木香生用理气煨熟止泻原理研究

【作者】 张旭；

【导师】 贾天柱； 杨静娴；

【作者基本信息】 辽宁中医药大学 ， 中药学， 2011， 博士

【摘要】 目的:木香是菊科植物木香Aucklandia lappa Decne.的干燥根,其性辛、苦、温,归脾、胃、大肠、三焦、胆经。《本草纲目》曰:云木香“凡入理气药只生用,不见火,若实大肠,宜面煨熟用”。生木香气芳香而辛散温通,擅长调中宣滞,行气止痛,多用于脘腹胀痛、泻痢后重、食积不消、不思饮食。“煨熟可止泻痢,因木香气味俱厚,且熟则无走散之性,惟觉香燥而守,故能实大肠,凡治泄泻恒用之”。由此可知,木香生用理气煨熟止泻作用在古代就有认识和临床应用,但其炮制原理尚无人进行深入研究。故本文在中医药理论的指导下,全面系统地考察木香生品、麸煨品、木香烃内酯和去氢木香内酯对胃肠动力作用的影响;结合麸煨前后化学成分的变化研究,寻求药效作用物质基础,探讨木香生用理气煨熟止泻的作用机制,解析木香炮制原理,从而规范木香炮制工艺,科学地指导临床用药。材料与方法:1.材料木香药材购自云南西双版纳勐海农科所,经辽宁中医药大学中药鉴定教研室翟延君教授鉴定为木香Aucklandia lappa Decne.的干燥根。木香烃内酯对照品(批号:111524-200503,纯度≥98%)、去氢木香内酯对照品(批号:111525-200706,纯度≥98%)均购自中国药品生物制品检定所。2.方法(1)采用HPLC法测定木香生品和麸煨品中木香烃内酯和去氢木香内酯两个指标性成分含量。(2)采用GC/MS法对木香生品和麸煨品挥发油化学成分进行测定分析。(3)建立利血平所致大鼠小鼠脾虚模型,考察木香生品和麸煨品对胃排空、小肠推进、D-木糖小肠吸收、胃蛋白酶活性等胃肠功能的影响。(4)建立番泻叶和蓖麻油致小鼠腹泻模型,考察木香生品和麸煨品抗小鼠腹泻作用;采用基线等比增减设计法筛选木香烃内酯和去氢木香内酯抗腹泻配比范围。(5)采用双抗体夹心酶联免疫吸附法(ELISA)测定脾虚大鼠胃肠激素水平和脾虚小鼠血清中细胞因子TNF-α和IL-10表达水平。(6)考察木香生品和麸煨品以及木香烃内酯、去氢木香内酯对正常大鼠和脾虚大鼠离体肠管平滑肌收缩的影响。(7)采用western-blot实验测定木香烃内酯和去氢木香内酯对利血平脾虚小鼠肠平滑肌组织肌球蛋白轻链激酶表达水平的影响。(8)采用正交设计配合药效指标综合筛选木香饮片切制工艺和麸煨工艺,采用HPLC法测定木香饮片稳定性。结果:1.木香生品和麸煨品中去氢木香内酯含量高于木香烃内酯含量,经麸煨后这两个指标性成分含量均降低,木香烃内酯含量下降幅度大于去氢木香内酯,从而导致两者的比例明显降低(P<0.01)。2.木香生品挥发油含量及油中主要成分木香烃内酯和去氢木香内酯含量均高于麸煨品,麸煨前后去氢木香内酯含量均高于木香烃内酯含量。生品挥发油共分离76个峰,鉴定了34个化合物;麸煨品挥发油共分离63个峰,鉴定了22个化合物。另外,有12个低沸点成分在麸煨木香挥发油中未能检出,还有三种含量较高的化合物尚未鉴定,其相对含量经麸煨后均明显增加。3.①正常小鼠胃排空和小肠推进实验结果:木香生品和麸煨品对正常小鼠胃排空无明显影响(P>0.05);木香生品的中、高剂量对小肠推进均有促进作用。利血平脾虚小鼠胃排空和小肠推进实验结果显示:木香麸煨品低、中剂量可明显促进脾虚小鼠胃排空;木香生品低、中、高三个剂量明显促进脾虚小鼠小肠推进。②正常大鼠D-木糖小肠吸收和胃蛋白酶活性测定结果显示:麸煨品低剂量对小肠吸收有明显的促进作用,并可提高胃液中胃蛋白酶活力。4.①抗小鼠腹泻实验腹泻指数结果表明:麸煨品止泻效果强于纸煨品和生品,且木香抗番泻叶腹泻作用比抗蓖麻油腹泻效果好。②基线等比增减设计法筛选出木香烃内酯和去氢木香内酯两者的比例在0.43-1.00范围内具有良好的抗腹泻作用。5.ELISA法测定胃肠激素和细胞因子结果显示:①木香生品和麸煨品对脾虚大鼠胃动素下降均有抑制作用,其中木香生品的中剂量对胃动素调节能力最强。木香生品的中、高剂量,木香麸煨品高剂量均能显著提高血清胃泌素浓度,降低血浆血管活性肠肽浓度,而各组血浆生长抑素浓度无明显差异。②木香烃内酯高剂量可以有效地提高脾虚小鼠血清中肿瘤坏死因子TNF-α浓度,而降低白介素IL-10浓度,而去氢木香内酯对这两种细胞因子调节作用不明显。6.离体肠管实验结果:①木香生品和麸煨品对正常大鼠自然状态的离体肠管平滑肌收缩均呈现先轻度抑制后较强兴奋作用,且呈剂量依赖关系。木香生品兴奋作用强于麸煨品。②木香生品和麸煨品对脾虚大鼠离体肠管平滑肌收缩均有先抑制后兴奋作用,总体表现为兴奋作用,生品作用强于麸煨品。③木香生品和麸煨品对由阿托品、心得安、高钙和低钙溶液所引起的肠管收缩异常均有调节作用,而生品对肠管收缩调节作用均强于麸煨品。④木香烃内酯对正常大鼠自然状态的离体肠管和脾虚大鼠离体肠管表现为兴奋作用,而去氢木香内酯对正常大鼠自然状态的离体肠管和脾虚大鼠离体肠管表现了抑制作用,且呈剂量依赖关系。7.Western-blot实验结果表明:高剂量木香烃内酯可显著提高脾虚小鼠肠平滑肌组织肌球蛋白轻链激酶表达水平,而去氢木香内酯作用较弱。8.炮制工艺研究结果:①木香最佳切制工艺参数:木香药材加入0.3倍量水,浸润10小时后切成1～2mm薄片。②最佳麸煨工艺参数:100 kg药材加30 kg麦麸,在110～120℃下煨制10 min。③不同厚度的木香饮片指标性含量均随放置时间出现先增加后降低的变化规律。结论:1.木香麸煨后,化学成分既发生了质变也发生了量变。麸煨后,两指标性成分木香烃内酯和去氢木香内酯含量比值发生了明显降低,可认为是木香煨熟止泻作用增强的主要原因之一。另外,木香生品和麸煨品挥发油中三种未鉴定的化合物也可能与木香煨熟止泻作用增强相关,还有待于以后进一步深入研究。2.木香生用理气作用机制:木香生品中木香烃内酯和去氢木香内酯含量比值较大,木香烃内酯促进肠蠕动作用明显,而去氢木香内酯对肠蠕动的抑制作用较弱,两者在生品中的协同作用整体表现为促进胃排空和小肠推进作用较强,有效地促进胃动素、胃泌素的分泌,降低血管活性肠肽分泌,增加胃肠平滑肌收缩频率,促进胃的血液循环,帮助消化吸收;并通过调节血清细胞因子TNF-α和IL-10含量而调节Th1/Th2细胞网络恢复至平衡状态,调节脾虚小鼠机体的免疫功能,提高肠平滑肌组织肌球蛋白轻链激酶表达水平而促进肠蠕动,调节了脾胃气机失调。3.木香煨熟止泻作用机制:木香麸煨品中木香烃内酯和去氢木香内酯含量比值与生品相比明显下降,即麸煨后木香烃内酯含量下降幅度大,而去氢木香内酯的含量下降幅度小,相对于木香烃内酯来说有所增加,因而导致两者的协同作用发生了改变,表现为对小肠蠕动促进作用降低,对小肠吸收促进作用增强。木香烃内酯和去氢木香内酯含量比值在一定的配比范围内协同治疗小鼠大肠性腹泻效果良好。4.木香生品和麸煨品对离体肠管收缩具有双向调节作用。5.考察工艺参数时,应配合药效指标来综合衡量。木香适宜现用现切片,且切薄片有效成分易于溶出,这与古籍记载木香“捣碎,薄切”符合。6.木香生用理气煨熟止泻的原理之一:木香生品木香烃内酯和去氢木香内酯含量比值较大,理气作用强;麸煨后,其两者含量比值降低,止泻作用增强。更多还原

【Abstract】 qPurpose:Radix Aucklandiae is the dry rhizome of Aucklandia lappa Decne.The drug is pungent and bitter in flavour, warm in nature, and acts on the spleen, stomach, large intestine, sanjiao and gallbladder channels. < Compendium of Materia Medica > said" Radix Aucklandiae only uses its crude samples when added into qi-regulating drugs, but no fire, and if solid the intestine, should be roasted with flour to cooked." Pungent and warm for dispersing, aromatic for enlivening the spleen, it is good at promoting circulation of Qi in the spleen, stomach and large intestine to remove stagnated food and alleviate pain. The herb is often used to treat Qi stagnation, gastro-intestinal distention, food retention, diarrhea, dysentery and abdominal pain.