汉桃叶有机酸类成分研究及软膏剂开发
摘要
目的：汉桃叶别名七叶莲、七加皮、七叶烂、龙手树等,为五加科广西鹅掌柴属植物,主要分布于我国广西、云南、贵州、福建、浙江等地,是我国苗族、彝族等少数民族地区常用的民间草药。临床应用止痛效果明显,对三叉神经痛、坐骨神经痛及胃肠绞痛等有明显的缓解作用,尤对某些神经痛明显有效,且副作用小。镇痛抗炎的有效成分是有机酸类物质。本课题以汉桃叶——广西鹅掌柴属植物(Schefflera kwangsiensis Merr.ex Li)为研究对象,建立完善汉桃叶药材的质量标准,利用现代分离和鉴定技术研究汉桃叶总有机酸的最佳提取工艺,并对汉桃叶有机酸的化学组分进行研究；制备汉桃叶软膏剂,进行镇痛、抗炎主要药效学的研究,为进一步开发中药5类新药汉桃叶软膏剂这一新剂型和临床安全合理用药提供理论和实验依据。
方法：1.运用HPLC分析方法,以反丁烯二酸为标准对照品,建立汉桃叶药材的质量标准,明确汉桃叶有机酸含量的测定方法；利用正交试验设计法研究汉桃叶有机酸的最佳提取工艺。
2.采用离子交换色谱法和高效液相色谱法相结合提取汉桃叶中的有机酸；并利用现代分离和鉴定的技术方法,分离、鉴定有机酸中的化学成分。
3.根据汉桃叶有机酸的理化性质,试制得到W/O型汉桃叶总提物软膏和汉桃叶总有机酸软膏。并建立镇痛抗炎主要药效学实验方法：(1)采用小鼠热刺痛法和醋酸致小鼠扭体法,研究汉桃叶软膏的镇痛作用；(2)通过二甲苯致小鼠耳廓肿胀、甲醛致小鼠足肿胀动物模型并通过测定小鼠炎性组织中PGE2含量,观察汉桃叶的抗炎作用,并初步探讨其作用机理。
结果：对汉桃叶进行HPLC的方法学考察,结果表明该方法准确、稳定、重现性、精密度、回收率良好。同时采用正交试验的设计方法,以反丁烯二酸的含量为指标,探究得到汉桃叶提取工艺的最佳条件即是,乙醇浓度为60%,提取时间为3h/次,提取次数为2次,料液体积比为10倍体积的溶剂。
通过离子交换色谱法得到汉桃叶药材中有机酸的得率为2.66%。通过熔点测定法和高效液相色谱法对从有机酸中分离的两种单体化合物进行鉴定得出：化合物Ⅰ为粘液酸(Mucic Acid)；化合物Ⅱ为反丁烯二酸(又名富马酸,Fumaric Acid).
汉桃叶各组软膏在连续外用涂抹给药7d后,各剂量组显示有镇痛作用。在小鼠热刺痛实验中,总提物高剂量组(80g/kg)、总有机酸高剂量组(80g/kg)在给药后60min时,与模型组相比有极显著性差异(P<0.01)；总有机酸中剂量组(40g/kg)与模型组相比有著性差异(P<0.05)。在给药后90min时,总有机酸高剂量组与模型组相比有显著性差异(P<0.05)；在小鼠扭体实验中,总有机酸高剂量组与模型组比较有极显著性差异,具有统计学意义(P<0.01)；总提物高剂量组与模型组比较有显著性差异,具有统计学意义(P<0.05)。
二四苯致小鼠耳廓肿胀实验中,总酸高组与模型组相比有极显著性差异(P<0.01),总提高组和总酸中组与模型组相比有显著性差异(P<0.05)。在甲醛致足肿胀的实验中结果显示,总酸高组和总酸中组与模型组相比有极显著性差异(P<0.01),总提高组与模型组相比有显著性差异(P<0.05)。测定小鼠炎性组织中PGE2含量的实验表明,总酸高组与模型组相比有极显著性差异(P<0.01),总提高组、总酸中组和总提中组与模型组相比有显著性差异(P<0.05)。
结论：对汉桃叶质量标准的研究是准确、有效的,汉桃叶具有镇痛抗炎作用,其有效部位是总有机酸,抗炎机制可能与抑制PGE2的生成、增强免疫功能等有关。新剂型汉桃叶软膏有较好的研究、开发和利用前景。
Objective:Hantaoye also called Qiyelian,Qijiapi,Qiyelan, Long-shoushu,and so on. It is belonged to Araliaceae section Schefflera kwangsiensis Merr.ex Li category plant,located in Guangxi,Yunnan, Guizhou, Fujian, Zhejiang Province of China. It is a traditional herbal medicine in the Ethnic minority areas such as Miao nationality and Li nationality, It is mainly used to relieve pain, such as trugeminal neuralgia, sciatica and gastrointestinal colic,especially to some neuralgia, and with the characteristics of little side effects.
