基于生物碱和多糖含量的仲景方中附子、乌头的古今用药差异性研究

【作者】 刘鹏；

【导师】 胡慧华；

【作者基本信息】 北京中医药大学 ， 中药炮制学， 2018， 硕士

【摘要】 1.目的现今临床使用附子、川乌多借鉴张仲景的用药经验,且张仲景使用附子是以枚为剂量单位,现在尚未有人研究过张仲景使用的南阳附子和现今使用的江油栽培附子之间的差异。根据前期本课题组研究张仲景附子“炮”法的炮制工艺,研究现今使用的江油附子及炮制品和张仲景使用的南阳附子及炮制品的生物碱和多糖在有机溶剂提取和水煎液中的含量差异;通过特征图谱整体评价现今使用的江油附子及炮制品和张仲景使用的南阳附子及炮制品在整体性上的差异;使用南阳的生、“炮”附子通过正交试验探究在张仲景煎煮条件下6种生物碱的含量差异;通过南阳的生、“炮”附子在煎煮过程中6种生物碱的含量变化探究张仲景煎煮附子时间的内涵;通过川乌、乌头和附子在水煎液中的生物碱和多糖含量,探究临床使用川乌、乌头和附子的差异;通过附子、乌头的煎煮实验,探究附子、乌头煎煮过程中生物碱变化的差异;通过测定南阳野生乌头整株植物不同部位的生物碱和多糖的含量探究张仲景选择药用部位的内涵。2.方法1.采用HPLC测定用有机溶剂和水煎液提取的现今使用的江油附子及炮制品和张仲景使用的南阳附子及炮制品中生物碱的含量,比较在6种单、双酯和总酯型生物碱含量方面的差异。2.采用HPLC法对有机溶剂和水煎液提取的江油附子及炮制品和南阳附子及炮制品进行特征图谱研究,对比相似度,进行整体差异性评价。3.采用HPLC法,参照张仲景煎煮条件下,使用南阳生附子及炮附子进行正交试验,比较6种单、双酯和总酯型生物碱含量方面的差异。4.采用HPLC法在南阳生、炮附子和乌头临床煎煮过程,测定6种单、双酯和总酯型生物碱含量和pH的动态变化。5.采用HPLC法和紫外分光光度计测定有机溶剂提取中南阳乌头整株植物不同部位的生物碱含量和多糖含量,比较在6种单、双酯和总酯型生物碱和多糖含量方面的差异。6.采用紫外分光光度计,测定现今使用的江油附子及炮制品和张仲景使用的南阳附子及炮制品在有机溶剂中和水煎液提取的多糖含量,比较多糖含量方面的差异。3.结果根据有机溶剂提取中测得的总酯型生物碱含量,得到理论剂量比为南阳生附子:江油生附片≈1:4,将该剂量比的南阳和江油附子投放入水煎液中,根据测得水煎液中总酯型生物碱含量,计算剂量比为,南阳生附子:江油生附片≈1:4,因此在实际临床煎煮中江油生附片32g左右才能与南阳生附子一枚具有相同的生物碱含量。南阳炮附子和江油附子的炮制品,参照有机溶剂提取中测得的总酯型生物碱含量,确定剂量比例,南阳炮附子:黑顺片:白附片:淡附片:炮附片:江油“炮”附子=1:25:25:25:25:2。将该剂量比例投入水煎液中,实际测得水煎液中总酯型生物碱含量,进而得到剂量比为,南阳炮附子:黑顺片:白附片:淡附片:炮附片:江油“炮”附子=1:13:14:14:10:2,因此实际煎煮中江油附子炮制品剂量的500g左右才能与南阳炮附子一枚具有相同的生物碱含量。说明现今的附子存在过度炮制的现象,在追求控制毒性的同时,使主要的生物碱成分流失,导致临床疗效不佳。通过特征图谱研究发现,南阳和江油附子的生品和炮制品在整体性上存在较大的差异,但同时也发现江油生附片经过张仲景的“炮”法后与南阳炮附子无论在整体上,还是在生物碱含量均接近。实测江油附子经过张仲景“炮”法后8g与南阳炮附子一枚具有相同的生物碱含量,因此可以考虑江油附子采用“炮”法来代替现在江油附子过度炮制的炮制品种。张仲景煎煮生附子多使用一枚、煎煮1次、煎煮比例为0.3、加水量(折算一剂)为300ml,这种条件下水煎液中单酯型生物碱含量最高,通过煎煮动态变化实验发现,在生附子煎煮30分钟左右,单酯型生物碱含量急速增加,此时可能具有较好的回阳救逆的作用。对于炮附子,张仲景在临床中多使用一枚、煎煮1次、煎煮比例为0.4、加水量(折算一剂)为500ml,这种条件下水煎液中双酯型生物碱含量最高,通过煎煮动态变化实验发现,在炮附子煎煮40分钟左右,双酯型生物碱含量高且随着时间变化稳定,此时可能具有较好的温里散寒的作用。研究南阳附子和江油附子在多糖含量上的差异,有机溶剂中通过多糖含量计算剂量比例,南阳生附子:江油生附片=1:0.91,南阳炮附子:黑顺片:白附片:淡附片:炮附片:江油“炮”附子=1:0.53:0.78:1.04:0.59:0.65,水提取中通过多糖含量计算剂量比例为南阳生附子:江油生附片=1:1.07,南阳炮附子:黑顺片:白附片:淡附片:炮附片:江油“炮”附子=1:1.33:1.36:1.81:1.40:1.51。因此实际临床煎煮中,南阳生附子一枚和江油生附片3g左右的多糖含量相同,南阳的炮附子一枚和江油附子炮制品的5g左右多糖含量相等。江油生附片经过“炮”法后,南阳的炮附子一枚约和江油“炮”附子3g的多糖含量相同,分析产生差异的原因可能与两者的生长环境和提取的溶剂有关。比较南阳和江油附子、南阳乌头和江油川乌在生物碱、多糖的差异,实验结果表明,两者由于生长环境的差异,在总酯型生物碱含量上,无论在有机溶剂还是水煎液中,南阳生附子>南阳乌头>江油生附片>江油川乌。在多糖含量上,有机溶剂中,江油川乌>江油生附片>南阳生附子>南阳乌头,水煎液中,南阳乌头>南阳生附子>江油生附片>江油川乌。在煎煮过程中两者由于生物碱含量不同,随着时间的变化,生物碱含量变化出现不同波动情况,附子生物碱含量变化波动大,乌头生物碱含量变化波动小。在水煎液中南阳附子和乌头在双酯型生物碱含量上相差近56倍,而江油附子和川乌在双酯型生物碱含量相差约15倍,这可能是张仲景临床附子用一枚、乌头用五枚的原因。但是两者的临床使用差异还需要进一步的研究。南阳野生乌头的整株植物不同部位的总酯型生物碱和多糖含量,发现在多糖含量上,附子>乌头>地上部分>须根,但是在生物碱含量上,附子>须根>乌头>地上部分,由于须根属于附子的一部分,因此其生物碱含量高于乌头,地上部分含量最低。因此,须根和地上部分是否在张仲景方中作为药用或者在炮制净制中除去,没有记载,需要进一步研究开发。4.结论通过实验发现现今使用的江油附子和张仲景使用的南阳附子在有机溶剂提取和水煎液中生物碱和多糖含量上相差很大,因此单靠有机溶剂换算剂量比存在着明显的误差,水煎液中含量更加符合临床实际情况。在水煎液中,对于生附子,研究发现生物碱方面,古今使用的生附子剂量差16倍,多糖含量接近,但是现今临床上生附子多不药用。对于附子炮制品,研究发现生物碱方面张仲景炮附子和江油附子的炮制品剂量相差250倍之多,多糖含量在水煎液中接近。