桃仁的质量标准研究
摘要
桃仁为蔷薇科植物桃Prunus persica (L.) Batsch或山桃Prunus davidiana (Carr.)Franch.的干燥成熟种子,果实成熟后采收,除去果肉和核壳,取出种子,晒干。性平味苦甘,归心、肝、大肠经。功能为活血祛瘀,润肠通便,止咳平喘,孕妇慎用。本品始载于《神农本草经》,现代研究表明桃仁对心脑血管疾病疗效显著,近年来受到广泛关注,但一直以来对桃仁的有效成分缺乏系统的研究,也没有具体的质量评价系统,致使桃仁药材市场流通处于混乱。故本试验采用TLC、HPLC以及指纹图谱法对采自山东、山西以及河北承德、安国等不同产地和不同品种的15份桃仁进行质量研究,为评价桃仁质量及临床合理用药提供科学依据。
目的:通过试验对不同产地和品种桃仁的研究,建立桃仁药材的薄层色谱、含量测定及指纹图谱的方法,为科学评价与有效控制桃仁的质量提供依据。
方法:1薄层色谱方法的建立(1)提取条件:考察不同的提取方法和提取溶剂对结果的影响,选用适宜的提取方法和提取溶剂。(2)展开条件:根据桃仁药材中主要指标成分化学性质的特点,确定适宜的固定相、展开系统、显色系统等。
2含量测定方法的建立(1)提取条件:考察不同提取方法、不同提取溶剂对提取桃仁药材中主要指标成分的影响,继而选取影响提取效率的三个主要因素:溶剂用量、提取时间、提取次数,采用正交设计对含量测定的前处理工艺进行优化,以选择提取效率较高的提取条件。(2)色谱条件:选择适宜的固定相,调整流动相的组成、配比、流速、进样量,调节柱温以使苦杏仁苷色谱峰与其它杂质峰完全分离。(3)系统适用性试验:在已确定的色谱条件下,考察苦杏仁苷色谱峰的理论板数、分离度。(4)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。(5)精密度试验:取同一份对照品溶液和供试品溶液,分别重复进样6次,测定峰面积。(6)重复性试验:取同一批桃仁药材6份,制备供试品溶液,分别进样,测定峰面积。(7)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。(8)回收率试验:取已知苦杏仁苷含量的桃仁药材适量,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。(9)最低限检测的测定: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。(10)样品测定:在上述色谱条件下,测定15批桃仁药材中苦杏仁苷的含量。
3指纹图谱方法的建立(1)提取条件:比较不同提取溶剂、不同提取方法及不同提取时间的提取效果,选择提取成分较多,提取率较高的提取条件。(2)色谱条件:选用合适的色谱柱、检测器及检测波长,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,考察苦杏仁苷色谱峰的理论板数、分离度。(4)精密度试验:制备样品溶液1份,重复进样6次,记录保留时间和峰面积,考察试验方法的精密度。(5)重复性试验:平行制备样品溶液6份,分别进样,记录保留时间和峰面积,考察试验方法的重复性。(6)稳定性试验:制备样品溶液1份,分别放置0,4,8,12,24,48 h后进样,记录保留时间和峰面积,考察试验方法的稳定性。(7)指纹图谱的建立:分别取同一品种不同产地桃仁药材10批,其他不同品种的桃仁药材5批,制备供试品溶液,进行指纹图谱分析,相似度评价。
结果:1薄层色谱方法:(1)提取方法:样品加甲醇10 ml,超声处理30 min,离心,取上清液作为供试品溶液。(2)展开条件:以三氯甲烷-乙酸乙酯-甲醇-水(15:40:22:10)5-10℃放置12 h的下层溶液为展开剂,展开,取出,挥干溶剂后以0.8%磷钼酸的15%硫酸乙醇溶液(现用现配)浸板,在105℃加热至斑点显色清晰,供试品色谱中,在与对照品色谱相应的位置上,显相同颜色的斑点。
2含量测定方法:(1)提取条件:确定了对桃仁进行含量测定的前处理工艺: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);以甲醇～水(15:85)为流动相;流速1.0 ml/min;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分别为0.71%、1.22%。(5)重复性试验:样品的重复性良好,RSD为0.53%。(6)稳定性试验:对照品和样品在48 h内均稳定,RSD分别为1.44%、0.96%。(7)回收率试验:样品平均回收率为99.57%,RSD为2.50%。(8)苦杏仁苷的最低检测限为40.1032 ng(。9)桃仁药材中苦杏仁苷的含量为2.332%～3.532%。3指纹图谱方法:(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 mm×250 mm,5μm);乙腈～水为流动相进行梯度洗脱,流速为1.0 ml/min,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,理论板数按苦杏仁苷峰计算约为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值分别为0.024%～0.137%和0.278%～1.621%,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0.069%～0.573%和1.075%～2.269%,重复性良好。(6)稳定性试验: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0.004%～0.245%和1.075%～1.951%,稳定性良好。(7)指纹图谱的建立:得到桃仁药材的指纹图谱及15个共有峰,同时得到不同品种来源桃仁药材的指纹图谱。(8)数据分析:运用“中药色谱指纹图谱相似度评价系统”2004年A版(国家药典委员会开发)对所得数据进行分析,所得结果能反映桃仁药材的质量。
结论:1薄层色谱:该方法显现斑点清晰,重复性和稳定性良好,能够更好的作为桃仁的定性鉴别方法。
2含量测定:以苦杏仁苷含量为其主要质量指标,建立了该药材定量分析方法,该方法稳定性,重复性和精密度良好,为科学评价与有效控制桃仁的质量提供了科学依据。
3指纹图谱:通过建立桃仁的HPLC指纹图谱,得到特征对照图谱,计算相似度,该方法重复性和精密度良好,有助于桃仁的质量控制,同时为桃仁药材的鉴别提供了新的依据。
Persicae Semen, dry mature seed of Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch, which is root in the family of Rosaceae. Mature fruit is harvested, pulp and nucleocapsid is removed, Taking out seeds and drying.The nature is calm and the taste is bitter and sweet which is ascriptioned the meridian of heart, liver and colon.It has many effects, such as removaling the stasis of blood, lubricating intestinal, relieving constipation, suppressing cough and relieving constipation. Pregnant using it shoud with caution. This product is contained in the“Shen Nong's Materia Medica”, Modern research shows that Persicae Semen effects on the cardio-cerebral