According to the civil application experience and preliminary reported in the literature,e activity constituent of analgesia and anti-inflammatory in Hantaoye is organic acid. The research object is Hantaoye(chefflera kwangsiensis Merr.ex Li).The purpose of research is to establish the quality standards of Hantaoye, and to study the optimum extraction of organic acid from Hantaoye. And apply to modern separation and identification techniques to study chemical composition of organic acid. Develop the crude ointment of Hantaoye and conduct a study of the analgesia and anti-inflammatory medicinal experiment to provide pharmacological experiments basis for the further development of the new formulation and reasonable security clinical medicine.
Method:1. Using HPLC, fumaric acid as the standard reference substance,to establish the quality standards, to clear the method for determination of organic acid and using orthogonal method to design the optimum extraction of organic acid from Hantaoye.
2. Combined with Ion exchange chromatography and HPLC to extract organic acids from Hantaoye, and using modern separation and identification techniques to separate and identify the chemical compositi-on of organic acid.
3. Based on the physical and chemical properties of organic acid, produce small scale W/O types ointments of extraction and organic acid. Establish the analgesia and anti-inflammatory medicinal experiment methods:(l) The hot tingling test and acetic acid writhing method were adopted in the experiment to study the analgestic effect of Hantaoye ointments;(2) xylene-induced ear swelling, formaldehyde-induced hind paw edema and the level of PGE2in the paw was examined were prepared to study the effects of Hantaoye ointments, to evaluate its mechanism.
Result:After the inspection of HPLC, the results showed tahat the method is accurate, stable, good reproducibility,precision and good recovery.
Using the orthogonal method, fumaric acid content as the index, explored the best extraction process is60%ethanol concentration, extraction time of3h,2times and10times of the liquid ratio.
The yield of organic acid in Hantaoye is2.655%by the Ion exchange chromatography. Derived by melting point determination and HPLC to identify the two compounds were separated from the organic acid:compound I is Mucic Acid,ompound II is Fumaric Acid.
After the7d of continuous topical smear administration by Hantaoye groups ointments, the results showed the analgestic effect. The hot tingling test showed that the high extraction group and the high acid group at60min after administration, compared with model group, there was a most significant difference(P<0.01), the middle acid group has a significant difference(P<0.05).At90min after administration, the high acid group, compared with model group, has a significant difference(P<0.05). In the mouse writhing test, the high acid group has a most significant difference(P<0.01), the high extraction group, compared with model group, there was a significant difference(P<05).
The xylene-induced ear swelling results showed that the high acid group has a most significant difference(P<0.01), the high extraction group and the middle acid group, compared with model group, there was a significant difference(P<0.05). In the formaldehyde-induced hind paw edema, the high acid group and the middle acid group has a most significant differenceP<0.01), the high extraction group there was a significant difference(P<0.05), compared with model group. From the level of PGE2in the paw showed that the high acid group has a most significant difference(P<0.01), the high extraction group,the middle extraction group and the middle acid group, compared with model group, there was a significant difference(p〉0.05). It showed that Hantaoye has the effect of anti-inflammatory.
