硫黄熏蒸对白芷化学成分、药效和安全性影响的研究
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摘要
自芷来源为伞形科植物白芷Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f.或杭白芷Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss) Shan et Yuan的干燥根,是一味常用中药。白芷主产于我国四川、河北、安徽、河南等省,一直以来,白芷的产地加工是一个突出的生产难题。白芷采收后若不及时干燥,极易腐烂变质,目前全国各地的白芷产地加工均采用硫黄熏蒸。硫黄熏蒸虽对不易干燥或易发霉、变质、生虫的中药材具有杀菌防腐、漂白、增加色泽美感等作用,但大量研究表明,熏硫造成中药材性味劣变,生物活性组分损失及有害物质残留,硫黄熏蒸对药材质量带来的负面影响己引起人们的高度关注。本文针对硫黄熏蒸前后及不同熏硫程度对白芷药材化学成分、药效和安全性的影响,二氧化硫残留量的变化规律及熏硫加工的规范化问题,在前期研究的基础上,运用多学科的技术方法,通过系统深入的研究,为白芷硫黄熏蒸加工方法的合理性、规范化及制定科学合理、客观可控的有效成分和外源性有害物质二氧化硫残留量的限量标准提供科学依据。研究结果如下：
1.不同熏硫程度白芷的加工及药材外观性状对比分析。
根据前期白芷主产区调研,进行了不同硫黄用量、不同熏蒸时间的加工试验,用产区传统搭灶的方式共制备不同熏硫程度白芷样品21个,为本文研究的顺利进行提供了保障。
通过对不同熏硫程度白芷外观性状的对比观察,发现熏硫与未熏硫白芷的主要区别在颜色、断面特征、气味等方面。硫黄熏蒸后的白芷表面颜色稍浅,断面类白色,粉性较强,形成层环和油点不甚明显,略带酸味,但不同熏硫程度的白芷上述特征存在一定差异,随着硫黄用量和熏蒸时间的增加,断面颜色逐渐变白,形成层环及棕色油点等特征逐渐消失。硫黄熏蒸对白芷的外观性状产生了一定的影响。从白芷的外观性状上,可以直观的判断白芷是否熏硫以及估计其硫黄熏蒸的程度。
2.首次进行了不同熏硫程度白芷二氧化硫残留量的动态检测
按照《中国药典》2010版附录项下的方法对不同熏硫程度白芷的二氧化硫残留量进行了动态检测。硫黄熏蒸过的样品二氧化硫残留量是自然晒干的样品的几百甚至上千倍,硫黄用量为25g、100g、200g/10kg药材和熏蒸时间为18h的样品其二氧化硫残留量符合国家规定的150mg/kg的限量标准。本研究为白芷硫黄熏蒸加工中熏硫程度的确定提供了参考依据。硫黄用量和硫黄熏蒸时间与二氧化硫残留量虽未呈现出良好的线性关系,但从总体上,随着硫黄用量的增加和熏蒸时间的延长,二氧化硫残留量也有所增加。
3.对不同熏硫程度白芷的化学成分进行了系统的对比分析
在薄层色谱鉴别方面,确立以氯仿-乙酸乙酯(10：1)和环己烷-氯仿-乙醚(4：1：2)进行二次展开的色谱条件,对22批不同程度硫黄熏蒸白芷及未熏硫白芷样品进行薄层色谱对比。氧化前胡素是白芷熏硫与否的一个指标性成分,从薄层色谱图上可较为直观的鉴别白芷是否熏硫。
在高效液相指纹图谱分析方面,建立22批不同程度硫黄熏蒸白芷HPLC指纹图谱,校正后得到的对照色谱中19个共有峰,其中确认了11个化合物。由于硫黄熏蒸程度的不同,在共有峰的峰面积上有差异。对比熏硫与未熏硫白芷的图谱,发现两者在色谱峰数量及峰面积上有差异。通过统计分析,未熏硫白芷与熏硫白芷聚为两类,主成分分析综合评分熏硫白芷得分较低,质量较差。从HPLC指纹图谱上也能直观的判断出白芷熏硫与否。
首次进行不同熏硫程度白芷LC-MS分析,建立不同熏硫程度白芷的药材、石油醚部位、乙酸乙酯部位、正丁醇部位的LC-MS分析方法,得到白芷香豆素类化合物不同的变化规律。三个不同的极性部位,硫黄熏蒸对石油醚、乙酸乙酯部位的影响与对药材的影响类似,对正丁醇部位的影响较大,新增加的峰多而明显。
首次进行不同程度硫黄熏蒸白芷GC-MS分析,在前期的研究基础上,建立了不同程度硫黄熏蒸白芷挥发油的GC-MS分析方法。不同程度硫黄熏蒸白芷的挥发油中共鉴定出67种化合物。其中有4种共有成分,分别为：2,6-二叔丁基对甲基苯酚,棕榈酸乙酯,亚油酸乙酯,十六碳烷基十四烷酸酯。随着硫黄熏蒸程度的不同,白芷中挥发油的种类也发生了变化,产生了很多新的化合物,四个共有成分变化不一。
对不同熏硫程度白芷的浸出物和指标性成分进行含量测定,随着硫黄用量和熏蒸时间的增加,,白芷的浸出物含量比未熏硫白芷下降28.5%-47.3%,氧化前胡素和欧前胡素的含量总体呈下降趋势。自然晒干样品的浸出物、氧化前胡素、欧前胡素含量明显高于其他经过硫黄熏蒸的样品。
4首次对熏硫与未熏硫白芷的进行体内血浆的HPLC图谱进行对比研究
通过采血时间、采血部位、给药剂量等方面的考察,建立了白芷血浆HPLC分析方法,对比了熏硫与未熏硫白芷体内血浆的HPLC图谱。未熏硫白芷给药后的血浆HPLC图谱中出现了12个色谱峰,其中有2个峰可能是代谢产生的峰。熏硫白芷入血成分较未熏硫白芷少,入血成分峰面积、总峰面积较低,并且尚未出现代谢产生的峰。本研究为白芷药效物质基础的研究奠定了基础。
5.首次采用药效学结合血浆药物化学的方法对熏硫与未熏硫白芷进行药效评价
分别采用经典的二甲苯致小鼠耳肿胀和醋酸致小鼠扭体法对硫黄熏蒸前后白芷进行抗炎、镇痛的实验。在药效学指标上未熏硫白芷高剂量表现出显著的抗炎、镇痛作用,而熏硫白芷部分剂量组显示出抗炎镇痛作用,但无统计学意义。各个剂量组的血浆成分类似但也有所不同。随着给药剂量的减小,各剂量组入血成分的峰面积也相应减小。推测水合氧化前胡内酯可能是白芷镇痛、抗炎的药效物质基础,为阐明白芷的谱效关系奠定了基础。
6进行了熏硫与未熏硫白芷急性毒性试验
通过对熏硫与未熏硫白芷的急性毒性试验,以评价其安全性。结果表明,单次灌胃给药未熏硫白芷LD50为52.41g生药/kg,即约相当于临床用量的353倍;单次灌胃给予硫黄熏蒸白芷的最大耐受量为123.6g生药/kg,即约相当于临床用量的832倍。虽然两者均在安全使用的范围,可以安全使用,但是未熏硫白芷的毒性明显比硫黄熏蒸白芷的毒性大。
7首次进行了不同熏硫程度白芷化学成分、药效及安全性的相关性分析
用曲线回归的方法探讨了二氧化硫残留量分别与氧化前胡素、欧前胡素的关系,建立了二者的模型方程,相关性良好。氧化前胡素与二氧化硫残留量二者呈对数相关为Y=0.085-0.0111nX；欧前胡素与二氧化硫残留量二者呈三次方相关,关系式为Y=0.176+3.150×10-7X2-1.666×10-10X3。
探讨了白芷抗炎、镇痛的谱效关系,用经典的二甲苯致小鼠耳肿胀法、醋酸致小鼠扭体法评价熏硫与未熏硫的白芷得到的药效数据,与各剂量组的血浆HPLC指纹图谱色谱峰峰面积进行多元相关分析。将各剂量组的色谱峰峰面积进行主成分分析,求得各组的综合评分后,用综合评分C作为自变量,药效学指标作为因变量Y做曲线回归,预测二者的关系,建立相关方程。综合抗炎、镇痛两个药理谱效关系结果,血浆主成分分析的综合评分与抗炎、镇痛的药效学指标呈正相关。