vascular disease significantly, which is received extensive attention in recent years. But systematic research on Active ingredients of Persicae Semen is penurious, and there is no specific quality evaluation system, which leding the circulation of Persicae Semen medicine market in disarray. Because the main ingredients of Persicae Semen is amygdalin, this study choose amygdalin as the major components, Respectively using TLC, HPLC as well as the fingerprint method, the content of amygdalin of the 15 samples which is taken from Shandong, Shanxi, and Hebei Chengde, anguo different origin and different varieties of Persicae Semen is qualitative and quantitative Studied, providing a scientific basis for evaluating the quality of Persicae Semen and rational drug using.
Objective: To establish a method of TLC, a method to detect the content of amygdalin in Persicae Semen and a method of fingerprint through experiments on different batches of Persicae Semen,Which providing a basis for scientific evaluation and effective control of Persicae Semen quality.
Methods: 1 The establishment of TLC: (1) Extracted conditions: Investigating the effects of different extraction methods and extraction solvents on the results, and selecting the best appropriate extraction method and extraction solvent. (2) The selection of development conditions: According to the chemistry of main indicators composition from Persicae Semen, determing the appropriate stationary phase, expand System, color systems.
2 The establishment of content determination by HPLC: (1) Extraction: to examine the influence of different extractions and different extraction solvents on distilling the effective ingredient from Persicae Semen by optimizing the beforehand handling procedure of quality control through experiment which focuses 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: Choose appropriate fixed phase, adjust different formulation and proportion of mobile phase , regulate flow rate,the injection volume and column temperature in order to separate the peak of amygdalin from others well. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Preparation of standard curve: Prepare a series of the reference solution, examine peak area, then the regress equation was obtained with the content of amygdalin as abscissa, the relevant peak area as ordinate. (5) In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant peak area of amygdalin, and then calculate the precision. (6) In the experiment of recovery, transfer 6 shares of Persicae Semen and add the reference solutions of amygdalin on same levels, detect the relevant peak area, then calculate the content of amygdalin and the RSD. (7) In the experiment of stability, transfer the sample solution from Semen Armeniacae Amarum to determine the relevant peak area at 0, 4, 8, 12, 24 and 48 h, then calculate the content and RSD. (8) In the experiment of recovery, transfer 9 shares of Persicae Semen and add the reference solutions of amygdalin on three levels and every level repeat three times, detect the relevant peak area , then calculate the content of amygdalin. (9) Determination of the lowest detection limitation: Dilute the reference solution of amygdalin until the value of S/N was more than or equal to 3. The relevant concentration was the lowest detection limitation. (10) Assay: Under above-mentioned conditions, determine the content of amygdalin in different batches of Persicae Semen.
