基于液质联用技术的温络方药效物质基础研究
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摘要
中药复方是中医辨证论治、整体观思维的具体表现,它是一组具有化学成分非常复杂、干扰因素众多等特点的典型复杂体系。中药复方通过君臣佐使各味药材之间的配伍组合,针对疾病的病因病机,通过适当的给药途径被吸收入血,整体协同互济达到治疗疾病的目的。温络方是类风湿性关节炎湿寒阻络证的代表方剂。
温络方由白术、卷柏、附子、桂枝组成,为治疗类风湿关节炎的验方。为揭示温络方的药效物质基础,提高整方的质量控制手段,本论文采用HPLC-DAD-MSn的技术手段研究了温络方主要的药效物质基础,具体研究内容如下所示：
第一部分文献综述
总结了温络方的临床应用和现代研究,并对温络方复方的四个单味药白术、卷柏、附子以及桂枝的化学成分进行了综述。共引用参考文献66篇。
第二部分温络方中卷柏化学成分的定性研究
首先建立了温络方中卷柏药材的液质联用定性分析方法。应用HPLC-DAD-MSn技术对卷柏中主要的双黄酮类化合物进行了鉴定。通过对含有三种双黄酮母核(穗花杉双黄酮、罗伯斯塔双黄酮、扁柏双黄酮)的7个标准品质谱裂解途径及紫外吸收光谱进行的研究,总结出了卷柏中主要三类双黄酮的质谱裂解规律与紫外吸收特征。并据此鉴定出卷柏药材中17个双黄酮化合物,其中5个化合物为卷柏中未见报道的成分。卷柏主要成分的鉴定为温络方全方定性以及其他富含双黄酮药材成分的快速鉴定奠定了基础。
第三部分温络方中白术化学成分的定性研究
建立了温络方中白术药材的液质联用定性分析方法。首先使用高分辨质谱对白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ的质谱裂解规律进行了深入的研究。后采用HPLC-DAD-MSn技术对白术药材中主要的化合物进行了鉴定。共检测出40个化合物,鉴定出其中的18个成分,其中3个未在白术的化学成分报道过,并推测出另外16个化合物的结构类型。鉴定出的化合物包括9个倍半萜内酯类成分以及9个聚烯炔类成分。白术HPLC-DAD-MSn分析条件的建立及主要化合物的分析鉴定,为温络方全方定性以及苍术属药材成分的快速鉴定奠定了基础。
第四部分温络方药效物质基础的研究
1、温络方化学成分的定性研究
在对白术及卷柏化学成分定性研究的基础上,采用HPLC-DAD-MSn技术对温络方的主要化学成分进行了分析,检测出48个化合物,鉴定推测出其中的44个化合物,包括12个来源于白术的倍半萜与聚乙炔成分；11个来源于卷柏的双黄酮与炔酚成分；15个来源于附子的生物碱类成分；6个来源于桂枝的苯丙素类小分子化合物。复方的定性研究工作为下一步复方入血研究及主要成分定量研究奠定了基础。
2、温络方活性部位入血移行成分初步研究
采用HPLC-DAD-MS11技术对温络方的入血移行成分进行了初步研究。通过比较含药血浆、空白血浆、温络方活性部位的质谱信息,发现了10个以原型入血的化合物,包括来自白术的倍半萜内酯成分、来自卷柏的双黄酮成分、来自于附子的二萜类生物碱成分以及来自于桂枝的苯丙素类小分子化合物,尤其以来自白术及桂枝的小分子成分含量高。
3、温络方中主要药效成分的定量研究
根据上述定性及入血分析的研究结果,采用了快速、灵敏度高的UPLC-DAD技术对温络方中含量较高且入血吸收明显的成分群进行了定量研究。包括白术中的3个白术内酯成分、桂枝中的4个小分子苯丙素类成分以及卷柏中的穗花杉双黄酮共8个化合物。定量研究不仅可以提高温络方的质量标准,而且使该复方的稳定性得到了有力的保障。
Treatment with traditional Chinese medicinal composite is one of the most important characteristics of traditional Chinese medicine (TCM). Studying material basis of TCM composite contributes a lot to modernizing the industry of Chinese medicinal herbs. In the present study, Wen-Luo-Fang, a multiherb medicine used to treat Rheumatoid Arthritis, is employed to address this key point. This dissertation is divided into four parts to study the effective substances of Chinese multiherb medicine Wen-Luo-Fang.
Part I:Summary of the related literature
The chemical constituents of Atractylodes macrocephala Kodiz, Aconitum carmichaeli Debx., Cinnamonun cassia Presl and Selaginella tamariscina (Beuav.) Spring which constitute Wen-Luo-Fang were summed up.66 references were cited.
Part II:Studies on the the components of Selaginella tamariscina (Beuav.) Spring by using HPLC-DAD-MS"
Biflavonoids are the main bioactive constituents of Selaginella tamariscina (Beuav.) Spring but few studies have reported the structural characterization method of biflavonoids with HPLC-MS". Firstly, The HPLC-DAD-MSn was used to study the fragmentation behavior of three main types of biflavonoids using seven biflavonoid reference compounds. And 17 biflavonoids were identified in an extract of Selaginella tamariscina based on the fragmentation behavior of different types of biflavonoids,5 of which have not been previously reported as the constituents of this plant. This result provides a fast and powerful approach for the online structural elucidation of biflavonoids from Selaginella plants and prescription Wen-Luo-Fang.
