药对麻黄-地龙抗哮喘药效物质基础与作用机制研究
摘要
为了研究药对麻黄-地龙抗哮喘药效物质基础与作用机制,首先对麻黄-地龙不同配比在体外模型上进行了活性考察。方法:观察麻黄、地龙3种用量配比(1:1,1:3,1:9)的水煎液对10μmol /L卡巴胆碱(carbachol,CCh)、10μmol/L组胺(histamine, His)引起离体气管平滑肌张力增加的松弛作用,以及对10μmol/L CCh引起的离体气管上皮短路电流增加的抑制作用。结论:麻黄、地龙药对具有对抗CCh和His引起离体气管平滑肌收缩的作用,并有对抗CCh引起气管上皮离子分泌的作用。1:3的配比组合是麻黄、地龙药对的较优组成。
对药对单煎合用、合煎两种配伍方式进行了比较。方法:测定指纹图谱,并结合定量考察指标化学成分。结果:两种配伍方式所得提取液的色谱图匹配度较高,全图谱的相似度大于0.9,有22个共有峰,峰面积相似度<0.9;合煎有利于麻黄生物碱溶出,单煎有利于地龙氨基酸溶出。
运用“整体法”对麻黄-地龙药对进行平喘活性研究,即将麻黄-地龙(质量比1:3)单煎合用的混合水提液作为一个整体,通过AB-8大孔吸附树脂划分成水、50%乙醇、95%乙醇三个洗脱部位,然后对三个极性部分进行平喘作用的考察。结果:三种极性部位均有对抗CCh和His引起的豚鼠气管平滑肌张力增加的作用,且水洗脱部位对抗CCh引起的大鼠气管上皮短路电流增加的作用较强,而含乙醇(50%乙醇和95%乙醇)洗脱部位均没有明显的作用。结论:药对中不同极性大小的物质可能通过不同的作用机制都有抗哮喘效果,但综合起来考虑,水洗脱高极性部位的平喘效果要优于其它部位。
运用“组合法”对麻黄-地龙药对的平喘活性进行研究,即分别筛选出两个药材的有效部位,再进行组合部位的药效考察。首先优化了麻黄总生物碱的提取分离工艺,结果用酸水提取、乙酸乙酯萃取方法得到含量大于50%的麻黄总碱(M)。接着优化了地龙的提取分离工艺,将地龙水提醇沉后的提取液通过离子交换树脂划分成地龙酸性部位(S)、地龙碱性部位物(J)、地龙醇沉部分(D),在解痉和抑制上皮离子分泌两种体外模型,筛选出地龙平喘有效部位为S。体内药效实验进一步显示S具有对抗乙酰胆碱和His混合液引起豚鼠平滑肌痉挛作用,并在氨水引咳的小鼠模型上具有镇咳作用。接着试图将S通过大孔吸附树脂进一步划分成高极性(S30)和低极性(S95)两个部位,并用两部位对卵蛋白诱发哮喘小鼠的抗炎和免疫调节作用进行考察,结果在降低支气管肺泡灌洗液(BALF)中EOS细胞数量方面,S优于S30和S95;在降低IGE,IL-4,IL-5,IL-13水平方面,S和S30、S95相比没有显著性差异。以上结果提示:划段分离后并不能得到活性比划分前更有效的部位。
筛选出地龙的有效部位后,再对M与S、J、D分别组合的组合物,以及单用M或单用S的活性进行考察比较。体外实验结果表明对于CCh和His引起的豚鼠离体气管平滑肌收缩的舒张作用,以及CCh引起大鼠气管黏膜短路电流上升的抑制作用,MS的组合均优于MJ和MD组合。体内实验结果进一步显示MS组合对于乙酰胆碱和His混合液致豚鼠哮喘的平喘作用优于单用M;MS对于氨水引咳的小鼠镇咳作用优于单用S。以上研究结果表明麻黄与地龙组合优于单独使用一种,且两种药的最佳组合部位为MS,即麻黄总碱与地龙的酸性部位组合。可能的平喘作用机制为解痉、抑制上皮离子分泌以及镇咳作用。
对于麻黄和地龙的有效部位中的化学成分进行了测定。利用酸性染料比色法测定了麻黄总碱中生物碱含量大于80%;用HPLC法测定了其中2个主要成分麻黄碱和伪麻黄碱的含量,在总碱中占80%。用茚三酮比色法测得地龙酸性部位总氨基酸含量在80%左右;利用HPLC、GC-MS、UPLC-MS等定性定量了该部位中的42个化学成分,包括氨基酸类(16个),嘌呤嘧啶类(5个),多羟基类(4个),脂肪酸类(17个)。即在麻黄、地龙中的最佳有效组合物中共有44个化学成分得到了确认。
To research on the anti-asthmatic ingredients and mechanisms of the combination of Herbal Ephedrae & Pheretima, firstly, we evaluated the dosage ratio and anti-asthmatic property. Methods: To observe the relaxant effect of E1L1、E1L3、E1L9(the dosage ratio between Ephedra and Pheretima is 1:1, 1:3, 1:9 respectively)on precontracted isolated tracheal rings by 10μmol/L Carbachol (CCh) and 10μmol/L Histamine (His); To study inhibitory effect of tracheal epithelium secretion by observation of the decrease effect on short- circuit current (Isc) increase induced by 10μmol/L CCh on isolated rat tracheal epithelium. Results: E1L3 was the optimized ratio for the anti-asthmatic drug partnership. The drug partnership had the relaxant effect on CCh and His precontracted isolated tracheal rings, and the inhibitory effect on CCh induced (Isc) increase on isolated rat tracheal epitheliums.
Secondly, we compared methods of extracting together or separately of the combination, using both fingerprint spectrum and quantification of index chemical components. The fingerprint spectrum indicated that the similarity of whole spectrum was more than 0.9, peak area with less than 0.9, there were 22 common peaks. Spectrophotometric analysis revealed that extract separately produced more amino acids than extract together, however, HPLC analysis revealed that more ephedrine alkaloids were obtained by extracting together.
