花椒品质评价方法及其应用研究
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摘要
花椒(Pericarpium Zanthoxyli)在我国资源丰富,且生物活性成分种类繁多,药理活性广泛。随着花椒药用价值,如消化系统、心血管系统、抑微生物作用、抗癌作用等研究以及化工、农药等领域的开发应用,花椒将不会停留在食品、香精香料等行业中,其他领域也将有着巨大的潜力和深远的意义。但我国对花椒基础性研究不够。如对不同品种、不同成熟度,同一品种产于不同地区的花椒有效成分缺乏系统深入的研究,这对花椒的精深加工和拓展应用领域极为不利。花椒现行质量等级标准中等级评定方法缺乏准确性和客观性,无法实现标准化和定量化；标准评定指标单一,能反映花椒内在质量的指标只有挥发油含量这一项,用这唯一指标来评价花椒的质量欠缺科学性和全面性。因此要深入研究花椒指标性成分,采用单一评价和整体评价方法相结合,对花椒进行相对宏观的质量考察,建立有效完整的品质评价方法。
本文以花椒为原料,利用现代有效成分分离纯化技术,结合仪器鉴定及分析测试技术,对花椒中麻味物质进行了分离、纯化和鉴定,并建立了花椒中麻味物质HPLC定量测定方法；深入研究了花椒中挥发油近红外光谱测定、总生物碱紫外分光光度法测定、花椒柠檬烯及芳樟醇GC测定、花椒香气成分分析等方法,并进行相应的方法学考察；采用原子荧光分光光度法、原子吸收分光光度法等常规方法对花椒中重金属指标进行了测定；利用指纹图谱技术建立了花椒挥发油GC-MS指纹图谱及花椒HPLC指纹图谱,并经过严格的方法学考察；依据上述建立的化学成分测定方法及指纹图谱评价等花椒品质评价方法,并结合色差仪分析花椒外观色泽和显微镜观察花椒粉末特征,充分挖掘数据信息,系统分析了不同品种、不同产地、不同采收时期花椒内在品质的差异性,并采用GC-MS质谱和高分辨率飞行时间质谱分别对挥发油GC-MS和HPLC指纹图谱中差异性色谱峰进行了鉴定。本文立足于花椒品质评价方法的建立及应用,为花椒质量标准的建立及其开发利用提供了依据。主要研究结果如下：
1、建立了花椒麻味物质的标准对照品制备技术及其HPLC定量测定方法：
(1)花椒麻味物质的分离纯化流程：花椒→花椒油树脂→麻味物质粗分离→逆流干柱层析→合并浸提液→硅胶色谱柱层析→热石油醚溶解→低温冷冻结晶→制备型液相色谱纯化→热石油醚溶解→低温冷冻结晶→花椒麻味物质纯品
(2)分析经制备型色谱分离纯化得到的P1、P2、P3纯度,用薄层色谱法鉴定,均只出现一个斑点,而用气相色谱归一化分析,纯度分别为75.25%、81.04%、72.13%；GC-MS分析纯度最高的P2晶体,分子离子峰质荷比均为263,依据碎片信息及参考文献,确定P2为花椒中的不饱和脂肪酰胺类物质。
(3)高分辨率质谱TOF-MS对纯度最高的P2晶体进行分析,总离子色谱图中出现的三个峰经TOF提取离子计算,分子量基本一致,为263.2,并推算化合物元素组成为C_(16)H_(25)NO_2.不饱和度为5,同位素分布及丰度比与理论值一致,初步推断为羟基-α-山椒素或羟基-β-山椒素及其同分异构体。
(4)建立高效液相色谱法测定花椒中麻味物质含量,方法线性范围为0-250μg/mL,回归方程为y=45.35x+31.7(R~2=0.9977)；花椒麻味物质加样回收率为96.10%(RSD=5.91%, n=6),回收率良好；方法专属性强、重复性好,但样品6h后测试不稳定,需低温下延长测试时间或尽快测试,最低检测浓度为0.1μg/mL。
2、建立了花椒其他特征化学成分的测定方法：
(1)近红外光谱法测定花椒中的挥发油,扫描分辨率为16cm~(-1),扫描次数为128时,所得扫描光谱建立的定标模型对验证集中的20个样品进行预测,决定系数达到0.973,RMSEP达到0.272,RPD为6.28,模型预测能力最好,能够较准确地检测完整花椒颗粒中挥发油的含量。
(2)花椒中柠檬烯、芳樟醇含量的测定方法,柠檬烯线性范围为0.4765-9.5300mg/mL,回归方程为Y=306213X-8925.9 (r=0.9997, n=7);芳樟醇线性范围为1.0535～23.1300mg/mL,回归方程Y=225875X+25659(r=0.9994, n=8)。所建立的方法重复性、稳定性好,加样回收率高,方法可行,可用于花椒质量的定量控制。