“Roasted product can stop the dysentery, both because its property and flavor are heavy, and cooked product has no dispersed property, dry and aromatic to keep, so it can solid large intestine, and often was used for diarrhea.”Thus it can be seen, the knowledge and clinical application of the raw Radix Aucklandiae with effect of regulating qi and roasetd Radix Aucklandiae with effect of antidiarrhea are early known in the ancient. But the depth study of processing principle is still undefined. Under the guidance of TCM theory, this thesis studied the effects of the raw Radix Aucklandiae, bran-roasted materials, costunolide and dehydrocostuslactone on gastro-intestinal motility sysmatically; combined with study of the change of chemical composition after bran roasted, find the therapeutic material basis and study the mechanism of the raw Radix Aucklandiae with effect of regulating qi and roasetd Radix Aucklandiae with effect of antidiarrhea, analysis the processing principles of Radix Aucklandiae, which will regulate processing technology and guide the clinical medicine.Material and method:1. MaterialRadix Aucklandiaes were purchased from the Menghai Research Institute of Agriculture in Xishuangbanna, Yunnan province. Zhai yanjun Professor of Liaoning University of traditional Chinese medicine identified them as the genuine. Costunolide reference substance (batch number: 111524-200503, purity≥98%), Dehydrocostuslactone reference substance (batch number: 111525-200706, purity≥98%) were purchased from national institutes for food and drug control.2. Method(1) The contents of the costunolide and the dehydrocostuslactone were estimated in raw and bran-roasted Radix Aucklandiae using HPLC.(2) Determinate the constituents of volatile oil in crude and bran-roasted Radix Aucklandia by GC-MS.(3) Established model of reserpine induced mouse spleen deficiency syndrome, to investigate the effect of the raw Radix Aucklandiae, bran-roasted materials on the gastric emptying, intestine propulsion, D-xylose intestinal absorption and the pepsin activity.(4) Establishment of senna and castor oil induced diarrhea model to study the anti-diarrhea action of bran–roasted materials and raw products; optimization of costunolide and dehydrocostuslactone effect on the experimental diarrhea with increase-decrease baseline geometric proportion design method.(5) By double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) measured levels of gastrointestinal hormones in spleen deficiency rats and in serum cytokine TNF-αand IL-10 expression of in spleen deficiency mice.(6) Investigate the effect of the raw Radix Aucklandiae, bran-roasted materials costunolide and dehydrocostuslactone on the vitro intestinal smooth muscle contraction of normal and deficiency rats.(7) Determinate the effect of costunolide and dehydrocostuslactone on the smooth muscle myosin light chain kinase expression of intestine tissue of mice induced by reserpine with the western-blot experiments.