Research On The Effects Of Chemical Composition,the Efficacy And Safety Of Angelica Dahurica By Sulfur-fumigation

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.