张仲景“炮”法作为一种新方法,操作简单,实验中江油附子通过“炮”法之后与南阳炮附子差异较小,且南阳和江油附子均存在炮制后双酯型生物碱含量降低,即毒性降低,2015版药典规定双酯型生物碱含量不超过0.010%,但是南阳附子经过“炮”法后双酯型生物碱含量显著高于0.010%。由于附子生长环境导致本身生物碱和多糖的含量存在差异,因此需要进一步研究江油附子采用张仲景“炮”法工艺的科学性。通过查阅文献研究张仲景的煎煮法,发现张仲景煎煮附子不用先煎且存在生、炮品均使用的情况,实验结果表明参照张仲景煎煮条件煎煮结束时,水煎液中单、双酯型生物碱均大量存在。通过文献整理发现张仲景使用附子保证安全不仅是煎煮方法,还在于其配伍减毒和辨证论治,还与当时“伤寒病”众多的环境有关。张仲景使用附子和乌头,而不用乌头的其他部位可能与生物碱和多糖含量低有关,也可能与其余部分占比小有关。本实验仅通过化学方面解释了古今附子和乌头的差异,在现今临床大量参照张仲景使用附子和乌头经验情况下,需要进一步用南阳附子和乌头通过药理、化学、临床方面探究张仲景使用附子和乌头的内涵,从而用来指导现在临床使用江油附子及炮制品替代南阳附子及炮附子。更多还原

【Abstract】 ObjectiveCurrent clinic use radix aconiti carmichaeli and radix aconite,which references the experience of Zhang Zhongjing and Zhang Zhongjing use the dose of radix aconiti carmichaeli is one.Now no one has studied the difference between Zhang Zhongjing used Nanyang radix aconiti carmichaeli and nowadays uses Jiangyou radix aconiti carmichaeli.According to project prophase studied Zhang Zhongjing "gun" processsing technology of Nanyang radix aconiti carmichaeli to study the difference of alkaloid and polysaccharides content between Jiangyou radix aconiti carmichaeli and its processed products and Zhang Zhongjing used Nanyang and processed products of radix aconiti carmichaeli by organic solvent and water extraction.Through feature maps evaluate overall difference between the Jiangyou processed and unprocessed products of radix aconiti carmichaeli and Nanyang radix aconiti carmichaeli and its processed product.Through orthogonal test to use the decoction condition of Nanyang unprocessed and processed radix aconiti carmichaeli to explore the differences of 6 kinds alkaloid content.With Nanyang unprocessed and processed radix aconiti carmichaeli to explore alkaloid content changes during the decoction process and inquire the connotation of Zhang Zhongjing decoction time.Through the alkaloid and polysaccharide content of Nangyang and Jangyou radix aconiti and radix aconiti carmichaeli to explore the differences between radix aconiti and radix aconiti carmichaeli.Through the processing of decocting between radix aconiti and radix aconiti carmichaeli to explore the difference change of them.Through measure the content of alkaloids and polysaccharide from different parts of the Nanyang radix aconiti to explore the connotation of Zhang Zhongjing chose the medicinal part.Methods1.We used the HPLC to measure organic solvent and water extract in Jiangyou and Nanyang radix aconiti carmichaeli and its processed products,comparing alkaloid content differences.2.By the method of HPLC to evaluate the similarity and the overall difference of organic solvent and water extract in Jiangyou unprocessed and processed products and Nanyang radix aconiti carmichaeli and its processed product.3.According