Conclusion:The study of quality standard of Hantaoye is accurate and effective. It has the effects of analgesic and anti-inflammatory, the active part is organic acid. The anti-inflammatory mechanism maybe related to the inhibition of PGE2production, enhance immune function. The new formulation of Hantaoye ointments is worth further research,development and utilization.
方法：1.运用HPLC分析方法,以反丁烯二酸为标准对照品,建立汉桃叶药材的质量标准,明确汉桃叶有机酸含量的测定方法；利用正交试验设计法研究汉桃叶有机酸的最佳提取工艺。
2.采用离子交换色谱法和高效液相色谱法相结合提取汉桃叶中的有机酸；并利用现代分离和鉴定的技术方法,分离、鉴定有机酸中的化学成分。
3.根据汉桃叶有机酸的理化性质,试制得到W/O型汉桃叶总提物软膏和汉桃叶总有机酸软膏。并建立镇痛抗炎主要药效学实验方法：(1)采用小鼠热刺痛法和醋酸致小鼠扭体法,研究汉桃叶软膏的镇痛作用；(2)通过二甲苯致小鼠耳廓肿胀、甲醛致小鼠足肿胀动物模型并通过测定小鼠炎性组织中PGE2含量,观察汉桃叶的抗炎作用,并初步探讨其作用机理。
结果：对汉桃叶进行HPLC的方法学考察,结果表明该方法准确、稳定、重现性、精密度、回收率良好。同时采用正交试验的设计方法,以反丁烯二酸的含量为指标,探究得到汉桃叶提取工艺的最佳条件即是,乙醇浓度为60%,提取时间为3h/次,提取次数为2次,料液体积比为10倍体积的溶剂。
通过离子交换色谱法得到汉桃叶药材中有机酸的得率为2.66%。通过熔点测定法和高效液相色谱法对从有机酸中分离的两种单体化合物进行鉴定得出：化合物Ⅰ为粘液酸(Mucic Acid)；化合物Ⅱ为反丁烯二酸(又名富马酸,Fumaric Acid).
汉桃叶各组软膏在连续外用涂抹给药7d后,各剂量组显示有镇痛作用。在小鼠热刺痛实验中,总提物高剂量组(80g/kg)、总有机酸高剂量组(80g/kg)在给药后60min时,与模型组相比有极显著性差异(P<0.01)；总有机酸中剂量组(40g/kg)与模型组相比有著性差异(P<0.05)。在给药后90min时,总有机酸高剂量组与模型组相比有显著性差异(P<0.05)；在小鼠扭体实验中,总有机酸高剂量组与模型组比较有极显著性差异,具有统计学意义(P<0.01)；总提物高剂量组与模型组比较有显著性差异,具有统计学意义(P<0.05)。
二四苯致小鼠耳廓肿胀实验中,总酸高组与模型组相比有极显著性差异(P<0.01),总提高组和总酸中组与模型组相比有显著性差异(P<0.05)。在甲醛致足肿胀的实验中结果显示,总酸高组和总酸中组与模型组相比有极显著性差异(P<0.01),总提高组与模型组相比有显著性差异(P<0.05)。测定小鼠炎性组织中PGE2含量的实验表明,总酸高组与模型组相比有极显著性差异(P<0.01),总提高组、总酸中组和总提中组与模型组相比有显著性差异(P<0.05)。
结论：对汉桃叶质量标准的研究是准确、有效的,汉桃叶具有镇痛抗炎作用,其有效部位是总有机酸,抗炎机制可能与抑制PGE2的生成、增强免疫功能等有关。新剂型汉桃叶软膏有较好的研究、开发和利用前景。
Objective:Hantaoye also called Qiyelian,Qijiapi,Qiyelan, Long-shoushu,and so on. It is belonged to Araliaceae section Schefflera kwangsiensis Merr.ex Li category plant,located in Guangxi,Yunnan, Guizhou, Fujian, Zhejiang Province of China. It is a traditional herbal medicine in the Ethnic minority areas such as Miao nationality and Li nationality, It is mainly used to relieve pain, such as trugeminal neuralgia, sciatica and gastrointestinal colic,especially to some neuralgia, and with the characteristics of little side effects.
