| Commercial specifications and grades of Chinese medicinal materials, the summary of practical experience for quality evaluation of traditional Chinese medicine, were the reflect of the quality of traditional Chinese medicine, which had played an important role in market circulation and ensuring clinical safety because it’s easy, fast and effective. However, most of these evaluation methods were still in the stage of experiential description which made it difficult to be inherited and applied. This research took Ligllsticum chuanxiong as the study subject, and systematically studied the scientific connotation of the division standards of commercial specifications and grades in L.chuanxiong from the aspects of sensory quality, physical and chemical quality, chemical composition analysis and biological evaluation. Based on the above indexes, werebuilt and improved the evaluation index system of commercial specifications and grades in L.chuanxiong, and formulated the industrial standard draft of commercial specifications and grades in L.chuanxiong. The main research results are as follows:1. Literature research, market and origin investigation1.1Study on the forming pattern of Sichuan genuine medicinal materials of L.chuanxiong and commercial specifications and grades in L.chuanxiongIn this study, it’s the first time that forming pattern of Sichuan genuine medicinal materials of L.chuanxiong and commercial specifications and grades in L.chuanxiong are studied, and we have come to the conclusion that the forming pattern of L.chuanxiong is the type of two determinants, which combined both unique ecological environment of genuine regions and advanced cultivation techniques. There were many classfication methods for commercial specifications and grades in L.chuanxiong in history, such as by producing areas, harvest time, origin processing method, processing method, etc. The current standards for commercial specifications and grades in L.chuanxiong carried out the standards of commercial specifications and grades in76Chinese medicinal materials published in1984, and mainly classified into3levels by weight.1.2Market investigation on commercial specifications and grades in L.chuanxiongIn this study, we investigated the current situation of commercial specifications and grades in L.chuanxiong from the aspects of origin planting and processing, marketing and clinical use. The results showed that the L.chuanxiong of gradeless and uniformly-priced goods were used in the whole process of from planting, circulation to clinical use. The planting area of L.chuanxiong has been shrinking every year in the traditional main producing area Dujiangyan. In recent years, Pengshan village has planted a lot L.chuanxiong and formed certain scales. However, the climatic environment, plant and harvest time in the new producing area are very different from traditional producing area, and further research is required to decide the quality consistency.2. Study on the evaluation index system for commercial specifications and grades in L.chuanxiong2.1Study on the seneory evalution of L.chuanxiong2.1.1Organization and standardization of the traditional experiential identification of L.chuanxiongThis research adopted the Delphi method to organize and standardize the traditional experiential identification of L.chuanxiong for the first time. The results showed that the main indexes of traditional experiential identification of L.chuanxiong were diameter of the central, weight, smell and shape. The reproducibility of five times experts’evaluation was an average of72%, which showed that the sensory evaluation of L.chuanxiong was objective and reliable. The analysis on the consistency of the four indexes in experts by Kender coefficients showed better total reproducibility in experts group in the indexes of diameter of central, weight and shape, while the Kender coefficient of smell index was0.349which showed its difficulty to control. The above identification indexes in the commercial specifications standards of L.chuanxiong should be underlined to reduce the interference of secondary information to experiential evaluation. It can improve the accuracy of identification and reproducibility.2.1.2Applied research of electronic nose in the quality evaluation of smell of L.chuanxiongThe results of Delphi method showed that Smell is an important index of traditional experiential identification of L.chuanxiong. However, its strong subjectivity and low reproducibility has limited its promotion and application in the quality evaluation of L.chuanxiong. In this research, we adopted electronic nose to study on the recognization of L.chuanxiong of different producing area and grade for the first time. The results of Dujiangyan, Pengzhou and Xindu were very close, while the result of new producing area Pengshan had a big difference from the others. The results of different grades in the same producing area were close to overlap and obviously different from off-grades.2.2Study on the conventional physical and chemical quality of L.chuanxiongMoisture, ash content, limit of harmful substance and other physical and chemical indexes are important parts in quality evaluation system of Chinese medicinal materials and are closely