Two routes of exploring drug partnership are developed. One is“integration route”, regarding the mixture of the two drugs as a whole, the other is“fraction combination route”, extracting individual drug separately, and then the combination of their fractions.
As an example of“integration route”, we evaluated the different fractions with various polarity separated from the mixture of Herbal Ephedrae & Pheretima (mass ratio1:3), namely, 0%E(water eluant), 50 %E (50% ethanol eluant) and 95% E (95% ethanol eluant ) over macrophorous resin. All fractions showed relaxant effects on CCh and His precontracted isolated tracheal rings, but only water eluant displayed decreasing Isc effects on CCh pretreated epitheliums. It was concluded that the anti-asthmatic constituents had a broad range of polarity, but large polar part was compromised to be the best.
As an example of“fraction combination route”, firstly,the alkaloid extract from Herbal Ephedrae (M) was obtained with alkaloid content up to 80%. Then the water extract of Pheretima was separated into three fractions of the ethanolic precipitate (D), the alkaline fraction (J) and the acidic fraction (S). S was screened to be the anti-asthmatic fraction in vitro using the aforementioned models. Further in vivo studies also displayed that the acidic fraction could protect experimental asthma model induced by the combination of His and acetylcholine chloride in guinea pigs to prolong the latent periods of asthma (P < 0.05) and significantly decrease the cough frequency caused by ammonia water in mice (P < 0.001). Furthermore, S was separated into two fractions of high polar fraction (S30) and low polar fraction (S95) over large pore resin to find the more active ingredients by observing the anti-inflammation and immune-regulation effects on ovalbumin (OVA)-sensitized BALB/c mice. The result indicated that S inhibited eosinophilia (EOS) secretion in BALF more efficiently than S30 and S95, and for the level of IGE, IL-4, IL-5, IL-13, S30 and S95 did not show better decreasing effect. So S was screened to be the final active fraction.
Furthermore, we combined M with D, J and S respectively, then evaluated their anti-asthmatic effects. MS was significantly more effective than MJ and MD in vitro; in vivo, MS displayed better protective effect than M alone against bronchospasm in guinea pigs challenged by mixture spray of His and acetylcholine chloride, and better antitussive effect than S alone in ammonia-induced mice. In conclusion: Combined Herbal Ephedrae and Pheretima was advantages over single one for asthma therapy; MS was the active constituent combination. M and S produced a synergistic anti-asthmatic effect, justifying the traditional usage of Herbal Ephedrae & Pheretima on the management of asthma.
