Study On Quality Evaluation System Of Pericarpium Zanthoxyli And Its Application

Pericarpium Zanthoxyli is rich in our country. Its bioactive components are variety, and it has wide range of pharmacological activity. With development and application of its medicinal value in different areaes, such as the digestive system, cardiovascular system, anti-microbial effect, anti-cancer role, Pericarpium Zanthoxyli will have great potential and far-reaching significance besides in the food, flavor and fragrance industries. However, there is an important problem in the research about Pericarpium Zanthoxyli that assessment method lack of accuracy and objectivity in Pericarpium Zanthoxyli quality grade standard can not be standardized and quantitative. Volatile oil content, as only one index, is short of scientific and comprehensive to evaluate Pericarpium Zanthoxyli quality. Therefore, it is needed to further study characteristic components as evaluation indexes of its quality, combine a single evaluation with overall evaluation, process the relative macro-pepper quality inspection, and establish an effective complete quality evaluation method.Using Pericarpium Zanthoxyli as raw materials, by applying advanced isolation and purification technique and testing technology.the referece standard sample of numb-tasted components were seperated and purified and the method for quantitative determination of numb-tasted components was established; The methods of volatile oil by rapid NIR inspection technology, total alkaloid of determing by ultra violet spectrophotometry, limonene, linalool and aroma components of quantitative determination by using GC and GC-MS were developed int this paper based on the elaborate methodological studies.Moreover, heavy metal elements in Pericarpium Zanthoxyli were determined by atomic fluorescence spectrophotometry (AFS) and atomic absorption spectrophotometry (AAS); GC-MS fingerprint and HPLC fingerprint of Pericarpium Zanthoxyli were established by fingerprinting technology. On the basis of the above established methods of quantitative determination of chemical constituents and fingerprint by GC-MS and HPLC combining with determing colour and microscopic characters, internal quality differences of Pericarpium Zanthoxyli from different species, origins and harvesting time were explored and characteristic peaks in HPLC fingerprint by analyzing the difference between Zanthoxylum Bungeanum Maxim and Zanthoxylum. Schinifolium Sieb.et Zucc were identified by TOF-MS. On the basis of establishment and application of Pericarpium Zanthoxyli quality evaluation method, the evaluation techniques were developed in order to make a foundation for establishing quality standard of Pericarpium Zanthoxyli, and its further exploiting. The main results obtained in this paper were as follows:1. The technology of separating and purifying numb-tasted components and the quantitative determination of numb-tasted compents by HPLC were established:(1) The route of separation and purification of numb-tasted components was determined: Zanthoxylum. Schinifolium Sieb.et Zucc→oil resin by supercritical extraction→separated in dry silica gel column with contrary-direction methods→collected the second and third groups→separated in silica gel column eluted with different solvents→dissolved in hot petroleum ether→freezing and crystallization→purified by pre-HPLC→crystallization.→dissolved in hot petroleum ether→freezing and crystallization(2) The purity of numb-tasted components, PI, P2 and P3 obtained by pre-HPLC were identified by thin layer chromatography (TLC) wih only one spot. The relative percentages of the constitutes were determined by GC method. The purity were 75.25%,81.04% and 72.13%. The ion mass of y of P2 was 263 by GC-MS, According to the fragments and referrences,then P2 was belonged to unsaturated aliphatic amides.(3) Crystal P2 with the highest purity was hydroxyl-a-sanshool, hydroxyl-(3-sanshool and their structural isomer, identified by GC-MS. And molecular weight of the three peaks in total Ion Chromatography were basically the same, determined and inferred by TOF-MS, molecular formula was C₁₆H₂₅NO₂, unsaturated degree was 5, Isotopic Ratio and Isotopic distribution were concordant with theroy. it should be hydroxyl-a-sanshool, hydroxyl-β-sanshool and their structural isomer.