PartⅢ:Studies on the the components of Atractylodes macrocephala Kodiz by using HPLC-DAD-MSn
The HPLC-DAD-MSn method was developed to study the sesquiterpenes and polyacetylenes from Atractylodes macrocephala Kodiz. AtractylenolideⅠ,ⅡandⅢwere used to study the fragmentation behavior of sesquiterpenes by HRMS firstly, and 9 sesquiterpenes were tentatively characterized based on their UV and MS spectra. Furthermore,9 polyacetylene compounds were identified by HPLC-MSn for the first time, 3 of which have not been previously reported as the constituent of this plant. The structural types of the other 16 compounds were also tentatively identified. This result makes it easier to analyze the effective substances in Wen-Luo-Fang and other plants from Atractylodes DC.
Part VI:Effective substances research of Chinese multiherb medicine Wen-Luo-Fang with HPLC-DAD-MS"
1 Qualitative analysis of the main constituents of Wen-Luo-Fang
The main constituents in Wen-Luo-Fang were identified by coupling technique HPLC-DAD-MS". A total of 44 compounds including aconitine-type alkaloids, sesquiterpenes, biflavonoids and other constituents were identified in Wen-Luo-Fang. The results lay a good foundation for further serum pharmacochemistry study and quantitative analysis of the major substances in Wen-Luo-Fang.
2 Initial studies on the constituents absorbed into blood after the administration of of Wen-Luo-Fang
HPLC-DAD-MS" method was employed in the analysis of the constituents absorbed into blood after the oral administration of Wen-Luo-Fang. After comparing the HPLC-MS information of blank plasma and Wen-Luo-Fang extract,10 original compounds of Wen-Luo-Fang were observed in the plasma including 4-benzoylmesaconine, mesaconitine, hypaconitine, hypaconitine, coumarin,2-hydroxy cinnamaldehyde, cinnamic acid, amentoflavone, atractylenolide I and atractylenolide III. The content of compounds from Cinnamonun cassia Presl and Atractylodes macrocephala Kodiz are higher than the others.
3 Quantitative determination of eight main constituents of Wen-Luo-Fang
According to the result above, a UPLC-DAD method for quantitative determination of 8 main effective substances which were the main constituents of the formula(including 5 compounds detected in serum) was established. The methord of quantitative determination of 8 major components in Wen-Luo-Fang not only elevates the quality control standard of the formula but also makes the clinic application of Wen-Luo-Fang more effective and reliable.
温络方由白术、卷柏、附子、桂枝组成,为治疗类风湿关节炎的验方。为揭示温络方的药效物质基础,提高整方的质量控制手段,本论文采用HPLC-DAD-MSn的技术手段研究了温络方主要的药效物质基础,具体研究内容如下所示：
第一部分文献综述
总结了温络方的临床应用和现代研究,并对温络方复方的四个单味药白术、卷柏、附子以及桂枝的化学成分进行了综述。共引用参考文献66篇。
第二部分温络方中卷柏化学成分的定性研究
首先建立了温络方中卷柏药材的液质联用定性分析方法。应用HPLC-DAD-MSn技术对卷柏中主要的双黄酮类化合物进行了鉴定。通过对含有三种双黄酮母核(穗花杉双黄酮、罗伯斯塔双黄酮、扁柏双黄酮)的7个标准品质谱裂解途径及紫外吸收光谱进行的研究,总结出了卷柏中主要三类双黄酮的质谱裂解规律与紫外吸收特征。并据此鉴定出卷柏药材中17个双黄酮化合物,其中5个化合物为卷柏中未见报道的成分。卷柏主要成分的鉴定为温络方全方定性以及其他富含双黄酮药材成分的快速鉴定奠定了基础。
第三部分温络方中白术化学成分的定性研究
建立了温络方中白术药材的液质联用定性分析方法。首先使用高分辨质谱对白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ的质谱裂解规律进行了深入的研究。后采用HPLC-DAD-MSn技术对白术药材中主要的化合物进行了鉴定。共检测出40个化合物,鉴定出其中的18个成分,其中3个未在白术的化学成分报道过,并推测出另外16个化合物的结构类型。鉴定出的化合物包括9个倍半萜内酯类成分以及9个聚烯炔类成分。白术HPLC-DAD-MSn分析条件的建立及主要化合物的分析鉴定,为温络方全方定性以及苍术属药材成分的快速鉴定奠定了基础。
第四部分温络方药效物质基础的研究
1、温络方化学成分的定性研究
在对白术及卷柏化学成分定性研究的基础上,采用HPLC-DAD-MSn技术对温络方的主要化学成分进行了分析,检测出48个化合物,鉴定推测出其中的44个化合物,包括12个来源于白术的倍半萜与聚乙炔成分；11个来源于卷柏的双黄酮与炔酚成分；15个来源于附子的生物碱类成分；6个来源于桂枝的苯丙素类小分子化合物。复方的定性研究工作为下一步复方入血研究及主要成分定量研究奠定了基础。
2、温络方活性部位入血移行成分初步研究
采用HPLC-DAD-MS11技术对温络方的入血移行成分进行了初步研究。通过比较含药血浆、空白血浆、温络方活性部位的质谱信息,发现了10个以原型入血的化合物,包括来自白术的倍半萜内酯成分、来自卷柏的双黄酮成分、来自于附子的二萜类生物碱成分以及来自于桂枝的苯丙素类小分子化合物,尤其以来自白术及桂枝的小分子成分含量高。
3、温络方中主要药效成分的定量研究
根据上述定性及入血分析的研究结果,采用了快速、灵敏度高的UPLC-DAD技术对温络方中含量较高且入血吸收明显的成分群进行了定量研究。包括白术中的3个白术内酯成分、桂枝中的4个小分子苯丙素类成分以及卷柏中的穗花杉双黄酮共8个化合物。定量研究不仅可以提高温络方的质量标准,而且使该复方的稳定性得到了有力的保障。
