【作者】 崔淑艳；

【导师】 郑玉光；

【作者基本信息】 河北医科大学 ， 中药学， 2010， 硕士

【摘要】 苦杏仁为多基源品种,《中华人民共和国药典》2005版一部规定苦杏仁来源于蔷薇科（Rosaceae）植物山杏（Prunus armeniaca L. Var. ansu Maxim.）、西伯利亚杏（Prunus sibirica L.）、东北杏（Prunus mandshurica （Maxim.） Koehne）或杏（Prunus armeniaca L.）的干燥成熟种子。主要有效成分为苦杏仁苷和脂肪油,具有降气、止咳、平喘、润肠通便之功效。由于其来源复杂,分布广泛,加之栽培及采收加工欠规范,常导致饮片质量不稳定,亦无有效的质量评价方法,致使其质量控制一直比较困难。据调查,目前市场上流通的作为苦杏仁入药的植物种子主要有6种,除山杏、西伯利亚杏、东北杏、杏与药典一致外,其余两种紫杏（Prunus armeniaca dasycarpa （Ehrh.） Borkh）、毛杏（Prunus armeniaca （L.） Lam. var. pubescens Kost）均未收入药典。本课题主要从薄层鉴别、含量测定和HPLC指纹图谱三个方面对上述苦杏仁进行系统的品种整理和质量研究,制定苦杏仁的质量评价方法。目的:通过薄层色谱法对苦杏仁药材进行定性鉴别,并采用高效液相色谱法测定苦杏仁中苦杏仁苷的含量,从而建立一种快速、高效的质量控制方法。同时建立苦杏仁药材的HPLC指纹图谱,获得对照图谱,并与不同品种苦杏仁药材指纹特征相对照,为科学评价和有效控制苦杏仁质量提供科学依据。方法:1薄层鉴别研究:采用薄层色谱法,选用合适的提取方法和提取溶剂,以及适宜的固定相、展开系统、显色系统,对苦杏仁进行定性鉴别。2苦杏仁的含量测定:（1）提取:考察不同提取方法、不同提取溶剂对提取苦杏仁药材中有效成分的影响,选取影响提取效率的溶剂用量、提取时间、提取次数三个主要因素,采用正交试验对质量分析测定的前处理工艺进行优化,选择提取效率较高的提取条件。（2）色谱条件:选择合适的固定相,调整流动相的组成、配比、流速,调节柱温,以使苦杏仁苷色谱峰与杂质峰分离度较好。（3）标准曲线的绘制:配制系列浓度的苦杏仁苷对照品溶液,分别进样,测定峰面积,以苦杏仁苷进样量为横坐标,相应峰面积为纵坐标,绘制标准曲线。（4）精密度试验:分别取同一份对照品溶液和供试品溶液,重复进样6次,测定峰面积,并计算RSD值。（5）重复性试验:取同一批苦杏仁药材6份,制备供试品溶液,分别进样,测定峰面积,并计算RSD值。（6）稳定性试验:分别取同一份对照品溶液和供试品溶液,分别于放置0,4,8,12,24,48 h后进样,测定峰面积,并计算RSD值。（7）回收率试验:取苦杏仁药材适量,分别加入等量的苦杏仁苷对照品,测定苦杏仁苷的含量,计算平均回收率和RSD值。（8）检测限的测定:将苦杏仁苷对照品溶液逐步稀释,当信噪比S/N≥3时确定为最低检测限。（9）样品测定:在上述色谱条件下,测定18批苦杏仁药材中苦杏仁苷的含量。3苦杏仁指纹图谱的建立:（1）提取:比较了不同提取方法、不同提取溶剂及不同提取时间的提取效果,选择提取成分较多,提取效率较高的提取条件。（2）色谱条件:选用合适的色谱柱及检测波长,调整流动相的组成、配比、流速,调节柱温,使色谱图符合指纹图谱研究的要求。（3）系统适用性试验:在已确定的色谱条件下,考察指纹图谱中苦杏仁苷峰的分离度和理论板数。（4）精密度试验:取同一份样品溶液,重复进样6次,记录保留时间和峰面积。（5）重复性试验:取同一批苦杏仁药材6份,平行制备样品溶液,进样,记录保留时间和峰面积。（6）稳定性试验:取样品溶液分别于0,4,8,12,24,48 h进样,记录保留时间和峰面积。（7）指纹图谱的建立:分别取同一品种不同产地苦杏仁药材10批,其他不同品种的苦杏仁药材8批,制备供试品溶液,进行指纹图谱分析。结果:1苦杏仁的薄层鉴别的研究:采用相应的薄层鉴别方法,薄层展开后,在供试品色谱中,与对照品色谱相应的位置上显相同颜色的斑点。2苦杏仁的主要成分苦杏仁苷的含量测定:（1）提取:确定了对苦杏仁中的苦杏仁苷进行含量测定的处理工艺:50倍甲醇超声提取一次,提取时间为30 min。（2）色谱条件:采用安捷伦C18色谱柱（4.6 mm×250 mm,5μm）;甲醇-水（15:85）为流动相;流速1.0 ml·min-1;柱温30℃;检测波长为210 nm;进样量10μl。在此色谱条件下,苦杏仁苷峰保留时间约为12 min,分离度大于1.5,理论塔板数不低于4000。（3）标准曲线的绘制:苦杏仁苷在0.138-13.8μg范围内,线性关系良好。回归方程为Y=513.57X+20.704,r=0.9999（n=6）（4）精密度试验:仪器和方法的精密度良好,RSD值分别为0.71%、0.78%。（5）重复性试验:样品的重复性好,RSD值为0.76%（6）稳定性试验:对照品溶液和样品溶液在48 h内稳定,RSD分别为1.44%、1.25%。（7）回收率试验:样品平均回收率为99.51%,RSD为1.81%。（8）苦杏仁苷的最低检测限为40.1032 ng。（9）苦杏仁药材中苦杏仁苷的含量为0.0724%-5.668%。3苦杏仁指纹图谱的建立:（1）提取:以70%甲醇超声60 min为佳,具有稳定和效率高等优点。（2）色谱条件:色谱柱为Waters Symmetry C18柱（4.6×250 mm,5μm）;乙腈-水为流动相进行梯度洗脱;流速为1.0 ml·min^-1;柱温为30℃;检测波长为225 nm;进样量10μl。（3）系统适用性试验:在此色谱条件下,苦杏仁苷的保留时间约为20 min,按苦杏仁苷峰计算理论板数约为50000,与其它色谱峰的分离度大于1.0。（4）精密度试验:以苦杏仁苷峰为内参比峰,计算各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积值,其RSD分别为0.009%-0.26%和1.01%-2.43%,精密度良好。（5）重复性试验:各主要色谱峰相对保留时间和占总峰面积5%以上色谱峰的相对峰面积均无明显变化,其RSD分别为0.009%-1.04%和1.76%-2.59%,重复性良好。（6）稳定性试验:在48 h内,各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积比值均无明显变化,其RSD分别为0.009%-0.32%和1.07%-2.35%,稳定性良好。（7）指纹图谱的建立:得到苦杏仁药材的指纹图谱,生成对照指纹图谱,同时得到不同品种苦杏仁药材的指纹图谱。（8）数据分析:运用相似度评价系统2004A版软件对所得数据进行分析,所得结果能反映苦杏仁药材的质量。结论:1薄层色谱法所得斑点清晰圆整,专属性强,重复性好,可用于苦杏仁的定性鉴别。2建立了苦杏仁样品中苦杏仁苷含量测定的HPLC法,方法专属性强,稳定性,重复性和精密度良好。3建立了不同产地同一品种来源苦杏仁药材的HPLC指纹图谱,得到对照图谱,并与不同品种苦杏仁药材指纹特征相比较,利用相似度分析对不同产地和品种的苦杏仁间的差异进行了研究,为科学评价与有效控制苦杏仁药材的质量提供了科学依据。更多还原