3 Establishment of fingerprint: (1) Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Precision test: In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant retention time and peak area, respectively. (5) Reproducibility test: In the experiment of reproducibility, prepare one concentration sample of Persicae Semen, repeat each concentration solution for six times in the same way, determine the relevant retention time and peak area, respectively. (6) Specificity test: In the experiment of stability, transfer the sample solution from Persicae Semen to determine the relevant retention time and peak area at 0, 4, 8, 12, 24 and 48 h, respectively. (7) Development of fingerprints: the test solution was prepared from different producing areas and different varieties and then their relevant fingerprint chromatograms were obtained.
Results: 1 The method of TLC: (1) Extraction: put 10ml of methanol in to the sample, and ultrasonic treated 30 min, take supernatant solution as for the solution for the test product. (2) Development condition: Take the lower solution of chloroform-ethyl-methanol-water (15:40:22:10) which is placed 12 hours on 5-10℃as for development solvent, developing,taking it out and drying, putting it in to the solution of 0.8% phosphomolybdate acid of 15% sulfate ethanol, heating and waiting the spot clearly on 105℃, 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 The method of content determination by HPLC: (1) Extraction: The experiment ascertains that the beforehand handling procedure of quality analysis of Persicae Semen is the ultrasonic extraction of the mixture of fifty portion of methanol and one portion of Persicae Semen, which are performed once and lasts for 30 minutes. (2) Chromatogram conditions: The HPLC system was performed on a C18 analytical column gradient eluted with a mixture consisting of methanol-water (15:85) at a flow rate of 1.0 ml/min.The temperature of column was 30℃. The UV detection wavelength was set at 210 nm. 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) The draw 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 1.22%. (5) Reproducibility: The RSD value of repeatability was 0.53%. (6) Stability: The control solution and test solution were sTable in 48 h and the RSD values of amygdalin were 1.44% and 0.96%. (7) Recovery: The average recoveries of amygdalin was 99.57% and the RSD value was 2.50%. (8) The lowest detection limitation was 40.1032 ng. (9) The results showed the contents of amygdalin in Persicae Semen of different sources were between 2.332%-3.532%.
3 The method of fingerprint chromatography: (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.The temperature of column was 30℃. The UV detection wavelength was set at 225 nm. 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 0.024%-0.137% and between0.278%-1.621%, respectively. (5) The reproducibility of sample was good and the RSD values of relative retention time and relative area were between0.069%-0.573% and between1.075%-2.269 %, respectively. (6) The test solution was sTable in 48 h and the relevant RSD values of relative retention time and relative area were between 0.004%-0.245% and between1.075%-1.951%, respectively. (7) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 15 batches of Persicae Semen were got. In addition, the fingerprint chromatograms of different varieties were also obtained. (8) Data analysis: similarity clustering analysis was performed and the result can significantly reflect the quality of Persicae Semen from different producing areas and different varieties.
Conclusion:1 TLC: This method revealed a clear spots, repeatability and stability is well, It is the better qualitative identification method to be used to differentiate Persicae Semen.
2 Content Determination: this study choose the content of amygdalin as the quality Index, the HPLC method was established to determine concentration of amygdalin in Persicae Semen. The method was found to be accurate, sensitive, quick and sTable It provides a scientific basis for scientific evaluation and effective control of the quality of Persicae Semen.
3 FingerprintChromatogram: Getting feature comparison Chromatogram though the establishment of the HPLC fingerprint of Persicae Semen.then calculation the similarity. The method was found to be accurate, repetitive.It contributes to the quality control of Persicae Semen, And provides a new basis to the identification of Persicae Semen at the same time.