根据熏硫与未熏硫白芷急性毒性结果,结合白芷体外、体内化学成分分析,推断引起白芷毒性的可能是与有效成分相关的内源性物质,至于是白芷中的原型成分还是代谢产物引起的毒性还需要进一步的研究。虽然熏硫白芷的毒性较未熏硫白芷低,但外源性有害物质二氧化硫残留量明显增加,影响用药的安全性,故应规范白芷硫黄熏蒸的加工方法。
8通过本文研究,为阐明白芷硫黄熏蒸加7-方法的合理性、确定合适的熏硫程度以及制定科学合理、客观可控的有效成分及二氧化硫限量标准提供了科学依据
研究发现,白芷熏硫后虽然影响有效成分和药效,且二氧化硫残留量增加,但便于干燥、贮藏,成本低,现阶段在白芷产区有一定的实用价值,只要控制好熏硫程度,能够保证白芷药材质量及临床用药的安全有效。根据硫黄熏蒸对白芷化学成分、药效及安全性影响的研究及相关性分析,初步建议,白芷硫黄熏蒸加工的硫黄用量不超过100g/10kg,熏蒸时间不超过8h;含量测定以欧前胡素、氧化前胡素为指标,前者不得低于0.14%、后者不得低于0.09%;二氧化硫残留量不得过400mg/kg。
Angelica dahurica root is from Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. or Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss) Shan et Yuan and a commonly used Chinese medicine. Angelica dahurica root cultivate in sichuan, hebei, anhui, henan and other provinces in China. All along, the original processing of Angelica dahurica is a prominent problem. It can easily spoile, if not dried timely. At present sulfur-fumigation is used in original processing of Angelica dahurica all over the country. Sulfur-fumigation can sterilizes, anticorrosive, bleach and increase the feeling color to traditional Chinese medicine which is not easy to dry or easy to mildew and deterioration. But a large number of studies have shown that sulfur-fumigation could cause loss of biological active component and harmful residues in traditional Chinese medicine. The negative impact of sulfur-fumigation has aroused great concern. This paper aimed at the effects of chemical composition, the efficacy and safety of Angelica dahurica by different degreed of sulfur-fumigation, changing rule of the sulfur dioxide residue and sulfur-fumigation processing standardization problems. Based on the previous studies and using multidisciplinary technology and methods, this paper provide a scientific basis for the rationality of sulfur-fumigation processing method and formulating scientific and rational, objective and controlled active ingredients and sulfur dioxide residue standard. The results are as follows:
1. The study took process on Angelica dahurica by different degrees of sulfur-fumigation and comparative analysis on appearance.
According to the preliminary investigation, different sulfur dosage and different time of fumigation processing was prepared with traditional region by way of the oven. A total of21samples with different sulfur smoked degree of angelica dahurica provided a guarantee for this study.
By comparative analysis of different degrees of sulfur-fumigation Angelica dahurica, the main difference between sulfur-fumigation and non-sulfur-fumigation is in color, section characteristics, smells, etc. After sulfur-fumigation the surface color of Angelica dahurica is a bit shallow, section class white,, cambium ring and oil not very clear, slightly sour. But there are some differences in the characteristics of different degrees of sulfur-fumigation of Angelica. With the increase of dosage of sulfur and fumigation time, cross section color gradually turned white, cambium ring and brown oil spots and other features gradually disappear. Sulfur-fumigation produced certain effect on appearance of angelica dahurica. From the appearance, we can intuitive judge Angelica dahurica whether sulfur-fumigation as well as to estimate the degree.