(3)试验中建立的酸性染料比色法测定花椒中总生物碱含量,线性范围为0.2-1.0mg,回归方程为y=0.917x-0.00188(r=0.9932),且方法重复性好,稳定性好,加样回收率高,方法可控。
(4)优化比较花椒中五种元素的测定方法,方法线性好,回收率在71%-102%之间,符合痕量分析的要求,并用建立方法测定国家级标准物质杨树叶中的各元素,结果各元素测定值均在允许值范围内,表明方法可用于花椒中重金属的控制。
3、建立了花椒挥发油GC-MS指纹图谱技术研究：
(1)建立的花椒挥发油指纹图谱分析方法,精密度、重现性、稳定性好,方法可行,符合指纹图谱评价技术的要求。
(2)建立的青花椒挥发油GC-MS特征指纹图谱中共有10个共有峰,青花椒(1-16号样品)挥发油GC-MS指纹图谱的相似度都不低于0.97,说明各地所产青花椒主要峰群的整体图貌基本一致,质量较稳定,相似度较高。
(3)建立的红花椒挥发油GC-MS特征指纹图谱中共有11个共有峰,红花椒(18-33号样品)挥发油GC-MS指纹图谱的相似度除18、19、20号甘肃产红花椒相似度较低外,其他红花椒具有较高相似度,说明各地所产红花椒主要峰群的整体图貌基本一致,质量较稳定,但存在个别地区性差别。
4、建立了花椒HPLC指纹图谱技术研究：
(1)建立的花椒HPLC指纹图谱方法具有良好的精密度、稳定性、重复性,符合指纹图谱评价技术要求。
(2)通过《中药色谱指纹图谱相似度评价系统A版》(研究版)提取青花椒HPLC图谱共有模式,组成HPLC特征指纹图谱并计算各批次样品的相似度,结果特征指纹图谱中有9个共有峰,18批青花椒样品相似度都很低,相似度为0.52～0.67之间,表明不同产地青花椒内在品质差异性很大。
(3)通过《中药色谱指纹图谱相似度评价系统A版》(研究版)提取红花椒HPLC图谱共有模式,组成HPLC特征指纹图谱并计算各批次样品的相似度,结果特征指纹图谱中有11个共有峰,15批红花椒样品中,除样品(甘肃秦安县郭家镇寺咀村大油椒,编号29)样本外,其他十四批样品相似度均大于0.97,表明不同产地红花椒内在品质很相似。
5、结合花椒色泽及粉末显微特征,利用上述建立方法对花椒内在品质进行评价：
(1)青花椒和红花椒的挥发油与麻味物质含量变异系数均较小(均小于5%),表明两个品质指标相对比较稳定；青花椒挥发油含量均值约为红花椒的2.4倍,分别为5.278mL/100g和2.211mL/100g,而麻味物质含量均值约为红花椒的1.6倍,分别为14.797mg/g和9.390mg/g；从挥发油和麻味物质两个特征指标来看,青花椒品质优于红花椒。
(2)青花椒、红花椒的挥发油和麻味物质含量两个特征指标与其颜色四个指标(△L、AA.△B、AE)之间无显著相关性；青花椒挥发油和麻味物质含量两个指标之间有极显著止相关(P≤0.01),而红花椒挥发油和麻味物质含量两个指标之间无显著相关；不同产地青花椒挥发油和麻味物质之间都存在显著性差异(P<0.05),而不同产地红花椒麻味物质之间不存在显著性差异,而挥发油含量有显著性差异(P<0.05)。
(3)青花椒、红花椒中柠檬烯、芳樟醇、生物碱含量都不稳定,含量变异系数大,均超过25%；由柠檬烯指标来看,青花椒平均值为12.89 mg/g,而红花椒平均值为14.04mg/g,经显著性差异分析,青花椒与红花椒之间无显著性差异(P>0.05)；由芳樟醇指标来看,青花椒平均值为72.53mg/g,而红花椒平均值为7.94 mg/g,经显著性差异分析,青花椒与红花椒之间存在极显著性差异(P<0.01)；由生物碱指标来看,青花椒平均值为1.44%,而红花椒平均值为0.96%,经显著性差异分析,红花椒与青花椒之间存在显著性差异(P<0.05)。
(4)重庆江津青花椒在6月8日左右采收,香气成分信息比较丰富,麻味物质含量最高,为13.65mg/g,但挥发油含量不是最高,为4.22mL/100g。
(5)青花椒挥发油共有峰中,萜烯类平均占共有峰的41.95%,醇类56.06%,酮类1.99%。红花椒挥发油共有峰中,萜烯类平均占共有峰的52.59%,醇类34.50%,酯类12.91%。