related with its purity, quality and safety. In this research, we have determined the moisture, total ash, acid-insoluble ash, ethanol-soluble extract, heavy metal and arsenic, and pesticide residues of L.chuanxiong from different producing areas with different grades. The results showed that there were no significant differences among different grades of L.chuanxiong. The results of total ash, acid-insoluble ash and ethanol-soluble extract in worm eaten L.chuanxiong had significantly decreased. All the samples in this research were not detected by BHC, PCNB and DDT. According to the Green standards of medicinal plants and preparations for foreign trade and economy, the heavy metals in the samples were out of limit, among which, the exceeding of copper and cadmium were more serious. The content of arsenic and mercury accorded with the limited standards. In order to ensure the safety and effectiveness in clinical use, the exceeding reasons of copper and cadmium should be further studied.2.3Study on the chemical quality of L.chuanxiongIn this study, we have determined the content of ferulic acid, total phenolic acids, po.lysaccharide, phthalides and fluorescent materials in L.chuanxiong, and compared the whole chemical compositions by GC-MS and HPLC fingerprint, in order to provide reference for the establishment of evaluation index system of commercial specifications and grades in L.chuanxiong.2.3.1Comparison on the content of ferulic acid in L.chuanxiongThe contents of ferulic acid in the samples of this research were determined by the method in Chinese pharmacopoeia (2010edition). The results showed that the contents of ferulic acid in red L.chuanxiong(different cultivation) and Ligusticum sinense Oliv.(counterfelt drug) have no significant differences; there are no significant differences between different producing areas and different grades of L.chuanxiong; the contents of ferulic acid in worm-eaten samples and burned samples have significantly decreased. The above results showed that the specificity was not strong to control the commercial specifications and grades in L.chuanxiong with ferulic acid as the evaluation index.2.3.2Comparison on the content of total phenolic acids in L.chuanxiongThe contents of total phenolic acids in the samples of this research were determined by the method of Appendix XB in Chinese pharmacopoeia (2010edition). The results showed that there are no significant differences in the content of total phenolic acids between different producing area except that Pengshan was a little higher; the content of total phenolic acids of different grades from the same producing area has no significant differences; compared with L.chuanxiong, the contents of total phenolic acids in worm-eaten samples do not reduce, while the content of total phenolic acids in burned samples has increased; the contents of total phenolic acids in Ligusticum sinense Oliv. were close to L.chuanxiong, while the contents of total phenolic acids in red L.chuanxiong was a little higher than L.chuanxiong. The above results showed that the contents of total phenolic acids could be used as an evaluation index of baking temperature in origin processing.2.3.3Comparison on the content of polysaccharide in L.chuanxiongThe contents of polysaccharides in L.chuanxiong were determined by sulfuric acid-phenol method. The results showed that there are significant differences between different producing areas of L.chuanxiong in the contents of polysaccharidesThe order of contents from high to low is Xiejia Pengshan, Aoping Pengzhou, Xinfan Xindu, commercial varieties and Xudu Dujiangyan; the contents of polysaccharides showed no significant differences between different grades of L.chuanxiong in the same producing area; the contents of polysaccharides in worm-eaten and burned samples significantly decreased when compared with L.chuanxiong; the contents of polysaccharides in red L.chuanxiong and Ligusticum sinense Oliv. were obviously less than L.chuanxiong. The above results showed that the contents of polysaccharides could be used as a chemical evaluation index of commercial specifications and grades in L.chuanxiong.2.3.4Comparison on the content of phthalides in L.chuanxiong The contents of ligustilide, senkyunolide A and levistilide A in different batches of L.chuanxiong samples were determined by HPLC. The results showed that there are some differences in the contents of the three from different producing areas, while there are no significant differences between different grades of L.chuanxiong in the same producing area; compared with L.chuanxiong, the contents of the three worm-eaten samples obviously decreased, while the contents of ligustilide and senkyunolide A in burned samples obviously decreased and the content of levistilide A increased; the contents of ligustilide and levistilide A in red L.chuanxiong were close to that of L.chuanxiong, while the content of senkyunolide A obviously decreased; the contents of ligustilide and levistilide A in Ligusticum sinense Oliv. were obviously lower than that of L.chuanxiong, while the content of senkyunolide A was obviously higer than that of L.chuanxiong. The above results showed that the contents of phthalides could be used as a chemical