2. The study took dynamic detection of sulfur dioxide residue in the different degrees of Angelica dahurica for the first time.
According to the appendix methods of "Chinese pharmacopoeia"2010version, sulfur dioxide residue of different degree of sulfur-fumigation angelica dahurica has carried on the dynamic test. The sulfur dioxide residue of sulfur-fumigated sample is the hundreds or even thousands of times higher than natural dried sample. When sulfur dosage is25g,100g,200g/10kg of medicinal materials and fumigation for18h, the sulfur dioxide residue sample conforms to the provisions of the state of the standard of set limit to150mg/kg. This study provides reference basis for determination the degrees of sulfur-fumigation.
Although dosage of sulfur and sulfur fumigation time and sulfur dioxide residue does not present a good linear relationship,but on the whole, with an increase in the dosage of sulfur and the extension of time, the sulfur dioxide residue also increased.
3. The study took comparative analysis of the chemical composition of the different degree of sulfur-fumigation Angelica dahurica systematically.
In TLC identification,22batches of different degrees of sulfur-fumigation Angelica dahurica and non-sulfur-fumigation sample comparison of TLC chromatography with establishing the quadratic expansion chromatography conditions with chloroform-ethyl acetate (10:1) and cyclohexane-chloroform-ether (4:1:2). Oxypeucedanin is an index components whether angelica dahurica is sulfur fumigated or not. From the thin-layer chromatogram can be more intuitive identify Angelica fumigated or not.
In HPLC fingerprint analysis, this study set up22batch of different degree of sulfur-fumigation angelica dahurica HPLC fingerprint with19common peak in the chromatogram, which confirmed that11compounds. Because of the different degree of sulfur fumigation, the peak area of common peak is differences. Contrasted sulfur-fumigation and non-sulfur-fumigation chromatogram of angelica dahurica, there are differences in chromatographic peak number and peak area. Sulfur-fumigation and non-sulfur-fumigation of angelica dahurica clustered into two categories by statistical analysis. Comprehensive score by principal component analysis shows that sulfur-fumigation angelica dahurica is poor quality. From the HPLC chromatogram can be more intuitive identify Angelica fumigated or not.
This study first carries LC-MS analysis on the different degrees of sulfur-fumigation Angelica dahurica. It set up LC-MS analysis method from petroleum ether parts, ethyl acetate parts and n-butanol parts of different degrees of sulfur-fumigation angelica dahurica to get variation of coumarin compounds. Influence from sulfur-fumigation in petroleum ether parts and ethyl acetate parts is similar with crude medicine. Influence in n-butanol parts is more obvious and increasing new peaks significantly.