(6)青花椒挥发油经聚类分析后,16批样品聚为4类,这4类青花椒产地分别是四川洪雅藤椒基地、云南昭通、重庆、四川金阳；红花椒挥发油经聚类分析后,16批样品也聚为4类,分别是甘肃天水市秦安县郭家镇大油椒、甘肃天水市秦安县和四川阿坝州金川县、四川汶川县、四川汉源和凉山州。
(7)四川汉源红花椒相似度很好,基本都达到1,表明在汉源不同乡镇种植的花椒内在品质基本一致；而四川金阳青花椒品质差异性很大,相似度很低；江津青花椒除一批样品外,相似度都达0.97以上。
(8)红花椒和青花椒HPLC特征指纹图谱中,差异性特征峰初步推断为不饱和五烯酰胺,即羟基-γ-山椒素或2-羟基-N-异丁基-2,4,8,10,12-十四烷五烯酰胺及其同分异构体。
本文首次建立花椒挥发油GC-MS指纹图谱及花椒HPLC指纹图谱,方法精密度、重现性、稳定性好,方法可行,符合指纹图谱评价技术的要求：首次系统地建立了花椒中柠檬烯、芳樟醇、生物碱及五种重金属元素多个指标含量测定方法,并经过严密的方法学验证；首次将单个成分评价与指纹图谱技术评价方法相结合,充分挖掘指纹图谱数据信息,结合质谱鉴定技术和数据统计分析理论,对不同产地、不同品种、不同采收时期花椒的各指标含量、指纹图谱组分、含量的差异性进行了系统深入的比较分析,为花椒内在品质评价提供化学研究基础；首次采用高分辨率飞行时间质谱对分离纯化制备的P2晶体进行了鉴定,分子式为C_16H_5NO_2,应为羟基-a-山椒素或羟基-β-山椒素及其同分异构体；首次利用高分辨率质谱对红花椒和青花椒HPLC指纹图谱中差异性特征峰进行了鉴定,推断为不饱和五烯酰胺,即羟基-Y-山椒素或2-羟基-N-异丁基-2,4,8,10,12-十四烷五烯酰胺及其同分异构体。
本文受国家白然科学基金项目“基于近红外光谱的花椒品质快速检测机理及模型优化方法研究”(30671198)和重庆市重点自然科学基金项目“花椒和生姜中功效成分的组成和功能性质及定量检测方法研究”(CSTC,2006BA1007)”的资助。
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 C_(16)H_(25)NO_2, 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 (R~2=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 C_(16)H_(25)NO_2, 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.
本文以花椒为原料,利用现代有效成分分离纯化技术,结合仪器鉴定及分析测试技术,对花椒中麻味物质进行了分离、纯化和鉴定,并建立了花椒中麻味物质HPLC定量测定方法；深入研究了花椒中挥发油近红外光谱测定、总生物碱紫外分光光度法测定、花椒柠檬烯及芳樟醇GC测定、花椒香气成分分析等方法,并进行相应的方法学考察；采用原子荧光分光光度法、原子吸收分光光度法等常规方法对花椒中重金属指标进行了测定；利用指纹图谱技术建立了花椒挥发油GC-MS指纹图谱及花椒HPLC指纹图谱,并经过严格的方法学考察；依据上述建立的化学成分测定方法及指纹图谱评价等花椒品质评价方法,并结合色差仪分析花椒外观色泽和显微镜观察花椒粉末特征,充分挖掘数据信息,系统分析了不同品种、不同产地、不同采收时期花椒内在品质的差异性,并采用GC-MS质谱和高分辨率飞行时间质谱分别对挥发油GC-MS和HPLC指纹图谱中差异性色谱峰进行了鉴定。本文立足于花椒品质评价方法的建立及应用,为花椒质量标准的建立及其开发利用提供了依据。主要研究结果如下：
1、建立了花椒麻味物质的标准对照品制备技术及其HPLC定量测定方法：
(1)花椒麻味物质的分离纯化流程：花椒→花椒油树脂→麻味物质粗分离→逆流干柱层析→合并浸提液→硅胶色谱柱层析→热石油醚溶解→低温冷冻结晶→制备型液相色谱纯化→热石油醚溶解→低温冷冻结晶→花椒麻味物质纯品
(2)分析经制备型色谱分离纯化得到的P1、P2、P3纯度,用薄层色谱法鉴定,均只出现一个斑点,而用气相色谱归一化分析,纯度分别为75.25%、81.04%、72.13%；GC-MS分析纯度最高的P2晶体,分子离子峰质荷比均为263,依据碎片信息及参考文献,确定P2为花椒中的不饱和脂肪酰胺类物质。