目的:通过试验对不同产地和品种桃仁的研究,建立桃仁药材的薄层色谱、含量测定及指纹图谱的方法,为科学评价与有效控制桃仁的质量提供依据。
方法:1薄层色谱方法的建立(1)提取条件:考察不同的提取方法和提取溶剂对结果的影响,选用适宜的提取方法和提取溶剂。(2)展开条件:根据桃仁药材中主要指标成分化学性质的特点,确定适宜的固定相、展开系统、显色系统等。
2含量测定方法的建立(1)提取条件:考察不同提取方法、不同提取溶剂对提取桃仁药材中主要指标成分的影响,继而选取影响提取效率的三个主要因素:溶剂用量、提取时间、提取次数,采用正交设计对含量测定的前处理工艺进行优化,以选择提取效率较高的提取条件。(2)色谱条件:选择适宜的固定相,调整流动相的组成、配比、流速、进样量,调节柱温以使苦杏仁苷色谱峰与其它杂质峰完全分离。(3)系统适用性试验:在已确定的色谱条件下,考察苦杏仁苷色谱峰的理论板数、分离度。(4)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。(5)精密度试验:取同一份对照品溶液和供试品溶液,分别重复进样6次,测定峰面积。(6)重复性试验:取同一批桃仁药材6份,制备供试品溶液,分别进样,测定峰面积。(7)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。(8)回收率试验:取已知苦杏仁苷含量的桃仁药材适量,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。(9)最低限检测的测定: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。(10)样品测定:在上述色谱条件下,测定15批桃仁药材中苦杏仁苷的含量。
3指纹图谱方法的建立(1)提取条件:比较不同提取溶剂、不同提取方法及不同提取时间的提取效果,选择提取成分较多,提取率较高的提取条件。(2)色谱条件:选用合适的色谱柱、检测器及检测波长,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,考察苦杏仁苷色谱峰的理论板数、分离度。(4)精密度试验:制备样品溶液1份,重复进样6次,记录保留时间和峰面积,考察试验方法的精密度。(5)重复性试验:平行制备样品溶液6份,分别进样,记录保留时间和峰面积,考察试验方法的重复性。(6)稳定性试验:制备样品溶液1份,分别放置0,4,8,12,24,48 h后进样,记录保留时间和峰面积,考察试验方法的稳定性。(7)指纹图谱的建立:分别取同一品种不同产地桃仁药材10批,其他不同品种的桃仁药材5批,制备供试品溶液,进行指纹图谱分析,相似度评价。
结果:1薄层色谱方法:(1)提取方法:样品加甲醇10 ml,超声处理30 min,离心,取上清液作为供试品溶液。(2)展开条件:以三氯甲烷-乙酸乙酯-甲醇-水(15:40:22:10)5-10℃放置12 h的下层溶液为展开剂,展开,取出,挥干溶剂后以0.8%磷钼酸的15%硫酸乙醇溶液(现用现配)浸板,在105℃加热至斑点显色清晰,供试品色谱中,在与对照品色谱相应的位置上,显相同颜色的斑点。
2含量测定方法:(1)提取条件:确定了对桃仁进行含量测定的前处理工艺: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);以甲醇～水(15:85)为流动相;流速1.0 ml/min;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分别为0.71%、1.22%。(5)重复性试验:样品的重复性良好,RSD为0.53%。(6)稳定性试验:对照品和样品在48 h内均稳定,RSD分别为1.44%、0.96%。(7)回收率试验:样品平均回收率为99.57%,RSD为2.50%。(8)苦杏仁苷的最低检测限为40.1032 ng(。9)桃仁药材中苦杏仁苷的含量为2.332%～3.532%。3指纹图谱方法:(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 mm×250 mm,5μm);乙腈～水为流动相进行梯度洗脱,流速为1.0 ml/min,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,理论板数按苦杏仁苷峰计算约为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值分别为0.024%～0.137%和0.278%～1.621%,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0.069%～0.573%和1.075%～2.269%,重复性良好。(6)稳定性试验: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0.004%～0.245%和1.075%～1.951%,稳定性良好。(7)指纹图谱的建立:得到桃仁药材的指纹图谱及15个共有峰,同时得到不同品种来源桃仁药材的指纹图谱。(8)数据分析:运用“中药色谱指纹图谱相似度评价系统”2004年A版(国家药典委员会开发)对所得数据进行分析,所得结果能反映桃仁药材的质量。
结论:1薄层色谱:该方法显现斑点清晰,重复性和稳定性良好,能够更好的作为桃仁的定性鉴别方法。
2含量测定:以苦杏仁苷含量为其主要质量指标,建立了该药材定量分析方法,该方法稳定性,重复性和精密度良好,为科学评价与有效控制桃仁的质量提供了科学依据。
3指纹图谱:通过建立桃仁的HPLC指纹图谱,得到特征对照图谱,计算相似度,该方法重复性和精密度良好,有助于桃仁的质量控制,同时为桃仁药材的鉴别提供了新的依据。
Persicae Semen, dry mature seed of Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch, which is root in the family of Rosaceae. Mature fruit is harvested, pulp and nucleocapsid is removed, Taking out seeds and drying.The nature is calm and the taste is bitter and sweet which is ascriptioned the meridian of heart, liver and colon.It has many effects, such as removaling the stasis of blood, lubricating intestinal, relieving constipation, suppressing cough and relieving constipation. Pregnant using it shoud with caution. This product is contained in the“Shen Nong's Materia Medica”, Modern research shows that Persicae Semen effects on the cardio-cerebral vascular disease significantly, which is received extensive attention in recent years. But systematic research on Active ingredients of Persicae Semen is penurious, and there is no specific quality evaluation system, which leding the circulation of Persicae Semen medicine market in disarray. Because the main ingredients of Persicae Semen is amygdalin, this study choose amygdalin as the major components, Respectively using TLC, HPLC as well as the fingerprint method, the content of amygdalin of the 15 samples which is taken from Shandong, Shanxi, and Hebei Chengde, anguo different origin and different varieties of Persicae Semen is qualitative and quantitative Studied, providing a scientific basis for evaluating the quality of Persicae Semen and rational drug using.