This study first carries GC-MS analysis on the different degrees of sulfur-fumigation Angelica dahurica. It set up GC-MS analysis method in volatile oil of different degrees of sulfur-fumigation Angelica dahurica. The volatile oil from different degrees of sulfur-fumigation angelica dahurica were identified67compounds. There are four common composition:butylated Hydroxytoluene, ethyl hexadecanoate,9,12-Octadecadienoic acid ethyl ester, myristylpalmitate. As the difference in the degrees of sulfur fumigation, the volatile oil of angelica dahurica in species have also changed and generates a lot of new compounds.
This study determined the content of extract and index components in different degree of sulfur-fumigation angelica dahurica. With the increase of dosage of sulfur and fumigation time, extract content decreased from15%to20%, oxypeucedanin and imperatorin was on the decline. Content of extract and oxypeucedanin&imperatorin in non-sulfur-fumigation sample was significantly higher than other samples.
4. The study took plasma HPLC of non-sulfur-fumigation and sulfur-fumigation angelica dahurica for the first time.
Through the investigation of the time, the blood collection site and dose, the study established plasma HPLC analysis methods of angelica dahurica with compared non-sulfur-fumigation sample and sulfur-fumigation samples. There are12peaks in plasma HPLC chromatographic and2of them are maybe metabolism. Sulfur-fumigation angelica dahurica has less components, lower peak area and total peak area into blood than non-sulfur-fumigation one, and has not yet appeared metabolism peak. This study laid the foundation in efficacy material for the further study of angelica dahurica.
5. The study evaluated efficacy of non-sulfur-fumigation and sulfur-fumigation angelica dahurica by pharmacodynamics combined with plasma chemical firstly.
The study take anti-inflammatory, analgesic experiment on non-sulfur-fumigation and sulfur-fumigation angelica dahurica with classic xylene induced ear swelling in mice and acetic acid in mice body torsion method. Non-sulfur-fumigation sample showed significant anti-inflammatory and analgesic effects. partial dose group showed anti-inflammatory and analgesic effects, but not statistically significant. Each dose group of plasma composition was similar but also different. With the decrease of the dosage, the peak area of different dose groups also decreased accordingly. Oxypeucedanin hydrate is speculated to be the material basis of anti-inflammatory efficacy of angelica dahurica, which laid a foundation on elucidated spectrum relationship of angelica dahurica.
6. The study took an acute toxicity test of non-sulfur-fumigation and sulfur-fumigation angelica dahurica.
In the safety evaluation, non-sulfur-fumigated Angelica of LD50is for52.41g crude drug/body weight (kg), which equals approximately clinical dosage of353 times. Sulfur-fumigated Angelica of MTD is for123.6g crude drug/body weight (kg), which equals approximately clinical dosage of832times. Although both are in the range of safe use,but non-sulfur-fumigated Angelica toxicity was significantly larger than the toxicity of sulfur-fumigated Angelica.
7. The study carried on correlation analysis in chemical composition, efficacy and safety of different degrees of sulfur-fumigated angelica dahurica for the first time.
Using the curve regression methods to explore the relationship of sulfur dioxide residue and oxypeucedanin&imperatorin with established the model equations. The correlation is good. Oxypeucedani and sulfur dioxide residue was ogarithmically related:Y=0.085-0.0111nX. Imperatorin and sulfur dioxide residue was cubic related:Y=0.176+3.150×10-7X2-1.666×10-10X3.
The study discussed the relationship of spectrum and efficacy of angelica dahurica. Efficacy data from experiment of xylene induced ear swelling in mice and acetic acid in mice body torsion, and plasma HPLC fingerprint chromatogram peak area with each dose group were multivariate analyzed. Using a composite score C from principal component analysis as the independent variable, the pharmacodynamic index as the dependent variable Y do curvilinear regression to predict the relationship between them and establish correlation equation. Composite score and pharmacodynamics index were positively correlated.
According to the result of acute toxicity test and chemical composition analysis in vitro and in vivo, the study concluded that the cause of toxicity of angelica dahurica could be related to the effective components. Is the prototype or metabolites component caused toxic need further study. Although toxicity of sulfur-fumigated angelica dahurica is lower than non-sulfur-fumigated one, sulfur dioxide increased significantly to affect the safety. So it should regulate sulfur-fumigation processing methods of Angelica.
8. This study provides a scientific basis to clarify the rationality of sulfur fumigation processing method, determine the appropriate degree of sulfur fumigation and formulate scientific and rational, objective and controlled effective composition and sulfur dioxide limits standard.