(3)高分辨率质谱TOF-MS对纯度最高的P2晶体进行分析,总离子色谱图中出现的三个峰经TOF提取离子计算,分子量基本一致,为263.2,并推算化合物元素组成为C_(16)H_(25)NO_2.不饱和度为5,同位素分布及丰度比与理论值一致,初步推断为羟基-α-山椒素或羟基-β-山椒素及其同分异构体。
(4)建立高效液相色谱法测定花椒中麻味物质含量,方法线性范围为0-250μg/mL,回归方程为y=45.35x+31.7(R~2=0.9977)；花椒麻味物质加样回收率为96.10%(RSD=5.91%, n=6),回收率良好；方法专属性强、重复性好,但样品6h后测试不稳定,需低温下延长测试时间或尽快测试,最低检测浓度为0.1μg/mL。
2、建立了花椒其他特征化学成分的测定方法：
(1)近红外光谱法测定花椒中的挥发油,扫描分辨率为16cm~(-1),扫描次数为128时,所得扫描光谱建立的定标模型对验证集中的20个样品进行预测,决定系数达到0.973,RMSEP达到0.272,RPD为6.28,模型预测能力最好,能够较准确地检测完整花椒颗粒中挥发油的含量。
(2)花椒中柠檬烯、芳樟醇含量的测定方法,柠檬烯线性范围为0.4765-9.5300mg/mL,回归方程为Y=306213X-8925.9 (r=0.9997, n=7);芳樟醇线性范围为1.0535～23.1300mg/mL,回归方程Y=225875X+25659(r=0.9994, n=8)。所建立的方法重复性、稳定性好,加样回收率高,方法可行,可用于花椒质量的定量控制。
(3)试验中建立的酸性染料比色法测定花椒中总生物碱含量,线性范围为0.2-1.0mg,回归方程为y=0.917x-0.00188(r=0.9932),且方法重复性好,稳定性好,加样回收率高,方法可控。
(4)优化比较花椒中五种元素的测定方法,方法线性好,回收率在71%-102%之间,符合痕量分析的要求,并用建立方法测定国家级标准物质杨树叶中的各元素,结果各元素测定值均在允许值范围内,表明方法可用于花椒中重金属的控制。
3、建立了花椒挥发油GC-MS指纹图谱技术研究：
(1)建立的花椒挥发油指纹图谱分析方法,精密度、重现性、稳定性好,方法可行,符合指纹图谱评价技术的要求。
(2)建立的青花椒挥发油GC-MS特征指纹图谱中共有10个共有峰,青花椒(1-16号样品)挥发油GC-MS指纹图谱的相似度都不低于0.97,说明各地所产青花椒主要峰群的整体图貌基本一致,质量较稳定,相似度较高。
(3)建立的红花椒挥发油GC-MS特征指纹图谱中共有11个共有峰,红花椒(18-33号样品)挥发油GC-MS指纹图谱的相似度除18、19、20号甘肃产红花椒相似度较低外,其他红花椒具有较高相似度,说明各地所产红花椒主要峰群的整体图貌基本一致,质量较稳定,但存在个别地区性差别。
4、建立了花椒HPLC指纹图谱技术研究：
(1)建立的花椒HPLC指纹图谱方法具有良好的精密度、稳定性、重复性,符合指纹图谱评价技术要求。
(2)通过《中药色谱指纹图谱相似度评价系统A版》(研究版)提取青花椒HPLC图谱共有模式,组成HPLC特征指纹图谱并计算各批次样品的相似度,结果特征指纹图谱中有9个共有峰,18批青花椒样品相似度都很低,相似度为0.52～0.67之间,表明不同产地青花椒内在品质差异性很大。
(3)通过《中药色谱指纹图谱相似度评价系统A版》(研究版)提取红花椒HPLC图谱共有模式,组成HPLC特征指纹图谱并计算各批次样品的相似度,结果特征指纹图谱中有11个共有峰,15批红花椒样品中,除样品(甘肃秦安县郭家镇寺咀村大油椒,编号29)样本外,其他十四批样品相似度均大于0.97,表明不同产地红花椒内在品质很相似。
5、结合花椒色泽及粉末显微特征,利用上述建立方法对花椒内在品质进行评价：
(1)青花椒和红花椒的挥发油与麻味物质含量变异系数均较小(均小于5%),表明两个品质指标相对比较稳定；青花椒挥发油含量均值约为红花椒的2.4倍,分别为5.278mL/100g和2.211mL/100g,而麻味物质含量均值约为红花椒的1.6倍,分别为14.797mg/g和9.390mg/g；从挥发油和麻味物质两个特征指标来看,青花椒品质优于红花椒。