to Zhang Zhongjing decocted condition method we use the HPLC to orthogonal experiment and explore the differences of alkaloid content.4.According to decoction process of Nanyang radix aconiti carmichaeli and radix aconiti to determinate the changes of alkaloid content and pH by HPLC.5.To explore the differences of alkaloid and polysaccharide content in Nanyang different parts of the radix aconiti by HPLC and ultraviolet spectrophotometer.6.Using ultraviolet spectrophotometer,we determinate to explore the differences of polysaccharide content by organic solvent and water extract in Jiangyou and Nanyang radix aconiti carmichaeli processed and unprocessed products.ResultsAccording to organic solvent extract alkaloid content proportion,the dose radix aconiti carmichaeli of Nanyang than Jiangyou is equal to 1:4.Then put 1:4 dose into the water decoction,the result was Nanyang than Jiangyou radix aconiti carmichaeli proportion is 1:4.So in actual decoction about 16 times(23g)dose of Jiangyou radix aconiti carmichaeli and Nanyang one radix aconiti carmichaeli with the same alkaloid content.According to organic solvent extract alkaloid content proportion,Nanyang processed radix aconiti carmichaeli:Jiangyou Heishunpian:Jiangyou Baifupian:Jangyou Danfupian:Jangyou Paofupian:Jiangyou"Pao" fuzi equal to 1:25:25:25:25:2,Then put the proportion into water decoction,the actually measured dose proportion,Nanyang processed radix aconiti carmichaeli:Jiangyou Heishunpian:Jiangyou Baifupian:Jiangyou Danfupian:Jiangyou Paofupian:Jiangyou "Pao" fuzi equal to 1:13:14:14:10:2.So in the actual decoction,Jiangyou radix aconiti carmichaeli about 250 times(500g)and Nanyang one processed with the same alkaloid content.Through feature maps found that processed and unprocessed Nanyang and Jiangyou radix aconiti carmichaeli have big differences on the whole,but also found that Jiangyou radix aconiti carmichaeli after Zhang Zhongjing "gun" processing method is close to Zhang Zhongjing Nanyang processed radix aconiti carmichaeli.Therefore Jiangyou 8 g radix aconiti carmichaeli is equal to Nanyang one processed radix aconiti carmichaeli with the same alkaloid content.Therefore,it is important to consider to use "gun" processing method to replace the over processed Jiangyou radix aconiti carmichaeli processed products.Research on the decoction method of radix aconiti carmichaeli of Zhang Zhongjing,Use a normal radix aconiti carmichaeli,decoction ratio is 0.3,adding water is 300 ml.At this water decoction method monoester alkaloidcontent in the highest,Through dynamic decoction experiment in the radix aconiti carmichaeli decoction 30 minutes,monoester alkaloid content increased rapidly.Maybe this time have good back to save inverse Yang