苹果花的质量控制方法研究

发布时间：2018-05-12 00:38

本文选题：苹果花 + 槲皮素　；参考：《河北医科大学》2017年硕士论文

【摘要】：苹果花来源于蔷薇科植物苹果Malus pumila Mill的干燥花。4-5月间采摘,阴干,放置干燥处贮存。苹果花性平,归脾、胃、肾经。苹果花茶有治疗神经痛,解毒,补血,明目,祛痘,美白的作用。经查阅,未找到有关苹果花的文献。目前市场上苹果花以花茶的形式销售,但未收载于任何质量标准中,其质量难以有效控制。因此,本试验对苹果花性状、显微鉴别、薄层鉴别、杂质、水分、总灰分、酸不溶性灰分、醇溶性浸出物(热浸法)等项目进行研究,并进行了有效成分根皮素的含量测定方法研究。在此基础上对8个省份收集到的12批苹果花进行了HPLC指纹图谱研究分析,可为苹果花的品种鉴别及质量标准制定提供一定的依据。目的:根据质量标准的制定方法要求,对各个项目进行研究,为苹果花的质量标准制定奠定基础。方法:1对8个省份收集到的12批苹果花样品进行性状鉴别、显微鉴别研究,测定12批样品中杂质、水分、总灰分、酸不溶性灰分、浸出物的含量。2薄层色谱方法的建立(1)提取条件:实验过程中摸索了不同的提取溶剂及提取方法,筛选最佳的提取溶剂和提取方法。(2)展开条件:根据苹果花成分的特点,确定合适的展开剂、固定相和显色剂等。3含量测定方法的建立(1)提取条件:考察不同提取方法、不同提取溶剂及其浓度、不同提取时间、不同浓度盐酸水解液、不同粉末粒度对提取苹果花中专属性有效成分根皮素的影响,选择根皮素提取率最高的提取条件。(2)色谱条件:筛选最佳检测波长,选择合适的固定相,调整流动相的组成、比例,选择槲皮素峰、根皮素峰、山柰素峰与相邻杂质峰分离度较好的色谱条件。(3)系统适用性试验:在上述色谱条件下,考察根皮素色谱峰的理论板数和分离度。(4)标准曲线:配制系列浓度的根皮素对照品溶液,分别进样,记录根皮素的峰面积,以根皮素浓度为横坐标,相应峰面积为纵坐标,绘制标准曲线。(5)精密度试验:分别取同一份对照品溶液与同一份供试品溶液,连续进样6次,测定根皮素的峰面积,计算RSD值。(6)重复性试验:精密称取同一批苹果花样品6份,配制供试品溶液,分别进样,测定根皮素的峰面积,计算RSD值。(7)稳定性试验:分别取同一份对照品溶液与同一份供试品溶液,分别于0、2、4、6、12、24、48h进样分析,测定根皮素峰面积,计算RSD值。(8)回收率试验:精密称取已知根皮素含量的苹果花适量,分别加入相当于样品中所含根皮素含量的80%、100%、120%的根皮素对照品溶液,配制供试品溶液,分别进样,测定根皮素的含量,计算其回收率和RSD值。(9)检测限与定量限的测定:将根皮素对照品溶液逐级稀释,当信噪比大于等于3时,为检测限;当信噪比大于等于10时,为定量限。(10)耐用性试验:采用三根不同品牌的色谱柱、两台不同品牌的高效液相色谱仪分别测定1批样品的含量,计算RSD值及RD值。(11)样品测定:在上述色谱条件下,测定12批苹果花中根皮素的含量。4指纹图谱方法的建立(1)提取条件和色谱条件的选择除检测波长为260nm外,其他条件同含量测定项下。(2)精密度试验:取同一份供试品溶液连续进样6次,记录保留时间和峰面积。(3)重复性试验:精密称取同一批苹果花6份,配制供试品溶液,分别进样,记录保留时间和峰面积。(4)稳定性试验:取同一份供试品溶液,分别在0、2、4、6、12、24、48h进样分析,记录保留时间和峰面积。(5)指纹图谱的建立:取不同省份来源的苹果花12个批次,配制供试品溶液,进行指纹图谱分析及相似度评价。结果:1确定了苹果花性状和粉末显微鉴别特征;杂质测定结果为1%~3%;水分测定结果为5.9%~7.9%;总灰分测定结果为6.2%~8.5%;酸不溶性灰分测定结果为0.6%~2.0%;醇溶性浸出物(热浸法)测定结果为34.9%~41.7%。