evaluation index of commercial specifications and grades in L.chuanxiong. However, the storage and origin processing influnce quite much on the contents of phthalides, so they could be used as the key index to standardize the origin processing of L.chuanxiong.2.3.5Comparison on the contents of fluorescent materials in L.chuanxiongIt is an important feature of traditional physical and chemical identification of L.chuanxiong that there is bright light blue fluorescence of the section of L.chuanxiong under UV.In this research, we adopted fluorometric spectrophotometry to determine the contents of total fluorescent materials for the first time. The results showed that the contents of total fluorescent materials were no big differences from different producing area, except that Xiejia Pengshan was a little higher; The contents of total fluorescent materials of different grades from the same producing area had no significant differences; compared with L.chuanxiong, the contents of total fluorescent materials in worm-eaten and burned samples obviously decreased; the contents in red L.chuanxiong had no significant difference with that of L.chuanxiong; the contents in Ligusticum sinense Oliv. were obviously lower than that of L.chuanxiong. The above results showed that the contents of total fluorescent materials could be used as a chemical evaluation index of commercial specifications and grades in L.chuanxiong. In this research, we adopted fluorometric spectrophotometry to determine the fluorescence intensity of the sample powder, and analyzed the correlation in the fluorescence intensity of the sample powder, the content of ligustilide and the contents of total fluorescent materials for the first time. The results showed that the intensity of the sample powder had a positive liner correlation with the content of ligustilide and the contents of total fluorescent materials, the correlation coefficient were0.838and0.940separately. The results showed that by determining the fluorescence intensity of the sample, the content of ligustilide could be predicted to achieve fast and nondestructive evaluation of the inner quality of L.chuanxiong.2.3.6Comparison on chemical fingerprint of L.xhuanxiong2.3.6.1GC-MS fingerprint Headspace solid-phase micro-extraction with GC-MS was used to analyze the volatile components in L.chuanxiong and angle cosine computation was used to evaluate the similarity of L.chuanxiong’s chromatography data of different producing areas and different commercial specifications. The results that the similarities between different producing areas and different commercial specifications were all more than0.9showed that there were no significant differences of volatile components in different commercial specifications and grades of L.chuanxiong. The similarity between red L.chuanxiong and L.chuanxiong more than0.9showed that the volatile components of the two had no significant differences. The similarity between Ligusticum sinense Oliv. and L.chuanxiong less than0.9showed that the volatile components of the two had significant differences. The contents of volatile components obviously decreased in worm-eaten L.chuanxiong and the similarity between worm-eaten L.chuanxiong and L.chuanxiong less than0.7showed that the volatile components obviously changed after worm-eaten. The types and contents of volatile components had changed after being burned and the similarity between burned L.chuanxiong and L.chuanxiong less than0.9showed that the storage and baking process influnced much on the quality of L.chuanxiong.2.3.6.2HPLC fingerprint In this research, we had established the HPLC fingerprint of L.chuanxiong. The similarity of the fingerprints between15batches of samples and standard was more than0.9showed that the fignerprints of different producing areas and different grades from same producing area were no significant differences. The similarity of the fingerprints between worm-eaten, burned and standard was all less than0.9, compared with the fingerprints, that worm-eaten mainly decreased the contents of some chemical components but not the types of chemical compositions. The contents and types of chemical compositions in burned L.chuanxiong had obviously changed. The similarity between red L.chuanxiong and standard was0.958, which showed that there were no significant differences between the two in the types and contents of chemical compositions. The similarity between Ligusticum sinense Oliv. and L.chuanxiong was0.608, which showed that there were big differences between the two in the types and contents of chemical compositions. The peak area ratio of ligustilide and senkyunolide A in L.chuanxiong was7.441, while Ligusticum sinense Oliv. was0.523, which showed that the phthalides in L.chuanxiong were primarily ligustilide and Ligusticum sinense Oliv. were senkyunolide A. It was similar to the previous contents determination results.2.4Study on the biological quality of L.chuanxiongBio metric is applicable to drugs with complex structure or thsoe that clinical effect couldn’t be reflected by physical and chemical determination. It has unique advantage in quality control and evaluation of traditional Chinese medicine. In this paper we estimated the anticoagulant