行修饰的酶的相关基因。为生物碱生物合成途径的研究提供了丰富的基因资源,进而为应用生物技术方法提高川贝母生物碱的含量,或寻找合适的中间体进行有机或生物合成及利用生物合成的方法直接进行生产奠定了基础。
The underground bulbs of six genus Fritillaria in Lilium. listed as "Chuan-beimu" in the2010edition of the Chinese Pharmacopoeia, have been widely used for thousands of years in traditional Chinese medicine as antitussive, anti-asthmatic and expectorant agents. Fritillariae cirrhosae bulbus is considerated as the highest value in genus Fritillaria. Currently, the sharp increased market demand pushed wild plants to the brink of extinction and break the equilibrium in the market. In order to control quality of Fritillariae cirrhosae bulbus, several gene Fritillaria species were analyzed in some aspect, including quantification of fat-soluble and water-soluble components, determination of mycotoxins, analysis on adaptive cultivated area and biosynthesis of isosteroidal alkaloids.
Fritillariae bulbus, Beimu in Chinese, derived from the bulbs of many Fritillaria species has been used as an antitussive and expectorant herb. Besides, the bulbs of more than30other species were utilized locally as substitutes for Fritillariae bulbus in the markert. Therefore, it is necessary to develop some methods to identify different Fritillaria species and their substitutes confused Fritillariae cirrhosae bulbus in market for the clinical safety of drugs. In this study,99patches Fritillariae cirrhosae bulbus from five Fritillaria species were analyzed by high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) and high-performance liquid chromatography coupled with a photodiode array detector (HPLC-DAD), respectively. Fingerprint analysis based on HPLC-ELSD was established. The result indicated that peimisine is a common constituent in all Fritillaria species investigated.10active nucleosides and nucleobases were detected in all samples, which could be useful for quality evaluation and control of all Fritillaria species in the future. In addition, take F. unibracteata for example. A simple and reliable method of ultra-performance liquid chromatography coupled with a photodiode array detector (UPLC-PAD) was developed for the quantitative analysis of10active nucleosides and nucleobases, including cytidine. uridine, guanosine, inosine. thymidine. cordycepin. adenosine. uracil, hypoxanthine and adenine, for quality control of F. unibracteata. The developed HPLC-ELSD for isosteroidal alkaloids and HPLC-DAD for nucleosides and nucleobases could be useful to control quality of Fritillariae cirrhosae bulbus.
Mycotoxins are widely distributed toxic secondary metabolites produced by several molds. The growth of molds under diverse environmental conditions could contaminate Fritillariae cirrhosae bulbus. Several studies have provided strong evidence that some mycotoxins have carcinogenic, teratogenic and neurotoxic effects. In the work, an effective method was established for simultaneous determination of aflatoxin B1and ochratoxin A in Fritillariae cirrhosae bulbus by solid-phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). And a rapid method was developed for simultaneously screening Fritillariae cirrhosae bulbus contaminated by DON, NIV, ZEN and T-2using LC-MS. These approaches could be implemented for the routine analysis to investigate the contamination of Fritillariae cirrhosae bulbus.
Owing to the increased demand for Fritillariae cirrhosae bulbus, wild Fritillaria species are threatened by overexploitation, habitat destruction and lack of proper cultivation practices. Thus, Ex-situ cultivation becomes an immediate action to sustain medicinal plant resources. The traditional Chinese medicine geographic information system (TCMGIS) was applied to predict the large-scale distribution of Fritillaria species. Adaptability analysis of producing area (with similarity rate>90%) was outputted, which gives insights into the discovery of potential habitats for Fritillaria species cultivation.
F. cirrhosa D. Don is the primary source of Fritillariae cirrhosae bulbus, which has been used as a traditional Chinese medicine for thousands of years as an antitussive apophlegmatic and expectorant. Previous pharmacological investigations indicated that isosteroidal alkaloids were responsible for the therapeutic effects. However, few studies have investigated how isosteroidal alkaloids are synthesized in the plant. To investigate the biosynthetic pathway of the isosteroidal alkaloids in F. cirrhosa, transcriptomic analysis was developed in the present work. A total of1,205,646high-quality sequence reads were generated from the cDNA library of F. cirrhosa bulbs and assembled into45,073unigenes. Many genes involved in the biosynthesis of isosteroidal alkaloids were discovered, including all the genes encoding enzymes catalyzing multiple reactions from acetyl-CoA to the first cyclic backbone, cycloartenol, in this pathway. Some candidate genes encode tailoring enzymes responsible for the modification of cycloartenol into various isosteroidal alkaloids. This study will lay the foundation for the elucidation of the mechanism underlying the biosynthesis of the isosteroidal alkaloids in F. cirrhosa and will also provide an important opportunity to further identify the uncharacterized compounds in the pathway.