More than 80% constituents in the effective fraction of Herbae Ephedrae were determined, including ephedrine and pseudoephedrine; 80% constituents in the effective fraction of Pheretima were identified, specifically, including 16 amino acids, 5 purines and pyrimidines, 4 multi-hydroxy compounds, 17 fatty acids using HPLC, GC-MS and UPLC-MS.`Total 44 chemical ingredients in the combination were identified
对药对单煎合用、合煎两种配伍方式进行了比较。方法:测定指纹图谱,并结合定量考察指标化学成分。结果:两种配伍方式所得提取液的色谱图匹配度较高,全图谱的相似度大于0.9,有22个共有峰,峰面积相似度<0.9;合煎有利于麻黄生物碱溶出,单煎有利于地龙氨基酸溶出。
运用“整体法”对麻黄-地龙药对进行平喘活性研究,即将麻黄-地龙(质量比1:3)单煎合用的混合水提液作为一个整体,通过AB-8大孔吸附树脂划分成水、50%乙醇、95%乙醇三个洗脱部位,然后对三个极性部分进行平喘作用的考察。结果:三种极性部位均有对抗CCh和His引起的豚鼠气管平滑肌张力增加的作用,且水洗脱部位对抗CCh引起的大鼠气管上皮短路电流增加的作用较强,而含乙醇(50%乙醇和95%乙醇)洗脱部位均没有明显的作用。结论:药对中不同极性大小的物质可能通过不同的作用机制都有抗哮喘效果,但综合起来考虑,水洗脱高极性部位的平喘效果要优于其它部位。
运用“组合法”对麻黄-地龙药对的平喘活性进行研究,即分别筛选出两个药材的有效部位,再进行组合部位的药效考察。首先优化了麻黄总生物碱的提取分离工艺,结果用酸水提取、乙酸乙酯萃取方法得到含量大于50%的麻黄总碱(M)。接着优化了地龙的提取分离工艺,将地龙水提醇沉后的提取液通过离子交换树脂划分成地龙酸性部位(S)、地龙碱性部位物(J)、地龙醇沉部分(D),在解痉和抑制上皮离子分泌两种体外模型,筛选出地龙平喘有效部位为S。体内药效实验进一步显示S具有对抗乙酰胆碱和His混合液引起豚鼠平滑肌痉挛作用,并在氨水引咳的小鼠模型上具有镇咳作用。接着试图将S通过大孔吸附树脂进一步划分成高极性(S30)和低极性(S95)两个部位,并用两部位对卵蛋白诱发哮喘小鼠的抗炎和免疫调节作用进行考察,结果在降低支气管肺泡灌洗液(BALF)中EOS细胞数量方面,S优于S30和S95;在降低IGE,IL-4,IL-5,IL-13水平方面,S和S30、S95相比没有显著性差异。以上结果提示:划段分离后并不能得到活性比划分前更有效的部位。
筛选出地龙的有效部位后,再对M与S、J、D分别组合的组合物,以及单用M或单用S的活性进行考察比较。体外实验结果表明对于CCh和His引起的豚鼠离体气管平滑肌收缩的舒张作用,以及CCh引起大鼠气管黏膜短路电流上升的抑制作用,MS的组合均优于MJ和MD组合。体内实验结果进一步显示MS组合对于乙酰胆碱和His混合液致豚鼠哮喘的平喘作用优于单用M;MS对于氨水引咳的小鼠镇咳作用优于单用S。以上研究结果表明麻黄与地龙组合优于单独使用一种,且两种药的最佳组合部位为MS,即麻黄总碱与地龙的酸性部位组合。可能的平喘作用机制为解痉、抑制上皮离子分泌以及镇咳作用。
对于麻黄和地龙的有效部位中的化学成分进行了测定。利用酸性染料比色法测定了麻黄总碱中生物碱含量大于80%;用HPLC法测定了其中2个主要成分麻黄碱和伪麻黄碱的含量,在总碱中占80%。用茚三酮比色法测得地龙酸性部位总氨基酸含量在80%左右;利用HPLC、GC-MS、UPLC-MS等定性定量了该部位中的42个化学成分,包括氨基酸类(16个),嘌呤嘧啶类(5个),多羟基类(4个),脂肪酸类(17个)。即在麻黄、地龙中的最佳有效组合物中共有44个化学成分得到了确认。
To research on the anti-asthmatic ingredients and mechanisms of the combination of Herbal Ephedrae & Pheretima, firstly, we evaluated the dosage ratio and anti-asthmatic property. Methods: To observe the relaxant effect of E1L1、E1L3、E1L9(the dosage ratio between Ephedra and Pheretima is 1:1, 1:3, 1:9 respectively)on precontracted isolated tracheal rings by 10μmol/L Carbachol (CCh) and 10μmol/L Histamine (His); To study inhibitory effect of tracheal epithelium secretion by observation of the decrease effect on short- circuit current (Isc) increase induced by 10μmol/L CCh on isolated rat tracheal epithelium. Results: E1L3 was the optimized ratio for the anti-asthmatic drug partnership. The drug partnership had the relaxant effect on CCh and His precontracted isolated tracheal rings, and the inhibitory effect on CCh induced (Isc) increase on isolated rat tracheal epitheliums.