Objective: To establish a method of TLC, a method to detect the content of amygdalin in Persicae Semen and a method of fingerprint through experiments on different batches of Persicae Semen,Which providing a basis for scientific evaluation and effective control of Persicae Semen quality.
Methods: 1 The establishment of TLC: (1) Extracted conditions: Investigating the effects of different extraction methods and extraction solvents on the results, and selecting the best appropriate extraction method and extraction solvent. (2) The selection of development conditions: According to the chemistry of main indicators composition from Persicae Semen, determing the appropriate stationary phase, expand System, color systems.
2 The establishment of content determination by HPLC: (1) Extraction: to examine the influence of different extractions and different extraction solvents on distilling the effective ingredient from Persicae Semen by optimizing the beforehand handling procedure of quality control through experiment which focuses 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: Choose appropriate fixed phase, adjust different formulation and proportion of mobile phase , regulate flow rate,the injection volume and column temperature in order to separate the peak of amygdalin from others well. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Preparation of standard curve: Prepare a series of the reference solution, examine peak area, then the regress equation was obtained with the content of amygdalin as abscissa, the relevant peak area as ordinate. (5) In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant peak area of amygdalin, and then calculate the precision. (6) In the experiment of recovery, transfer 6 shares of Persicae Semen and add the reference solutions of amygdalin on same levels, detect the relevant peak area, then calculate the content of amygdalin and the RSD. (7) In the experiment of stability, transfer the sample solution from Semen Armeniacae Amarum to determine the relevant peak area at 0, 4, 8, 12, 24 and 48 h, then calculate the content and RSD. (8) In the experiment of recovery, transfer 9 shares of Persicae Semen and add the reference solutions of amygdalin on three levels and every level repeat three times, detect the relevant peak area , then calculate the content of amygdalin. (9) Determination of the lowest detection limitation: Dilute the reference solution of amygdalin until the value of S/N was more than or equal to 3. The relevant concentration was the lowest detection limitation. (10) Assay: Under above-mentioned conditions, determine the content of amygdalin in different batches of Persicae Semen.
3 Establishment of fingerprint: (1) Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Precision test: In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant retention time and peak area, respectively. (5) Reproducibility test: In the experiment of reproducibility, prepare one concentration sample of Persicae Semen, repeat each concentration solution for six times in the same way, determine the relevant retention time and peak area, respectively. (6) Specificity test: In the experiment of stability, transfer the sample solution from Persicae Semen to determine the relevant retention time and peak area at 0, 4, 8, 12, 24 and 48 h, respectively. (7) Development of fingerprints: the test solution was prepared from different producing areas and different varieties and then their relevant fingerprint chromatograms were obtained.