市场上贝母类药材种类繁多,外观形态相似,鉴别难度较大。由于川贝具有很高的开发价值,价格较高,市场上经常出现贝母混售或以其他伪品充当川贝母出售的现象,致使川贝母的质量控制难度加大。为有效监管川贝母质量,保证临床疗效,本文分别对几种川贝母药材的脂溶性成分(主要是生物碱类)和水溶性成分(主要是核苷类成分)进行了检测和分析。采用HPLC-ELSD建立了可同时检测川贝母药材中4种生物碱(贝母辛、西贝素、贝母素甲和贝母素乙)含量的方法,并利用该方法对99批川贝母药材进行了含量测定。分别建立了5种川贝母药材的HPLC-ELSD指纹图谱,对共有峰进行了标定,并通过相似度对药材品质进行了评价。建立了HPLC-DAD方法同时检测川贝母药材10个核苷类成分含量的方法。99批川贝母样品含量测定结果表明尿嘧啶、胞苷、尿苷、次黄嘌呤、肌苷、鸟苷、胸苷、腺苷、腺嘌吟、2-脱氧腺苷10种核苷是川贝母药材共有成分,这对更好地评价川贝母品质具有重要的参考价值。同时以暗紫贝母为例建立了UPLC快速测定川贝母药材中10种核苷成分含量的技术体系,代替了传统的液相法进行核苷测定。川贝母中脂溶性和水溶性成分含量测定方法的建立,对有效区分贝母种类,科学评价川贝母品质,保证临床疗效具有重要的现实意义。
川贝母药材在生长、储存、运输过程中都有可能受到霉菌感染,受到真菌毒素的污染,对临床用药安全造成严重的威胁。本文采用HPLC-MS对黄曲霉毒素(aflatoxins, AF)、赭曲霉毒素A (Ochratoxin A, OTA)、雪腐镰刀菌烯醇(nivalenol, NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)、玉米赤霉烯酮(zearalenone, ZEN)、T-2毒素(T-2toxin)等毒素进行检测。分别建立了利用HPLC-MS/MS同时定性定量测定AFB1和OTA含量和采用HPLC-MS同时检测DON、NIV、ZEN及T-2毒素的方法,严格控制川贝母药材质量,为临床用药安全提供保障。
市场需求的快速增长使川贝母野生资源处于濒危境地,寻找川贝母潜在的生态适宜区进行野生抚育和人工种植是缓解资源紧张行之有效的方法。本文采用《中药材产地适宜性分析地理信息系统》(TCMGIS)对川贝母类群的生态适宜区进行研究,计算出与川贝母生态相似度为95%～100%和90%-95%的区域,科学、准确地分析出川贝母类群潜在的生产适宜区,为川贝母的引种栽培和生产布局合理化进行指导。
川贝母作为传统的化痰止咳药,甾体生物碱是其主要活性成分。但川贝母中生物碱的含量很低,提取分离对自然资源消耗很大,这在一定程度上限制了川贝母进一步的研究与开发。为解决这一难题,很多化学家已经将目光转向天然产物的遗传分析,通过构建cDNA文库来获悉编码生物合成关键酶的编码基因,从而解析生物合成的机制,进而达到通过合成生物学的手段来获得特定单一产物的目的。本文利用高通量测序技术对中药川贝母的基原植物之一——川贝母(ritillaria cirrhosa D.Don)转录组进行研究,获得了45,073条单一序列。发现了许多编码川贝母生物碱生物合成途径关键酶的相关基因,包括编码参与甲羟戊酸(MVA)途径所有酶和可能对下游反应进行修饰的酶的相关基因。为生物碱生物合成途径的研究提供了丰富的基因资源,进而为应用生物技术方法提高川贝母生物碱的含量,或寻找合适的中间体进行有机或生物合成及利用生物合成的方法直接进行生产奠定了基础。
The underground bulbs of six genus Fritillaria in Lilium. listed as "Chuan-beimu" in the2010edition of the Chinese Pharmacopoeia, have been widely used for thousands of years in traditional Chinese medicine as antitussive, anti-asthmatic and expectorant agents. Fritillariae cirrhosae bulbus is considerated as the highest value in genus Fritillaria. Currently, the sharp increased market demand pushed wild plants to the brink of extinction and break the equilibrium in the market. In order to control quality of Fritillariae cirrhosae bulbus, several gene Fritillaria species were analyzed in some aspect, including quantification of fat-soluble and water-soluble components, determination of mycotoxins, analysis on adaptive cultivated area and biosynthesis of isosteroidal alkaloids.