The study found that sulfur fumigation effect the active ingredients and efficacy and increase in sulfur dioxide residue. But it has practical value in angelica dahurica region at present, which is easy of drying, storage, and low cost. As long as the good control the of degree sulfur fumigation, we can guarantee the quality of angelica dahurica and the safety of clinical use. According to the research on the effects of chemical composition, the efficacy and safety of Angelica dahurica by sulfur-fumigation and correlation analysis, preliminary Suggestions as follow. Sulfur dosage is not exceed100g/10kg and fumigation time not more than8h. Oxypeucedanin is not less than0.09%and imperatorin is not less than0.14%. Sulfur dioxide residue should not exceed400mg/kg.
1.不同熏硫程度白芷的加工及药材外观性状对比分析。
根据前期白芷主产区调研,进行了不同硫黄用量、不同熏蒸时间的加工试验,用产区传统搭灶的方式共制备不同熏硫程度白芷样品21个,为本文研究的顺利进行提供了保障。
通过对不同熏硫程度白芷外观性状的对比观察,发现熏硫与未熏硫白芷的主要区别在颜色、断面特征、气味等方面。硫黄熏蒸后的白芷表面颜色稍浅,断面类白色,粉性较强,形成层环和油点不甚明显,略带酸味,但不同熏硫程度的白芷上述特征存在一定差异,随着硫黄用量和熏蒸时间的增加,断面颜色逐渐变白,形成层环及棕色油点等特征逐渐消失。硫黄熏蒸对白芷的外观性状产生了一定的影响。从白芷的外观性状上,可以直观的判断白芷是否熏硫以及估计其硫黄熏蒸的程度。
2.首次进行了不同熏硫程度白芷二氧化硫残留量的动态检测
按照《中国药典》2010版附录项下的方法对不同熏硫程度白芷的二氧化硫残留量进行了动态检测。硫黄熏蒸过的样品二氧化硫残留量是自然晒干的样品的几百甚至上千倍,硫黄用量为25g、100g、200g/10kg药材和熏蒸时间为18h的样品其二氧化硫残留量符合国家规定的150mg/kg的限量标准。本研究为白芷硫黄熏蒸加工中熏硫程度的确定提供了参考依据。硫黄用量和硫黄熏蒸时间与二氧化硫残留量虽未呈现出良好的线性关系,但从总体上,随着硫黄用量的增加和熏蒸时间的延长,二氧化硫残留量也有所增加。
3.对不同熏硫程度白芷的化学成分进行了系统的对比分析
在薄层色谱鉴别方面,确立以氯仿-乙酸乙酯(10：1)和环己烷-氯仿-乙醚(4：1：2)进行二次展开的色谱条件,对22批不同程度硫黄熏蒸白芷及未熏硫白芷样品进行薄层色谱对比。氧化前胡素是白芷熏硫与否的一个指标性成分,从薄层色谱图上可较为直观的鉴别白芷是否熏硫。
在高效液相指纹图谱分析方面,建立22批不同程度硫黄熏蒸白芷HPLC指纹图谱,校正后得到的对照色谱中19个共有峰,其中确认了11个化合物。由于硫黄熏蒸程度的不同,在共有峰的峰面积上有差异。对比熏硫与未熏硫白芷的图谱,发现两者在色谱峰数量及峰面积上有差异。通过统计分析,未熏硫白芷与熏硫白芷聚为两类,主成分分析综合评分熏硫白芷得分较低,质量较差。从HPLC指纹图谱上也能直观的判断出白芷熏硫与否。
首次进行不同熏硫程度白芷LC-MS分析,建立不同熏硫程度白芷的药材、石油醚部位、乙酸乙酯部位、正丁醇部位的LC-MS分析方法,得到白芷香豆素类化合物不同的变化规律。三个不同的极性部位,硫黄熏蒸对石油醚、乙酸乙酯部位的影响与对药材的影响类似,对正丁醇部位的影响较大,新增加的峰多而明显。
首次进行不同程度硫黄熏蒸白芷GC-MS分析,在前期的研究基础上,建立了不同程度硫黄熏蒸白芷挥发油的GC-MS分析方法。不同程度硫黄熏蒸白芷的挥发油中共鉴定出67种化合物。其中有4种共有成分,分别为：2,6-二叔丁基对甲基苯酚,棕榈酸乙酯,亚油酸乙酯,十六碳烷基十四烷酸酯。随着硫黄熏蒸程度的不同,白芷中挥发油的种类也发生了变化,产生了很多新的化合物,四个共有成分变化不一。
对不同熏硫程度白芷的浸出物和指标性成分进行含量测定,随着硫黄用量和熏蒸时间的增加,,白芷的浸出物含量比未熏硫白芷下降28.5%-47.3%,氧化前胡素和欧前胡素的含量总体呈下降趋势。自然晒干样品的浸出物、氧化前胡素、欧前胡素含量明显高于其他经过硫黄熏蒸的样品。
4首次对熏硫与未熏硫白芷的进行体内血浆的HPLC图谱进行对比研究
通过采血时间、采血部位、给药剂量等方面的考察,建立了白芷血浆HPLC分析方法,对比了熏硫与未熏硫白芷体内血浆的HPLC图谱。未熏硫白芷给药后的血浆HPLC图谱中出现了12个色谱峰,其中有2个峰可能是代谢产生的峰。熏硫白芷入血成分较未熏硫白芷少,入血成分峰面积、总峰面积较低,并且尚未出现代谢产生的峰。本研究为白芷药效物质基础的研究奠定了基础。
5.首次采用药效学结合血浆药物化学的方法对熏硫与未熏硫白芷进行药效评价
分别采用经典的二甲苯致小鼠耳肿胀和醋酸致小鼠扭体法对硫黄熏蒸前后白芷进行抗炎、镇痛的实验。在药效学指标上未熏硫白芷高剂量表现出显著的抗炎、镇痛作用,而熏硫白芷部分剂量组显示出抗炎镇痛作用,但无统计学意义。各个剂量组的血浆成分类似但也有所不同。随着给药剂量的减小,各剂量组入血成分的峰面积也相应减小。推测水合氧化前胡内酯可能是白芷镇痛、抗炎的药效物质基础,为阐明白芷的谱效关系奠定了基础。
6进行了熏硫与未熏硫白芷急性毒性试验