Secondly, we compared methods of extracting together or separately of the combination, using both fingerprint spectrum and quantification of index chemical components. The fingerprint spectrum indicated that the similarity of whole spectrum was more than 0.9, peak area with less than 0.9, there were 22 common peaks. Spectrophotometric analysis revealed that extract separately produced more amino acids than extract together, however, HPLC analysis revealed that more ephedrine alkaloids were obtained by extracting together.
Two routes of exploring drug partnership are developed. One is“integration route”, regarding the mixture of the two drugs as a whole, the other is“fraction combination route”, extracting individual drug separately, and then the combination of their fractions.
As an example of“integration route”, we evaluated the different fractions with various polarity separated from the mixture of Herbal Ephedrae & Pheretima (mass ratio1:3), namely, 0%E(water eluant), 50 %E (50% ethanol eluant) and 95% E (95% ethanol eluant ) over macrophorous resin. All fractions showed relaxant effects on CCh and His precontracted isolated tracheal rings, but only water eluant displayed decreasing Isc effects on CCh pretreated epitheliums. It was concluded that the anti-asthmatic constituents had a broad range of polarity, but large polar part was compromised to be the best.
As an example of“fraction combination route”, firstly,the alkaloid extract from Herbal Ephedrae (M) was obtained with alkaloid content up to 80%. Then the water extract of Pheretima was separated into three fractions of the ethanolic precipitate (D), the alkaline fraction (J) and the acidic fraction (S). S was screened to be the anti-asthmatic fraction in vitro using the aforementioned models. Further in vivo studies also displayed that the acidic fraction could protect experimental asthma model induced by the combination of His and acetylcholine chloride in guinea pigs to prolong the latent periods of asthma (P < 0.05) and significantly decrease the cough frequency caused by ammonia water in mice (P < 0.001). Furthermore, S was separated into two fractions of high polar fraction (S30) and low polar fraction (S95) over large pore resin to find the more active ingredients by observing the anti-inflammation and immune-regulation effects on ovalbumin (OVA)-sensitized BALB/c mice. The result indicated that S inhibited eosinophilia (EOS) secretion in BALF more efficiently than S30 and S95, and for the level of IGE, IL-4, IL-5, IL-13, S30 and S95 did not show better decreasing effect. So S was screened to be the final active fraction.
Furthermore, we combined M with D, J and S respectively, then evaluated their anti-asthmatic effects. MS was significantly more effective than MJ and MD in vitro; in vivo, MS displayed better protective effect than M alone against bronchospasm in guinea pigs challenged by mixture spray of His and acetylcholine chloride, and better antitussive effect than S alone in ammonia-induced mice. In conclusion: Combined Herbal Ephedrae and Pheretima was advantages over single one for asthma therapy; MS was the active constituent combination. M and S produced a synergistic anti-asthmatic effect, justifying the traditional usage of Herbal Ephedrae & Pheretima on the management of asthma.
More than 80% constituents in the effective fraction of Herbae Ephedrae were determined, including ephedrine and pseudoephedrine; 80% constituents in the effective fraction of Pheretima were identified, specifically, including 16 amino acids, 5 purines and pyrimidines, 4 multi-hydroxy compounds, 17 fatty acids using HPLC, GC-MS and UPLC-MS.`Total 44 chemical ingredients in the combination were identified
引文
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