Treatment with traditional Chinese medicinal composite is one of the most important characteristics of traditional Chinese medicine (TCM). Studying material basis of TCM composite contributes a lot to modernizing the industry of Chinese medicinal herbs. In the present study, Wen-Luo-Fang, a multiherb medicine used to treat Rheumatoid Arthritis, is employed to address this key point. This dissertation is divided into four parts to study the effective substances of Chinese multiherb medicine Wen-Luo-Fang.
Part I:Summary of the related literature
The chemical constituents of Atractylodes macrocephala Kodiz, Aconitum carmichaeli Debx., Cinnamonun cassia Presl and Selaginella tamariscina (Beuav.) Spring which constitute Wen-Luo-Fang were summed up.66 references were cited.
Part II:Studies on the the components of Selaginella tamariscina (Beuav.) Spring by using HPLC-DAD-MS"
Biflavonoids are the main bioactive constituents of Selaginella tamariscina (Beuav.) Spring but few studies have reported the structural characterization method of biflavonoids with HPLC-MS". Firstly, The HPLC-DAD-MSn was used to study the fragmentation behavior of three main types of biflavonoids using seven biflavonoid reference compounds. And 17 biflavonoids were identified in an extract of Selaginella tamariscina based on the fragmentation behavior of different types of biflavonoids,5 of which have not been previously reported as the constituents of this plant. This result provides a fast and powerful approach for the online structural elucidation of biflavonoids from Selaginella plants and prescription Wen-Luo-Fang.
PartⅢ:Studies on the the components of Atractylodes macrocephala Kodiz by using HPLC-DAD-MSn
The HPLC-DAD-MSn method was developed to study the sesquiterpenes and polyacetylenes from Atractylodes macrocephala Kodiz. AtractylenolideⅠ,ⅡandⅢwere used to study the fragmentation behavior of sesquiterpenes by HRMS firstly, and 9 sesquiterpenes were tentatively characterized based on their UV and MS spectra. Furthermore,9 polyacetylene compounds were identified by HPLC-MSn for the first time, 3 of which have not been previously reported as the constituent of this plant. The structural types of the other 16 compounds were also tentatively identified. This result makes it easier to analyze the effective substances in Wen-Luo-Fang and other plants from Atractylodes DC.
Part VI:Effective substances research of Chinese multiherb medicine Wen-Luo-Fang with HPLC-DAD-MS"
1 Qualitative analysis of the main constituents of Wen-Luo-Fang
The main constituents in Wen-Luo-Fang were identified by coupling technique HPLC-DAD-MS". A total of 44 compounds including aconitine-type alkaloids, sesquiterpenes, biflavonoids and other constituents were identified in Wen-Luo-Fang. The results lay a good foundation for further serum pharmacochemistry study and quantitative analysis of the major substances in Wen-Luo-Fang.
2 Initial studies on the constituents absorbed into blood after the administration of of Wen-Luo-Fang
HPLC-DAD-MS" method was employed in the analysis of the constituents absorbed into blood after the oral administration of Wen-Luo-Fang. After comparing the HPLC-MS information of blank plasma and Wen-Luo-Fang extract,10 original compounds of Wen-Luo-Fang were observed in the plasma including 4-benzoylmesaconine, mesaconitine, hypaconitine, hypaconitine, coumarin,2-hydroxy cinnamaldehyde, cinnamic acid, amentoflavone, atractylenolide I and atractylenolide III. The content of compounds from Cinnamonun cassia Presl and Atractylodes macrocephala Kodiz are higher than the others.
3 Quantitative determination of eight main constituents of Wen-Luo-Fang
According to the result above, a UPLC-DAD method for quantitative determination of 8 main effective substances which were the main constituents of the formula(including 5 compounds detected in serum) was established. The methord of quantitative determination of 8 major components in Wen-Luo-Fang not only elevates the quality control standard of the formula but also makes the clinic application of Wen-Luo-Fang more effective and reliable.
引文
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