通过对熏硫与未熏硫白芷的急性毒性试验,以评价其安全性。结果表明,单次灌胃给药未熏硫白芷LD50为52.41g生药/kg,即约相当于临床用量的353倍;单次灌胃给予硫黄熏蒸白芷的最大耐受量为123.6g生药/kg,即约相当于临床用量的832倍。虽然两者均在安全使用的范围,可以安全使用,但是未熏硫白芷的毒性明显比硫黄熏蒸白芷的毒性大。
7首次进行了不同熏硫程度白芷化学成分、药效及安全性的相关性分析
用曲线回归的方法探讨了二氧化硫残留量分别与氧化前胡素、欧前胡素的关系,建立了二者的模型方程,相关性良好。氧化前胡素与二氧化硫残留量二者呈对数相关为Y=0.085-0.0111nX；欧前胡素与二氧化硫残留量二者呈三次方相关,关系式为Y=0.176+3.150×10-7X2-1.666×10-10X3。
探讨了白芷抗炎、镇痛的谱效关系,用经典的二甲苯致小鼠耳肿胀法、醋酸致小鼠扭体法评价熏硫与未熏硫的白芷得到的药效数据,与各剂量组的血浆HPLC指纹图谱色谱峰峰面积进行多元相关分析。将各剂量组的色谱峰峰面积进行主成分分析,求得各组的综合评分后,用综合评分C作为自变量,药效学指标作为因变量Y做曲线回归,预测二者的关系,建立相关方程。综合抗炎、镇痛两个药理谱效关系结果,血浆主成分分析的综合评分与抗炎、镇痛的药效学指标呈正相关。
根据熏硫与未熏硫白芷急性毒性结果,结合白芷体外、体内化学成分分析,推断引起白芷毒性的可能是与有效成分相关的内源性物质,至于是白芷中的原型成分还是代谢产物引起的毒性还需要进一步的研究。虽然熏硫白芷的毒性较未熏硫白芷低,但外源性有害物质二氧化硫残留量明显增加,影响用药的安全性,故应规范白芷硫黄熏蒸的加工方法。
8通过本文研究,为阐明白芷硫黄熏蒸加7-方法的合理性、确定合适的熏硫程度以及制定科学合理、客观可控的有效成分及二氧化硫限量标准提供了科学依据
研究发现,白芷熏硫后虽然影响有效成分和药效,且二氧化硫残留量增加,但便于干燥、贮藏,成本低,现阶段在白芷产区有一定的实用价值,只要控制好熏硫程度,能够保证白芷药材质量及临床用药的安全有效。根据硫黄熏蒸对白芷化学成分、药效及安全性影响的研究及相关性分析,初步建议,白芷硫黄熏蒸加工的硫黄用量不超过100g/10kg,熏蒸时间不超过8h;含量测定以欧前胡素、氧化前胡素为指标,前者不得低于0.14%、后者不得低于0.09%;二氧化硫残留量不得过400mg/kg。
Angelica dahurica root is from Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. or Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss) Shan et Yuan and a commonly used Chinese medicine. Angelica dahurica root cultivate in sichuan, hebei, anhui, henan and other provinces in China. All along, the original processing of Angelica dahurica is a prominent problem. It can easily spoile, if not dried timely. At present sulfur-fumigation is used in original processing of Angelica dahurica all over the country. Sulfur-fumigation can sterilizes, anticorrosive, bleach and increase the feeling color to traditional Chinese medicine which is not easy to dry or easy to mildew and deterioration. But a large number of studies have shown that sulfur-fumigation could cause loss of biological active component and harmful residues in traditional Chinese medicine. The negative impact of sulfur-fumigation has aroused great concern. This paper aimed at the effects of chemical composition, the efficacy and safety of Angelica dahurica by different degreed of sulfur-fumigation, changing rule of the sulfur dioxide residue and sulfur-fumigation processing standardization problems. Based on the previous studies and using multidisciplinary technology and methods, this paper provide a scientific basis for the rationality of sulfur-fumigation processing method and formulating scientific and rational, objective and controlled active ingredients and sulfur dioxide residue standard. The results are as follows:
1. The study took process on Angelica dahurica by different degrees of sulfur-fumigation and comparative analysis on appearance.