activity and the activity to antagonize the uterine smooth muscle spasm induced by oxytocin in L.chuanxiong based on its effect of moving Blood and Qi and expelling wind to relieve pain. The results are as follows:2.4.1Estimation of anticoagulant activity In this paper, we have established the bioassay method of anticoagulant activity in L.chuanxiong with low molecular weight heparin used as comparison. The samples of L.chuanxiong all have the biological activity of elongating the clotting-time of rabbits. There were no significant differences of the biological potency of anticoagulant activity in the samples of L.chuanxiong form different producing areas and from different grades in the same producing area. The biological potency of anticoagulant activity in worm-eaten samples was slightly higher than L.chuanxiong in the same producing area, while the biological potency of anticoagulant activity in burned samples was2times of L.chuanxiong in the same producing area. The biological potency of anticoagulant activity in red L.chuanxiong was a little higher than that of L.chuanxiong, while the biological potency of anticoagulant activity in Ligusticum sinense Oliv. was obviously lower than that of L.chuanxiong. 5.4.2Estimation of the activity to antagonize the uterine smooth muscle spasm induced by oxytocin in L.chuanxiong This research initially established the bioassay method of the activity in L.chuanxiong to antagonize the uterine smooth muscle spasm induced by oxytocin, with magnesium sulfate as comparison. The samples of L.chuanxiong all have the biological activity of antagonizing the uterine smooth muscle spasm induced by oxytocin. The biological potencies showed no significant differences in the samples of L.chuanxiong form different producing areas and from different grades in the same producing area. The biological potencies in worm-eaten samples and burned samples obviously decreased, while the biological potencies in red L.chuanxiong and Ligusticum sinense Oliv. were obviously lower than that of L.chuanxiong2.5Correlation research on the index system2.5.1Correlation analysis on chemical evaluation and biological evaluationIn order to further study on the correlation of pharmacological activity and chemical compositions, we analyzed the correlation of biological potency and the contents of chemical components in L.chuanxiong in this research. The contents of total phenolic acids and levistilide A had a positive liner correlation with the biological potency of anticoagulant activity(P<0.01), which showed that total phenolic acids might be the main active ingredients of anticoagulant activity inL.chuanxiong. The contents of ligustilide and total fluorescent materials had a positive liner correlation with the biological potency of the activity in L.chuanxiong to antagonie the uterine smooth muscle spasm induced by oxytocin(P<0.05), which showed that the main active ingredients of spasmolytic activity in L.chuanxiong might be phthalides with ligustilide as a representative.2.5.2Correlation analysis on HPLC fingerprint and biological potencyMultiple linear correlation was used to analyze the relationship between the peak areas of shared peaks in HPLC fingerprint of L.chuanxiong and the biological potency of anticoagulant activity and the activity to antagonize the uterine smooth muscle spasm induced by oxytocin. The peak areas of NO.11peak and NO.13peak (levistilide A) had a positive correlation with the biological potency of anticoagulant activity. The peak area of NO.10peak (ligustilide) had a positive correlation with the biological potency of the activity to antagonize the uterine smooth muscle spasm induced by oxytocin. The peak area of ligustilide was the largest in the HPLC fingerprint and owned more than50%of the total peak areas, which showed that the HPLC fingerprint established in this research could reflect the biological activity of L.chuanxiong.3. formulation of the industrial standard draft of commercial specifications and grades in L.chuanxiongThe research results showed that, there were no significant correlation between the current standards of commercial specifications and grades in L.chuanxiong divided by weight and the inner quality of L.chuanxiong. However, the division of commercial specifications and grades in Chinese medicinal materials should be the reelect of its quality. In this research we have optimized the sensory quality index, conventional physical and chemical quality index, chemical evaluation index and biological evaluation index, and established the quality control method and index system accorded with the properties of Chinese medicinal materials, then initially formulated the industrial standard draft of commercial specifications and grades in L.chuanxiong referd to Chinese pharmacopoeia (2010edition) and the standards of commercial specifications and grades in76Chinese medicinal materials. The research results play an important role in ensuring L.chuanxiong’s safety and effectiveness in clinical use, and have significances for the study of commercial specifications and grades in other medicinal materials and promotion of the standard system in Chinese medicinal materials. |
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