(8) Optimization of the cutting technology and the bran-roased technology associated with pharmacodynamic index for Radix Aucklandiae by orthogonal design; using HPLC to determinate the stability of Radix Aucklandiae pieces. Results:1. In the raw and bran-roasted Radix Aucklandiae, the contents of dehydrocostuslactone were higher than that of costunolide, after bran roasted, the contents of the two index components were reduced, costunolide content declined more than dehydrocostuslactone, which leads to significantly lower the ratio of the two index components (P <0.01).2. The content of volatile oil, costunolide and dehydrocostuslactone in crude drug were higher than that of bran-roasted Aucklandia lappa Decne. The crude products essential oil were isolated 76 peaks, of which 34 compounds were identified; 63 peaks of the bran-roasted products essential oil were separated, identified 22 compounds. In addition, 12 low boiling point components in the bran-roasted material are not detected in the essential oil, there are three higher levels of compounds are not identified, and the relative contents of them increase significantly after roasted.3.①The results of gastric emptying and intestine propulsion experments in normal mice suggested that the effect of the raw and bran-roasted Radix Aucklandiae on gastric emptying had no effect (P> 0.05); Raw materials of the secondary dose and high dose promoted the intestine propulsion. Spleen reserpine gastric emptying results show that: the roasted materials of low dose, secondary dose can significantly contribute to gastric emptying; small intestine results show that: the raw materials of low, secondary and high doses of the small intestine porpulsion significantly promoted.②Normal rat intestinal absorption of D-xylose showed that low doses of bran-roasted materials promoted the intestinal absorption and increaseed pepsin activity significantly.4. In Anti-diarrhea experment, diarrhea index results showed that the Anti-diarrhea effect of bran-roasted materials are stronger than paper-roasted products and raw materials, and the Anti-diarrhea effect for senna of Radix Aucklandiae was better than that of castor oil. The ratio 0.43-1.00 of costunolide and dehydrocostuslactone effect on the experimental diarrhea is better by increase-decrease baseline geometric proportion design method.5. Gastrointestinal hormone and cytokine ELISA, the results showed that:①the raw and bran-roasted Radix Aucklandiae on the motilin’s fallen has inhibited action, in which raw materials of the middle dose for motilin regulation is strongest. Raw products of middle dose, high dose of bran roasted materials can significantly increase serum gastrin levels, lower plasma vasoactive intestinal peptide, and each group showed no significant difference in plasma somatostatin.②Costunolide high doses can increase serum tumor necrosis factor TNF-αlevel, while reducing interleukin IL-10 levels of the deficency mice, while the dyhydrocustuslactone’s regulation of these two cytokines was not obvious.6. The results of isolated intestine experments showed that:①bran-roasted materials and raw products on the natural state of normal rat intestinal smooth muscle in vitro showed a slight inhibition of the first after a strong stimulant, and a dose-dependent. Excitatory effects of raw materials are stronger than that of bran-roasted products.②Bran-roasted materials and raw products on the spleen rat intestinal smooth muscle stimulant were first inhibited, the overall performance is stimulant, raw materials are stronger than bran-roasted products.③Bran-roasted materials and raw products on the atropine, propranolol, high calcium and low calcium solution caused both abnormal regulation of intestinal contractions, and raw products Regulation of intestinal contractions are stronger than the bran-roasted materials.