function.For Nanyang processed radix aconiti carmichaeli,Zhang Zhongjing in clinic used a normal radix aconiti carmichaeli,decoction ratio is 0.4,adding water is 500 ml.The double ester alkaloid content in the highest level.Through dynamic decoction experiment has proved that the processed radix aconiti carmichaeli decocted in 40 minutes,the ester alkaloids content of it might have good temperature in the body.The polysaccharide content in radix aconiti carmichaeli of Nanyang and Jiangyou is differenct.Organic solvent extracted polysaccharide dose rate,Nanyang than Jiangyou is equal to 1:1.07.Nanyang processed radix aconiti carmichaeli Jiangyou Heishunpian:Jiangyou Baifupian Jiangyou Danfupian:Jiangyou Paofupian:Jiangyou "Pao" fuzi equal tol:0.53:0.78:1.04:0.59:0.65.Water extracted polysaccharide dose rate,Nanyang than Jiangyou is equal to 1:0.91.Nanyang processed radix aconiti carmichaeli:Jiangyou Heishunpian:Jiangyou Baifupian:Jiangyou Danfupian:Jiangyou Paofupian:Jiangyou "Pao" fuzi equal to1:1.33:1.36:1.81:1.40:1.51.Analysis the causes of differences may be related to the growth of environment and extracting solvent.So in the actual clinical decoction,Nanyang one unprocessed is equal to Jiangyou 3g radix aconiti carmichaeli with the same polysaccharide content.The Nanyang one processed is equal to Jiangyou 5g radix aconiti carmichaeli with the same polysaccharide content.Jiangyou 3g radix aconiti carmichaeli after "gun" method processing have the same polysaccharide content with Nanyang one processed.Comparing the differences in alkaloids and polysaccharides content in Nanyang and Jiangyou radix aconiti carmichaeli and radix aconite.The experiment results shows that the differences in environment causes the content of alkaloids in Nanyang radix aconiti carmichaeli and radix aconiti above Jiangyou.But the contents of polysaccharide in Nanyang radix aconiti carmichaeli and radix aconiti below Jiangyou by organic solvent extraction.While the contents of polysaccharide in Nanyang radix aconiti carmichaeli and radix aconiti higher than Jiangyou by water extraction.During decoction process shows the different change rule because of the alkaloids content is unlike.Nanyang radix aconiti carmichaeli and radix aconiti in water decoction on double ester alkaloids content is nearly 56 times,and Jiangyou radix aconiti carmichaeli and radix aconite on double ester alkaloids content is about 15 times.So this is Zhang Zhongjing clearly differently use one radix aconiti carmichaeli and five radix aconiti in clinic.The difference need to futher study.Nanyang wild radix aconiti whole plant differenet parts on alkaloid content order is radix aconiti carmichaeli,radix aconiti,fibrous root and floor section part.The polysaccharide