(4) Using crystal P2 as standard sample, HPLC method was built up to decide the quantities of numb-tasted components. Linearal range was 0-250μg/mL, regression equation was y=45.35x +31.7 (R²=0.9977), The average rate of recovery was 96.10%(RSD=5.91%). Specificity and repeatability was great. But after test 6h, the result was unstable. It need to delay test time under low temperature or test as soon as possible. This method can br used for quality control of Pericarpium Zanthoxyli.2. Measurement methods of other chemical compositions were built:(1) Volatile oil contents were determined by NIR. Twenty samples were predicted by calibration model established when scanning resolution was 16cm-1 and scan number was 128. The result showed coefficient of determination was 0.973, RMSEP was 0.272, RPD was 6.28. predictive capability of the model was good. The calibration model can accurately detect volatile oil content of the integrated Pericarpium Zanthoxyli particles.(2) The method for content determination of limonene and linalool was established. Linear range of limonene was 0.4765-9.5300mg/mL, the regression equation was Y=306213X-8925.9(r=0.9997, n=7). Linear range of linalool was 1.053～23.1300mg/mL, the regression equation was Y=225875X+25659(r=0.9994 n=8).The method has good repeatability and stability with high recovery rate, and it can be used for quantity control of Pericarpium Zanthoxyli quality.(3) The method for content determination of total alkaloids in Pericarpium Zanthoxyli was established by acid-dye colorimetry. Linear range was 0.2-1.0mg, the regression equation was y=0.917x-0.00188(r=0.9932). The method with good repeatability, stability and recovery rate, was controllable.(4) The methods of determination of trace heavy metal elements (Five kinds) in Pericarpium Zanthoxyli were established by optimization of experiments of experiments.The average recoveries of standard addition were in the range of 71%～102%.The accuracy of methods developed was evaluated by analsysis of corresponding trace heavy metal elements in standard reference material (GBW07064).It has showed the methods were suitable for control the heavy metal elements in Pericarpium Zanthoxyli.3. GC-MS fingerprint technology of Pericarpium Zanthoxyli was built:(1) The established GC-MS method for establshment of fingerprint of volatile oil had good repeatability, stability and recovery rate. The method was controllable and accorded with the requirements of fingerprint assessment techniques.(2) The established GC-MS characterstic fingerprint of Zanthoxylum Bungeanum Maxim had eleven common peaks, and had high similarity except 18,19 and 20 sample. The result indicated that the main peaks of Zanthoxylum Bungeanum Maxim from different origins were basically the same, their quality was stable. But there was difference in individual areaes.(3) The established GC-MS characterstic fingerprint of Zanthoxylum Schinifolium Sieb.et Zucc had teen common peaks. Similarity value of GC-MS fingerprint of Zanthoxylum Schinifolium Sieb.et Zucc was not lower than 0.97. The result indicated the main peaks of Zanthoxylum Schinifolium Sieb.et Zucc anum from different origins were basically consistent. And their quality was stable.4. HPLC fingerprint technology of Pericarpium Zanthoxyli was established.(1) The established method had favorable accuracy, stability and repeatability, and conformed to technical requirements to evaluate fingerprinting.(2) The characterstic Pericarpium Zanthoxyli fingerprint was established by《Evaluation Systems of similarity of the chromatographic fingerprint of traditional Chinese medicine, version A》(researchful version). Fingerprint of Zanthoxylum Bungeanum Maxim has eleven common peaks, fingerprint of Zanthoxylum Schinifolium Sieb.et Zucc has nine common peaks.(3) Referring to common peaks, similarity value of Zanthoxylum Bungeanum Maxim and Zanthoxylum Schinifolium Sieb.et Zucc was calculated. The results showed fingerprint similarity of Zanthoxylum Bungeanum Maxim was high (amount similarity>0.97), but fingerprint of Zanthoxylum Schinifolium Sieb.et Zucc was low similarity (0.52～0.67). Quality of Zanthoxylum Bungeanum Maxim from different origins was more consistent, while Zanthoxylum Schinifolium Sieb.et Zucc has big difference.5. Using the established methods to evaluate the internal quality of Pericarpium Zanthoxyli:(1) The coefficient of variation of volatile oil and numb-tasted components were both lower than 5% and the contents of volatile oil in Zanthoxylum schinifolium Sieb. et Zucc and Zanthoxylum bungeanum Maxim were 5.278 mL/100 g and 2.211 mL/100 g, respectively while the contents of numb-tasted components were 14.797 mg/g and 9.390 mg/g, separately.