Results: 1 The method of TLC: (1) Extraction: put 10ml of methanol in to the sample, and ultrasonic treated 30 min, take supernatant solution as for the solution for the test product. (2) Development condition: Take the lower solution of chloroform-ethyl-methanol-water (15:40:22:10) which is placed 12 hours on 5-10℃as for development solvent, developing,taking it out and drying, putting it in to the solution of 0.8% phosphomolybdate acid of 15% sulfate ethanol, heating and waiting the spot clearly on 105℃, 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 The method of content determination by HPLC: (1) Extraction: The experiment ascertains that the beforehand handling procedure of quality analysis of Persicae Semen is the ultrasonic extraction of the mixture of fifty portion of methanol and one portion of Persicae Semen, which are performed once and lasts for 30 minutes. (2) Chromatogram conditions: The HPLC system was performed on a C18 analytical column gradient eluted with a mixture consisting of methanol-water (15:85) at a flow rate of 1.0 ml/min.The temperature of column was 30℃. The UV detection wavelength was set at 210 nm. 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) The draw 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 1.22%. (5) Reproducibility: The RSD value of repeatability was 0.53%. (6) Stability: The control solution and test solution were sTable in 48 h and the RSD values of amygdalin were 1.44% and 0.96%. (7) Recovery: The average recoveries of amygdalin was 99.57% and the RSD value was 2.50%. (8) The lowest detection limitation was 40.1032 ng. (9) The results showed the contents of amygdalin in Persicae Semen of different sources were between 2.332%-3.532%.
3 The method of fingerprint chromatography: (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.The temperature of column was 30℃. The UV detection wavelength was set at 225 nm. 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 0.024%-0.137% and between0.278%-1.621%, respectively. (5) The reproducibility of sample was good and the RSD values of relative retention time and relative area were between0.069%-0.573% and between1.075%-2.269 %, respectively. (6) The test solution was sTable in 48 h and the relevant RSD values of relative retention time and relative area were between 0.004%-0.245% and between1.075%-1.951%, respectively. (7) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 15 batches of Persicae Semen were got. In addition, the fingerprint chromatograms of different varieties were also obtained. (8) Data analysis: similarity clustering analysis was performed and the result can significantly reflect the quality of Persicae Semen from different producing areas and different varieties.
Conclusion:1 TLC: This method revealed a clear spots, repeatability and stability is well, It is the better qualitative identification method to be used to differentiate Persicae Semen.
2 Content Determination: this study choose the content of amygdalin as the quality Index, the HPLC method was established to determine concentration of amygdalin in Persicae Semen. The method was found to be accurate, sensitive, quick and sTable It provides a scientific basis for scientific evaluation and effective control of the quality of Persicae Semen.
3 FingerprintChromatogram: Getting feature comparison Chromatogram though the establishment of the HPLC fingerprint of Persicae Semen.then calculation the similarity. The method was found to be accurate, repetitive.It contributes to the quality control of Persicae Semen, And provides a new basis to the identification of Persicae Semen at the same time.
引文
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3颜永刚,裴瑾,万德光.桃仁脂肪酸GC-MS指纹图谱研究.中草药,2008,39(9):1415-1416
4袁丹,胡爽,杜慧琴,等.橘红丸中苦杏仁苷的定性、定量分析方法的研究,中国药学杂志,2002,37(11):857-860
5易俊.三种不同产地的桃仁生药学研究.福建教育学院学报,2000,2:67-69
6戴敬,段吉平.中华人民共和国药典中药材薄层色谱彩色图集第二册.人民卫生出版社,2009.1:445-447
1袁丹,胡爽,毕开顺,等.桃仁药材质量评价法的研究.中国药学杂志,2003,38(1):53
2朱友平,顾冠彬,苏中武,等.杏仁和桃仁的电泳鉴别研究,中药通报,1988,13(8):6
3张贵军.常用中药鉴定大全.黑龙江科学技术出版社,660
4王道芳.浅述桃仁与苦杏仁的药理及临床应用.基层中药杂志,2002,16(6):61
5王友兰,李红兵,华玉琴.HPLC法测定桃仁中苦杏仁苷的含量. China Pharmacist,2002,5(9):550
6林小明,姚燕平.正交实验优选桃仁煎煮工艺的研究.时珍国医国药,2006,17(4):597
1范丽芳.河北道地药材色谱指纹图谱研究.34
2谢培山.中药质量控制模式的发展趋势.中药新药与临床药理,2001,12(3):188-191
3姜波,沙吾列,范圣第.桃仁油中脂肪酸的GC-MS分析.中国油脂,2008,33(11):71
4袁丹,胡爽,毕开顺,等.桃仁药材质量评价法的研究.中国药学杂志,2003,38(1):53
5林凌,郑梅,邓思珊,等.生桃仁中苦杏仁甙提取工艺研究.海峡药学,2002,14(3):42
1有地滋,永本典生.第四回天然药物の开发と应用,シンポジウム要旨集,1982.7
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