【Abstract】 Semen Armeniacae Amarum, the semen of Prunus armeniaca L. Var. ansu. Maxim., Prunus. sibirica. L., Prunus.mandshurica (Maxim.) Koehne, or Prunus armeniaca L., is multi-origin variety in the family of Rosaceae. It is reported that amygdalin and fatty acid are the main bioactive components of this drug, as a result, many effects, such as repessing upward perversion of Qi, suppressing cough, relieving dyspnea and relieving constipation with laxatives, have been proved thoutsand years ago. Their complex origin, wide distribution, unstandadd cultivation and elaboration lead to its unsTable quality, and there is no effective method for its quality evaluation, for this reason, it is difficult for people to control its quality. According to the researches, besides the ones which have been recorded in the Chinese Pharmacopoeia (2005 Edition) , there are also some varieties used as Semen Armeniacae Amarum in the market, like Prunus armeniaca dasycarpa (Ehrh.) Borkh and Prunus armeniaca (L.) Lam. var. pubescens Kost. In this study, the methods of thin-layer chromatography, content determination of Amygdalin and HPLC fingerprint were used to study the variety and quality control of Semen Armeniacae Amarum, and its quality evaluation method was also established.Objective:To establish a TLC method for qualitative identification, and a method to determine the contents of amygdalin in Semen Armeniacae Amarum, which can provide fast and efficient guideline to identify Semen Armeniacae Amarum. Besides, to establish HPLC fingerprints of Semen Armeniacae Amarum and get reference fingerprint, to compare the fingerprints of Semen Armeniacae Amarum collected from different varieties, so as to establish a scientific method to control the quality of Semen Armeniacae Amarum.Methods:1 Studies on TLC of Semen Armeniacae Amarum: By selecting suiTable extraction solvent, extraction approach, eligible fixed phase, development system, and colouration system, Semen Armeniacae Amarum was identified with TLC.2 Determination of amygdalin in Semen Armeniacae Amarum by HPLC: (1) Extraction: To examine the influence of different extractions and different extraction solvents on distilling the effective ingredient from Semen Armeniacae Amarum, and to optimize the beforehand handling procedure of quality control, the orthogonal experiment was performed, which focused on three major factors affecting the efficiency of extraction, including the rates of solvent and medicine material, extraction time and extraction times. (2) Chromatogram conditions: The appropriate fixed phase, the different formulation and proportion of mobile phase, the flow rate and the column temperature were studied to make the separation of the peak for amygdalin better. (3) Preparation of standard curve: Different concentrations of reference solution were prepared, and the peak area was examined, then the regress equation was obtained with the content of amygdalin as abscissa, the relevant peak area as ordinate. (4) Precision: The same test solution was determined for six times, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (5) Reproducibility: The test samples of Semen Armeniacae Amarum were prepeared for six times used the same group of this drugs in the same way, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (6) Stability: The same test solution was determined at 0, 4, 8, 12, 24 and 48 h, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (7) Recovery: Six shares of Semen Armeniacae Amarum were took, and the same amount of reference substance was added respectively, then the test samples were prepeared in the same way, and the relevant peak areas of amygdalin was recorded to calculate the RSD value. (8) Determination of the lowest detection limitation: The reference solution of amygdalin was diluted until the value of S/N was more than or equal to 3. The relevant concentration was the lowest detection limitation. (9) Assay: Under above-mentioned conditions, the content of amygdalin in different batches of Semen Armeniacae Amarum was determined.3 Establishment of fingerprint: (1) Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: The appropriate column, the different formulation and proportion of mobile phase, the flow rate and the column temperature were studied to make the separation of the peak for amygdalin better. (3) System suitability test: The separation and theoretical plate of amygdalin peak were calculated in the chromatographic condition. (4) Precision: The same test solution was determined for six times, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (5) Reproducibility: The test samples of Semen Armeniacae Amarum were prepeared for six times used the same group of this drugs in the same way, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (6) Stability: The same test solution was determined at 0, 4, 8, 12, 24 and 48 h, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (7) Development of fingerprints: The samples of different producing areas and different varieties were prepared in the same way, and then their relevant fingerprint chromatograms were obtained.Results:1 Studies on TLC of Semen Armeniacae Amarum: TLC methods were established for the identification of Semen Armeniacae Amarum, the principal spot in the chromatogram obtained from the test solution was similar in position, colour and intensity to the principal spot in the chromatogram obtained from the reference solution.2 Determination of amygdalin in Semen Armeniacae Amarum by HPLC: (1) Extraction: The experiment ascertained that the beforehand handling procedure of quality analysis of Semen Armeniacae Amarum was the ultrasonic extraction of the mixture of fifty portion of methanol and one portion of Semen Armeniacae Amarum, which were performed once and lasted for 30 minutes. (2) Chromatogram conditions: The HPLC system was performed on a C18 analytical column eluted with a mixture consisting of methanol-water (15:85) at a flow rate of 1.0 ml·min-1, the temperature of column was 30℃, the UV detection wavelength was 210 nm, and the injection volume was 10μl. Under the above condition, the peaks of samples were separated well with the resolution of not less than 1.5. The retention