(2) There were not significant correlations between the content of volatile oil or the content of numb-tasted components and the four color characteristics (L, A, B, and E) for both Zanthoxylum schinifolium Sieb. etZucc and Zanthoxylum bungeanum Maxim. However, a significant correlation (P≤0.01) was observed between the contents of volatile oil and numb-taste components in Zanthoxylum schinifolium Sieb. et Zucc while no correlation was found in Zanthoxylum bungeanum Maxim. Samples of Zanthoxylum bungeanum Maxim from different areas showed significant difference in the content of volatile oil, but not in the content of numb-taste components while samples of Zanthoxylum schinifolium Sieb. et Zucc from different areas showed significant differences both in the contents of volatile oil and numb-taste components.(3) Limonene.linalool and alkaloid content of Zanthoxylum Bungeanum Maxim and Zanthoxylum Schinifolium Sieb.et Zucc were all unstable and variation coefficient was high.all over 25%. As limonene for example, average 12.89 mg/g in Zanthoxylum Schinifolium Sieb.et Zucc, but 14.04mg/g in Zanthoxylum Bungeanum Maxim, was not statistically significant between them(P>0.05);For linalool average 72.53 mg/g in Zanthoxylum Schinifolium Sieb.et Zucc, but 7.94mg/g in Zanthoxylum Bungeanum Maxim, there was very significant difference(P<0.01). For total alkaloids, there was significent difference(P<0.05) between them, average 0.96% in Zanthoxylum Bungeanum Maxim and 1,44% in Zanthoxylum Schinifolium Sieb.et Zucc.(4) The best season for harvest Zanthoxylum Schinifolium Sieb.et Zucc produce from Jinjiang, Chongqin was in 8 June. It was rich in aroma components, and had the highest content of numb-tasted components, but the content of volatile oil was not the highest.(5) Among all the common peaks of volatile oil in Zanthoxylum Schinifolium Sieb.et Zucc, terpenes owned 41.95%, alcohols owned 56.06%, and ketones owned 1.99%. While terpenes owned 52.59%, alcohols owned 34.50%, and ester owned 12.91% in Zanthoxylum Schinifolium Sieb.et Zucc. Alcohols accounted for more than half part of the common peak in volatile oil Zanthoxylum Schinifolium Sieb.et Zucc, while terpenes accounted for more than half part of volatile oil Zanthoxylum Bungeanum Maxim.(6) By clustering analysis,16 batches of Zanthoxylum Schinifolium Sieb.et Zucc samples were divided into 4 categories, including Hongya Sichuan,Zhaotong Yunnan,Chongqing and Jinyang Sichuan.16 batches of Zanthoxylum Bungeanum Maxim samples were also divided into 4 caregories, including Qing an Town Tianshui Gansu,Jinchuan Aba Sichuan,Wenchuan Sichuan,Liangshan and Hanyuan of Sichuan.(7) The similarity of Zanthoxylum Bungeanum Maxim from Hanyuan of Sichuan province was good, all similarity values were next to 1, this indicated internal quality of Zanthoxylum Bungeanum Maxim from different towns of Hanyuan was basically same. The similarity value of Zanthoxylum Schinifolium Sieb.et Zucc from Jiangjin was higher than 0.97, except a set of sample. While there existed great difference in Zanthoxylum Schinifolium Sieb.et Zucc from Jinyang of Sichuan province with the low similarity.(8) Identified by TOF:characteristic peak in HPLC fingerprint of Zanthoxylum Bungeanum Maxim and Zanthoxylum Schinifolium Sieb.et Zucc was unsaturated fatty acid amide, hydroxyl-y-sanshol or 2'-hydroxyl-N-isobutyl-2,4,8,10,12-tetradepentaenoateamide or their structural isomer.In this paper, volatile oil GC-MS and HPLC fingerprint of Pericarpium Zanthoxyli was established for the first time. The method had good accuracy, repeatability and stability, and conformed to technical requirements to evaluate fingerprinting. Also, evaluation of individual components systematically combined with assessment methodology of fingerprint for the first time. Measurement methods of color, essential oil, limonene, linalool, alkaloid, aroma components and heavy metal in Pericarpium Zanthoxyli were established. And using established evaluation methods, the intrinsic quality of Pericarpium Zanthoxyli with different species, origins and harvesting time was analyzed and compared. The method was feasible. Furthermore, crystal P2 was identified firstly by TOF/MS with high resolution, molecular formula was C₁₆H₂₅NO₂, it should be hydroxyl-a-sanshool, hydroxyl-β-sanshool and their structural isomer. In addition, characteristic difference between peak of HPLC fingerprint of Zanthoxylum Bungeanum Maxim and Zanthoxylum. Schinifolium Sieb.et Zucc was identified firstly by high resolution mass spectrometer, and was inferred to hydroxyl-y-sanshool or 2'-hydroxyl-N-isobutyl-2,4,8.10,12-tetradepentaenoateamide or their structural isomer.