content order is radix aconiti carmichaeli,fibrous root,radix aconiti and floor section part.Because fibrous root is one part of radix aconiti carmichaeli,so its polysaccharide content is higher than radix aconiti.So it is important to study the fibrous root and floor section part and investigate whether ZhangZhongjing use them for medicinal part.ConclusionThrough the experiment find that there is widely different in alkaloid and polysaccharide content between Jiangyou radix aconiti carmichaeli and Nanyang radix aconiti carmichaeli.In the organic solvent extraction and water decoction is more different,but water decoction is more losely to the clinical practice.The study found that the alkaloid content of Nanyang radix aconiti carmichaeli more than Jiangyou radix aconiti carmichaeli 16 times,polysaccharide content is equal.But now clinic does not use unprocessed radix aconiti carmichaeli as medicine.The study found that alkaloids content in Nanyang unprocessed radix aconiti carmichaeli and Jiangyou varieties of processed is 250 times as much,polysaccharide content in water decoction is close.Zhang Zhongjing "gun" method as a kind of new method,the operation is simple.Jangyou radix aconiti carmichaeli after "gun" method is close to Nanyang processed radix aconiti carmichaeli difference.2015 edition of pharmacopoeia double ester alkaloids content less than 0.010%,but in Nanyang after "gun" method of radix aconiti carmichaeli double ester alkaloids content is significantly higher than 0.010%.because of the growing environment causes the alkaloid and polysaccharide content is different,so it needs to further study on Zhang Zhongjing "gun" method.By studying Zhang Zhongjing decoction method,we found that Zhang Zhongjing decoction without first decocted,radix aconiti carmichaeli decocted with most single,double ester alkaloids.Through literature found that Zhang Zhongjing use radix aconiti carmichaeli is security.It is not only the radix aconiti carmichaeli decoction,also lies in its compatibility,syndrome different treatment,the numerous "typhoid fever" environment to reduce the poison.Zhang Zhongjing use radix aconiti carmichaeli and radix aconiti without other parts may be because of other parts alkaloid and polysaccharide content is small and poor proportion.This experiment uses chemistry to explain the difference of Nanyang and Jiangyou radix aconiti carmichaeli.Today a large number of clinic reference to the use of experience of radix aconiti carmichaeli in Zhang Zhongjing prescriptions.So it is need to use pharmacology,chemistry to guide clinic use radix aconiti carmichaeli experience of Zhang Zhongjing and process Jiangyou radix aconiti carmichaeli instead of Nanyang radix aconiti carmichaeli.更多还原