According to the civil application experience and preliminary reported in the literature,e activity constituent of analgesia and anti-inflammatory in Hantaoye is organic acid. The research object is Hantaoye(chefflera kwangsiensis Merr.ex Li).The purpose of research is to establish the quality standards of Hantaoye, and to study the optimum extraction of organic acid from Hantaoye. And apply to modern separation and identification techniques to study chemical composition of organic acid. Develop the crude ointment of Hantaoye and conduct a study of the analgesia and anti-inflammatory medicinal experiment to provide pharmacological experiments basis for the further development of the new formulation and reasonable security clinical medicine.
Method:1. Using HPLC, fumaric acid as the standard reference substance,to establish the quality standards, to clear the method for determination of organic acid and using orthogonal method to design the optimum extraction of organic acid from Hantaoye.
2. Combined with Ion exchange chromatography and HPLC to extract organic acids from Hantaoye, and using modern separation and identification techniques to separate and identify the chemical compositi-on of organic acid.
3. Based on the physical and chemical properties of organic acid, produce small scale W/O types ointments of extraction and organic acid. Establish the analgesia and anti-inflammatory medicinal experiment methods:(l) The hot tingling test and acetic acid writhing method were adopted in the experiment to study the analgestic effect of Hantaoye ointments;(2) xylene-induced ear swelling, formaldehyde-induced hind paw edema and the level of PGE2in the paw was examined were prepared to study the effects of Hantaoye ointments, to evaluate its mechanism.
Result:After the inspection of HPLC, the results showed tahat the method is accurate, stable, good reproducibility,precision and good recovery.
Using the orthogonal method, fumaric acid content as the index, explored the best extraction process is60%ethanol concentration, extraction time of3h,2times and10times of the liquid ratio.
The yield of organic acid in Hantaoye is2.655%by the Ion exchange chromatography. Derived by melting point determination and HPLC to identify the two compounds were separated from the organic acid:compound I is Mucic Acid,ompound II is Fumaric Acid.
After the7d of continuous topical smear administration by Hantaoye groups ointments, the results showed the analgestic effect. The hot tingling test showed that the high extraction group and the high acid group at60min after administration, compared with model group, there was a most significant difference(P<0.01), the middle acid group has a significant difference(P<0.05).At90min after administration, the high acid group, compared with model group, has a significant difference(P<0.05). In the mouse writhing test, the high acid group has a most significant difference(P<0.01), the high extraction group, compared with model group, there was a significant difference(P<05).
The xylene-induced ear swelling results showed that the high acid group has a most significant difference(P<0.01), the high extraction group and the middle acid group, compared with model group, there was a significant difference(P<0.05). In the formaldehyde-induced hind paw edema, the high acid group and the middle acid group has a most significant differenceP<0.01), the high extraction group there was a significant difference(P<0.05), compared with model group. From the level of PGE2in the paw showed that the high acid group has a most significant difference(P<0.01), the high extraction group,the middle extraction group and the middle acid group, compared with model group, there was a significant difference(p〉0.05). It showed that Hantaoye has the effect of anti-inflammatory.
Conclusion:The study of quality standard of Hantaoye is accurate and effective. It has the effects of analgesic and anti-inflammatory, the active part is organic acid. The anti-inflammatory mechanism maybe related to the inhibition of PGE2production, enhance immune function. The new formulation of Hantaoye ointments is worth further research,development and utilization.
引文
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