time is about 12 min. The theoretical plates were more than 4000. (3) Preparation of standard curve: The liner range for amygdalin was 0.138-13.8μg. Regression equation was Y=513.57X+20.704, r=0.9999 (n=6). (4) Precision: The precision of instrument and method were good and the RSD values of amygdalin were 0.71% and 0.78% respectively. (5) Reproducibility: The RSD value of repeatability was 0.76%. (6) Stability: The control solution and test solution were sTable in 48 h and the RSD values of amygdalin were 1.44% and 1.25% respectively. (7) Recovery: The average recovery of amygdalin was 99.51% and the RSD value was 1.81%. (8) The lowest detection limitation was 40.1032 ng. (9) The results showed the contents of amygdalin in Semen Armeniacae Amarum of different sources were between 0.0724%-5.668%.3 Establishment of fingerprint: (1) Extraction: The method of supersonic wave-extraction with 70% methanol for 60 min was simple, quick and sTable. (2) The HPLC system was performed on a Waters Symmetry-C18 analytical column gradient eluted with a mixture consisting of acetonitrile, water at a flow rate of 1.0 ml·min-1, the temperature of column was 30℃, the UV detection wavelength was set at 225 nm, and the injection volume was 10μl. (3) System suitability test: Under the above condition, the peak corresponding to amygdalin of the test solution was separated well with the resolution of more than 1.0 and about 50000 of theoretical plate. (4) The precision of sample was good, amygdalin peak as reference peak, the RSD values of relative retention time and relative area were between 0.009%-0.26% and between 1.01%-2.43%, respectively. (5) The reproducibility of sample was good and the RSD values of relative retention time and relative area were between 0.009%-1.04% and between 1.76%-2.59%, respectively. (6) The test solution was sTable in 48 h and the relevant RSD values of relative retention time and relative area were between 0.009%-1.04% and between 1.07%-2.59%, respectively. (7) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 10 batches of Semen Armeniacae Amarum were got. In addition, the fingerprint chromatograms of different varieties were also obtained. (8) Data analysis: Similarity clustering analysis was performed and the results could significantly reflect the qualities of Semen Armeniacae Amarum from different producing areas and different varieties.Conclusion:1 In identification, the relevant spots were clear and characteristic and could be used to differentiate Semen Armeniacae Amarum.2 The HPLC method was established to determine concentration of amygdalin in Semen Armeniacae Amarum. The method was found to be accurate, sensitive, quick and sTable.3 It was the first time to establish the HPLC fingerprint chromatogram of Semen Armeniacae Amarum, get reference fingerprint chromatogram. We compared the fingerprints of 8 samples which were from different varieties and studied their difference with similarity. This method provides a scientific basis for the scientific evaluation and utility control of the quality of Semen Armeniacae Amarum.更多还原