According to the preliminary investigation, different sulfur dosage and different time of fumigation processing was prepared with traditional region by way of the oven. A total of21samples with different sulfur smoked degree of angelica dahurica provided a guarantee for this study.
By comparative analysis of different degrees of sulfur-fumigation Angelica dahurica, the main difference between sulfur-fumigation and non-sulfur-fumigation is in color, section characteristics, smells, etc. After sulfur-fumigation the surface color of Angelica dahurica is a bit shallow, section class white,, cambium ring and oil not very clear, slightly sour. But there are some differences in the characteristics of different degrees of sulfur-fumigation of Angelica. With the increase of dosage of sulfur and fumigation time, cross section color gradually turned white, cambium ring and brown oil spots and other features gradually disappear. Sulfur-fumigation produced certain effect on appearance of angelica dahurica. From the appearance, we can intuitive judge Angelica dahurica whether sulfur-fumigation as well as to estimate the degree.
2. The study took dynamic detection of sulfur dioxide residue in the different degrees of Angelica dahurica for the first time.
According to the appendix methods of "Chinese pharmacopoeia"2010version, sulfur dioxide residue of different degree of sulfur-fumigation angelica dahurica has carried on the dynamic test. The sulfur dioxide residue of sulfur-fumigated sample is the hundreds or even thousands of times higher than natural dried sample. When sulfur dosage is25g,100g,200g/10kg of medicinal materials and fumigation for18h, the sulfur dioxide residue sample conforms to the provisions of the state of the standard of set limit to150mg/kg. This study provides reference basis for determination the degrees of sulfur-fumigation.
Although dosage of sulfur and sulfur fumigation time and sulfur dioxide residue does not present a good linear relationship,but on the whole, with an increase in the dosage of sulfur and the extension of time, the sulfur dioxide residue also increased.
3. The study took comparative analysis of the chemical composition of the different degree of sulfur-fumigation Angelica dahurica systematically.
In TLC identification,22batches of different degrees of sulfur-fumigation Angelica dahurica and non-sulfur-fumigation sample comparison of TLC chromatography with establishing the quadratic expansion chromatography conditions with chloroform-ethyl acetate (10:1) and cyclohexane-chloroform-ether (4:1:2). Oxypeucedanin is an index components whether angelica dahurica is sulfur fumigated or not. From the thin-layer chromatogram can be more intuitive identify Angelica fumigated or not.
In HPLC fingerprint analysis, this study set up22batch of different degree of sulfur-fumigation angelica dahurica HPLC fingerprint with19common peak in the chromatogram, which confirmed that11compounds. Because of the different degree of sulfur fumigation, the peak area of common peak is differences. Contrasted sulfur-fumigation and non-sulfur-fumigation chromatogram of angelica dahurica, there are differences in chromatographic peak number and peak area. Sulfur-fumigation and non-sulfur-fumigation of angelica dahurica clustered into two categories by statistical analysis. Comprehensive score by principal component analysis shows that sulfur-fumigation angelica dahurica is poor quality. From the HPLC chromatogram can be more intuitive identify Angelica fumigated or not.
This study first carries LC-MS analysis on the different degrees of sulfur-fumigation Angelica dahurica. It set up LC-MS analysis method from petroleum ether parts, ethyl acetate parts and n-butanol parts of different degrees of sulfur-fumigation angelica dahurica to get variation of coumarin compounds. Influence from sulfur-fumigation in petroleum ether parts and ethyl acetate parts is similar with crude medicine. Influence in n-butanol parts is more obvious and increasing new peaks significantly.
This study first carries GC-MS analysis on the different degrees of sulfur-fumigation Angelica dahurica. It set up GC-MS analysis method in volatile oil of different degrees of sulfur-fumigation Angelica dahurica. The volatile oil from different degrees of sulfur-fumigation angelica dahurica were identified67compounds. There are four common composition:butylated Hydroxytoluene, ethyl hexadecanoate,9,12-Octadecadienoic acid ethyl ester, myristylpalmitate. As the difference in the degrees of sulfur fumigation, the volatile oil of angelica dahurica in species have also changed and generates a lot of new compounds.