Fritillariae bulbus, Beimu in Chinese, derived from the bulbs of many Fritillaria species has been used as an antitussive and expectorant herb. Besides, the bulbs of more than30other species were utilized locally as substitutes for Fritillariae bulbus in the markert. Therefore, it is necessary to develop some methods to identify different Fritillaria species and their substitutes confused Fritillariae cirrhosae bulbus in market for the clinical safety of drugs. In this study,99patches Fritillariae cirrhosae bulbus from five Fritillaria species were analyzed by high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) and high-performance liquid chromatography coupled with a photodiode array detector (HPLC-DAD), respectively. Fingerprint analysis based on HPLC-ELSD was established. The result indicated that peimisine is a common constituent in all Fritillaria species investigated.10active nucleosides and nucleobases were detected in all samples, which could be useful for quality evaluation and control of all Fritillaria species in the future. In addition, take F. unibracteata for example. A simple and reliable method of ultra-performance liquid chromatography coupled with a photodiode array detector (UPLC-PAD) was developed for the quantitative analysis of10active nucleosides and nucleobases, including cytidine. uridine, guanosine, inosine. thymidine. cordycepin. adenosine. uracil, hypoxanthine and adenine, for quality control of F. unibracteata. The developed HPLC-ELSD for isosteroidal alkaloids and HPLC-DAD for nucleosides and nucleobases could be useful to control quality of Fritillariae cirrhosae bulbus.
Mycotoxins are widely distributed toxic secondary metabolites produced by several molds. The growth of molds under diverse environmental conditions could contaminate Fritillariae cirrhosae bulbus. Several studies have provided strong evidence that some mycotoxins have carcinogenic, teratogenic and neurotoxic effects. In the work, an effective method was established for simultaneous determination of aflatoxin B1and ochratoxin A in Fritillariae cirrhosae bulbus by solid-phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). And a rapid method was developed for simultaneously screening Fritillariae cirrhosae bulbus contaminated by DON, NIV, ZEN and T-2using LC-MS. These approaches could be implemented for the routine analysis to investigate the contamination of Fritillariae cirrhosae bulbus.
Owing to the increased demand for Fritillariae cirrhosae bulbus, wild Fritillaria species are threatened by overexploitation, habitat destruction and lack of proper cultivation practices. Thus, Ex-situ cultivation becomes an immediate action to sustain medicinal plant resources. The traditional Chinese medicine geographic information system (TCMGIS) was applied to predict the large-scale distribution of Fritillaria species. Adaptability analysis of producing area (with similarity rate>90%) was outputted, which gives insights into the discovery of potential habitats for Fritillaria species cultivation.
F. cirrhosa D. Don is the primary source of Fritillariae cirrhosae bulbus, which has been used as a traditional Chinese medicine for thousands of years as an antitussive apophlegmatic and expectorant. Previous pharmacological investigations indicated that isosteroidal alkaloids were responsible for the therapeutic effects. However, few studies have investigated how isosteroidal alkaloids are synthesized in the plant. To investigate the biosynthetic pathway of the isosteroidal alkaloids in F. cirrhosa, transcriptomic analysis was developed in the present work. A total of1,205,646high-quality sequence reads were generated from the cDNA library of F. cirrhosa bulbs and assembled into45,073unigenes. Many genes involved in the biosynthesis of isosteroidal alkaloids were discovered, including all the genes encoding enzymes catalyzing multiple reactions from acetyl-CoA to the first cyclic backbone, cycloartenol, in this pathway. Some candidate genes encode tailoring enzymes responsible for the modification of cycloartenol into various isosteroidal alkaloids. This study will lay the foundation for the elucidation of the mechanism underlying the biosynthesis of the isosteroidal alkaloids in F. cirrhosa and will also provide an important opportunity to further identify the uncharacterized compounds in the pathway.
引文
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