④costunolide for the natural state of normal rat intestine and spleen deficiency rat intestine showed excitatory effects, while the dehydrocostuslactone for the natural state of normal rats in vitro rat intestine and spleen deficiency intestine demonstrated inhibition in a dose dependent manner.7. The result of Western-blot experment showed that: the high dose costunolide for the deficiency mice can significantly increase the expression of intestinal smooth muscle MLCK, and the weak action of dehydrocostuslactone.8. Processing technology results:①the best cutting technology of Radix Aucklandiae is cutting 1-2mm thin pieces after soaking 10h with 0.3 times water.②The optimum processing technology was Radix Aucklandiae mixed with 30% wheat brans stir-frying for 10 minutes at 110～120℃.③The index components of pieces of different thickness increased with the storage time firstly and then decreased. Conclusion:1. After bran-roasted, the chemical composition of Radix Aucklandiae occurred qualitative changes and quantitative changes, the ratio of the two index components costunolide and dehydrocostuslactone lowering significantly can be considered as to one of the main reason for enhancing the antidiarrheal effect of bran-roasted Radix Aucklandiae. In addition, in the essential oil of the raw materials and bran-roasted products, there are three unidentified compounds which maybe have relative with the antidiarrheal effect of bran-roasted Radix Aucklandiae, yet to be further studied.2. Regulating-qi mechanism of the raw Radix Aucklandiae: the content ratio of costunolide and dehydrocostuslactone is large in raw materials, and costunolide has significance act on promoting intestine, but the dehydrocostuslactone has weak inhibition for intestine movement. the synergistic reaction of these two components exhibite the stronger promoting gastric emptying and intestinal propulsion, promote the secretion of motilin and gastrin, reduce the secretion of vasoactive intestinal peptide to increase gastrointestinal smooth muscle contraction frequency, and promote blood circulation of the stomach to help digestion and absorption; and by regulating the serum cytokine TNF-αand IL-10 levels and adjust the network back to the Th1/Th2 cell balance and regulate the body’s immune mouse spleen Functions, improve intestinal smooth muscle myosin light chain kinase expression and promote expression of bowel movements, regulate the spleen and stomach qi disorders.3. Antidiarrheal mechanism of the bran-roasted Radix Aucklandiae: the content ratio of costunolide and dehydrocostuslactone decreased significantly compared with the raw materials, namely the descending scope of costunolide content is rather obvious, while the content of dehydrocostuslactone relative to costunolide increase, resulting in synergistic reaction between the two has changed, showing reduced the action of promoting the small bowel, enhanced the action of the intestinal absorption. The content ratio of costunolide and dehydrocostuslactone in a certain proportion treated large intestine diarrhea well.4. The raw and bran-roasted Radix Aucklandiae act on the contraction of isolated intestine with two-way adjustment.5. Investigated process parameters should be integrated with the indicators to measure efficacy. Radix Aucklandiae is suitable for current use, and easy dissolution of active ingredients cut thin slices, which consistent with historical records "broken, thin slice". 6. One of the processing principle of Radix Aucklandiae is that the larger the content ratio of costunolide and dehydrocostuslactone, the stronger regulating-qi; after bran-roasted, the contentration of them decrease and the antidiarrheal effect enhanced.更多还原