This study determined the content of extract and index components in different degree of sulfur-fumigation angelica dahurica. With the increase of dosage of sulfur and fumigation time, extract content decreased from15%to20%, oxypeucedanin and imperatorin was on the decline. Content of extract and oxypeucedanin&imperatorin in non-sulfur-fumigation sample was significantly higher than other samples.
4. The study took plasma HPLC of non-sulfur-fumigation and sulfur-fumigation angelica dahurica for the first time.
Through the investigation of the time, the blood collection site and dose, the study established plasma HPLC analysis methods of angelica dahurica with compared non-sulfur-fumigation sample and sulfur-fumigation samples. There are12peaks in plasma HPLC chromatographic and2of them are maybe metabolism. Sulfur-fumigation angelica dahurica has less components, lower peak area and total peak area into blood than non-sulfur-fumigation one, and has not yet appeared metabolism peak. This study laid the foundation in efficacy material for the further study of angelica dahurica.
5. The study evaluated efficacy of non-sulfur-fumigation and sulfur-fumigation angelica dahurica by pharmacodynamics combined with plasma chemical firstly.
The study take anti-inflammatory, analgesic experiment on non-sulfur-fumigation and sulfur-fumigation angelica dahurica with classic xylene induced ear swelling in mice and acetic acid in mice body torsion method. Non-sulfur-fumigation sample showed significant anti-inflammatory and analgesic effects. partial dose group showed anti-inflammatory and analgesic effects, but not statistically significant. Each dose group of plasma composition was similar but also different. With the decrease of the dosage, the peak area of different dose groups also decreased accordingly. Oxypeucedanin hydrate is speculated to be the material basis of anti-inflammatory efficacy of angelica dahurica, which laid a foundation on elucidated spectrum relationship of angelica dahurica.
6. The study took an acute toxicity test of non-sulfur-fumigation and sulfur-fumigation angelica dahurica.
In the safety evaluation, non-sulfur-fumigated Angelica of LD50is for52.41g crude drug/body weight (kg), which equals approximately clinical dosage of353 times. Sulfur-fumigated Angelica of MTD is for123.6g crude drug/body weight (kg), which equals approximately clinical dosage of832times. Although both are in the range of safe use,but non-sulfur-fumigated Angelica toxicity was significantly larger than the toxicity of sulfur-fumigated Angelica.
7. The study carried on correlation analysis in chemical composition, efficacy and safety of different degrees of sulfur-fumigated angelica dahurica for the first time.
Using the curve regression methods to explore the relationship of sulfur dioxide residue and oxypeucedanin&imperatorin with established the model equations. The correlation is good. Oxypeucedani and sulfur dioxide residue was ogarithmically related:Y=0.085-0.0111nX. Imperatorin and sulfur dioxide residue was cubic related:Y=0.176+3.150×10-7X2-1.666×10-10X3.
The study discussed the relationship of spectrum and efficacy of angelica dahurica. Efficacy data from experiment of xylene induced ear swelling in mice and acetic acid in mice body torsion, and plasma HPLC fingerprint chromatogram peak area with each dose group were multivariate analyzed. Using a composite score C from principal component analysis as the independent variable, the pharmacodynamic index as the dependent variable Y do curvilinear regression to predict the relationship between them and establish correlation equation. Composite score and pharmacodynamics index were positively correlated.
According to the result of acute toxicity test and chemical composition analysis in vitro and in vivo, the study concluded that the cause of toxicity of angelica dahurica could be related to the effective components. Is the prototype or metabolites component caused toxic need further study. Although toxicity of sulfur-fumigated angelica dahurica is lower than non-sulfur-fumigated one, sulfur dioxide increased significantly to affect the safety. So it should regulate sulfur-fumigation processing methods of Angelica.
8. This study provides a scientific basis to clarify the rationality of sulfur fumigation processing method, determine the appropriate degree of sulfur fumigation and formulate scientific and rational, objective and controlled effective composition and sulfur dioxide limits standard.
The study found that sulfur fumigation effect the active ingredients and efficacy and increase in sulfur dioxide residue. But it has practical value in angelica dahurica region at present, which is easy of drying, storage, and low cost. As long as the good control the of degree sulfur fumigation, we can guarantee the quality of angelica dahurica and the safety of clinical use. According to the research on the effects of chemical composition, the efficacy and safety of Angelica dahurica by sulfur-fumigation and correlation analysis, preliminary Suggestions as follow. Sulfur dosage is not exceed100g/10kg and fumigation time not more than8h. Oxypeucedanin is not less than0.09%and imperatorin is not less than0.14%. Sulfur dioxide residue should not exceed400mg/kg.
引文
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