(2)青花椒、红花椒的挥发油和麻味物质含量两个特征指标与其颜色四个指标(△L、AA.△B、AE)之间无显著相关性；青花椒挥发油和麻味物质含量两个指标之间有极显著止相关(P≤0.01),而红花椒挥发油和麻味物质含量两个指标之间无显著相关；不同产地青花椒挥发油和麻味物质之间都存在显著性差异(P<0.05),而不同产地红花椒麻味物质之间不存在显著性差异,而挥发油含量有显著性差异(P<0.05)。
(3)青花椒、红花椒中柠檬烯、芳樟醇、生物碱含量都不稳定,含量变异系数大,均超过25%；由柠檬烯指标来看,青花椒平均值为12.89 mg/g,而红花椒平均值为14.04mg/g,经显著性差异分析,青花椒与红花椒之间无显著性差异(P>0.05)；由芳樟醇指标来看,青花椒平均值为72.53mg/g,而红花椒平均值为7.94 mg/g,经显著性差异分析,青花椒与红花椒之间存在极显著性差异(P<0.01)；由生物碱指标来看,青花椒平均值为1.44%,而红花椒平均值为0.96%,经显著性差异分析,红花椒与青花椒之间存在显著性差异(P<0.05)。
(4)重庆江津青花椒在6月8日左右采收,香气成分信息比较丰富,麻味物质含量最高,为13.65mg/g,但挥发油含量不是最高,为4.22mL/100g。
(5)青花椒挥发油共有峰中,萜烯类平均占共有峰的41.95%,醇类56.06%,酮类1.99%。红花椒挥发油共有峰中,萜烯类平均占共有峰的52.59%,醇类34.50%,酯类12.91%。
(6)青花椒挥发油经聚类分析后,16批样品聚为4类,这4类青花椒产地分别是四川洪雅藤椒基地、云南昭通、重庆、四川金阳；红花椒挥发油经聚类分析后,16批样品也聚为4类,分别是甘肃天水市秦安县郭家镇大油椒、甘肃天水市秦安县和四川阿坝州金川县、四川汶川县、四川汉源和凉山州。
(7)四川汉源红花椒相似度很好,基本都达到1,表明在汉源不同乡镇种植的花椒内在品质基本一致；而四川金阳青花椒品质差异性很大,相似度很低；江津青花椒除一批样品外,相似度都达0.97以上。
(8)红花椒和青花椒HPLC特征指纹图谱中,差异性特征峰初步推断为不饱和五烯酰胺,即羟基-γ-山椒素或2-羟基-N-异丁基-2,4,8,10,12-十四烷五烯酰胺及其同分异构体。
本文首次建立花椒挥发油GC-MS指纹图谱及花椒HPLC指纹图谱,方法精密度、重现性、稳定性好,方法可行,符合指纹图谱评价技术的要求：首次系统地建立了花椒中柠檬烯、芳樟醇、生物碱及五种重金属元素多个指标含量测定方法,并经过严密的方法学验证；首次将单个成分评价与指纹图谱技术评价方法相结合,充分挖掘指纹图谱数据信息,结合质谱鉴定技术和数据统计分析理论,对不同产地、不同品种、不同采收时期花椒的各指标含量、指纹图谱组分、含量的差异性进行了系统深入的比较分析,为花椒内在品质评价提供化学研究基础；首次采用高分辨率飞行时间质谱对分离纯化制备的P2晶体进行了鉴定,分子式为C_16H_5NO_2,应为羟基-a-山椒素或羟基-β-山椒素及其同分异构体；首次利用高分辨率质谱对红花椒和青花椒HPLC指纹图谱中差异性特征峰进行了鉴定,推断为不饱和五烯酰胺,即羟基-Y-山椒素或2-羟基-N-异丁基-2,4,8,10,12-十四烷五烯酰胺及其同分异构体。
本文受国家白然科学基金项目“基于近红外光谱的花椒品质快速检测机理及模型优化方法研究”(30671198)和重庆市重点自然科学基金项目“花椒和生姜中功效成分的组成和功能性质及定量检测方法研究”(CSTC,2006BA1007)”的资助。
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 C_(16)H_(25)NO_2, 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 (R~2=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 C_(16)H_(25)NO_2, 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.
引文
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