2薄层色谱法(1)提取方法:取本品粉末0.5g,置具塞三角烧瓶中,加乙醇40m L,密塞,超声处理20分钟,放冷,摇匀,滤过。取续滤液20 m L,置250 m L三角烧瓶中,加乙醇20 m L,25%盐酸溶液10 m L,摇匀,置85℃水浴中加热回流30分钟,蒸干。残渣加乙醇4m L使溶解,作为供试品溶液。(2)展开条件:薄层板为硅胶G板,以甲苯-乙酸乙酯-甲酸(7∶2∶1)的上层溶液为展开剂,展开,取出,晾干,喷以3%三氯化铝乙醇溶液,在105℃加热约10分钟,置紫外光灯(365nm)下检视,供试品色谱中,在与对照样品色谱和对照品色谱相应的位置上,显相同颜色的荧光斑点。3含量测定方法(1)提取条件:取本品粉末(过四号筛)约0.2g,精密称定,置具塞三角烧瓶中,精密加入乙醇20m L,密塞,称定重量,超声处理20分钟,放冷,再称定重量,用乙醇补足减失的重量,摇匀,滤过,精密量取续滤液10m L,置100m L三角烧瓶中,加乙醇10m L,25%盐酸溶液5m L,摇匀,置85℃水浴中加热回流1小时,冷却至室温,转移至50m L量瓶中,用乙醇稀释至刻度,摇匀,滤过,即得。(2)色谱条件:采用YMC ODS-A C18(250 mm*4.6 mm,5μm)色谱柱;流动相:甲醇-0.4%磷酸溶液(50:50);检测波长为286nm;流速1.0m L·min-1;柱温为30℃,进样量为10μL。(3)系统适用性试验:在此色谱条件下,理论板数按根皮素峰计算应不低于5000,分离度大于1.5。(4)标准曲线的绘制:根皮素在1.23137~246.274μg/m L范围内,线性关系良好,回归方程为y=40466x-6026,r=1.0000(n=6)。(5)精密度试验:仪器和方法精密度良好,RSD均为0.1%。(6)重复性试验:样品重复性良好,RSD为0.2%。(7)稳定性试验:对照品溶液和供试品溶液在48h内稳定性良好,RSD值分别为0.2%和0.1%。(8)回收率试验:根皮素的平均回收率为104%,RSD为1.7%。(9)根皮素的检测限为0.4ng,定量限为1.2ng。(10)耐用性试验:色谱柱和仪器耐用性良好。(11)苹果花中根皮素的含量结果为2.3%~4.2%。4指纹图谱方法(1)提取条件和色谱条件的选择除检测波长为260nm外,其他条件同含量测定项下。(2)精密度试验:根皮素峰为参照峰,计算各共有峰相对保留时间和占总峰面积2%以上共有峰的相对峰面积,其RSD值分别为0.03%~0.11%和0.25%~0.98%,精密度良好。(3)重复性试验:12个主要共有峰相对保留时间和占总峰面积2%以上色谱峰的相对峰面积均无明显变化,其RSD值分别为0.02%~0.10%和0.19%~1.36%,重复性良好。(4)稳定性试验:样品在48h内,12个共有峰相对保留时间和占总峰面积2%以上色谱峰的相对峰面积均无明显变化,其RSD值分别为0.04%~0.19%和0.10%~1.04%,稳定性良好。(5)指纹图谱的建立:得到不同产地苹果花的指纹图谱及12个共有峰。(6)数据分析:运用“中药色谱指纹图谱相似度评价系统软件”2004年A版(国家药典委员会开发)”对所得数据进行分析,所得结果能为苹果花的品种鉴别及质量标准的制定提供依据。结论:1性状鉴别、显微鉴别、检查项、醇溶性浸出物方法稳定。2薄层色谱该方法斑点清晰可见,分离度、重复性、稳定性良好,能够很好的作为苹果花的定性鉴别手段。3含量测定以专属性有效成分根皮素的含量为主要指标,建立了苹果花的定量分析方法,该方法精密度、重复性、稳定性良好,可用于评价苹果花质量,为苹果花的质量标准的制定奠定基础。4指纹图谱通过建立苹果花的高效液相色谱指纹图谱,生成对照图谱,计算相似度,该方法精密度和重复性良好,能够控制苹果花的质量,同时为苹果花的真伪鉴别提供了数据支持。
[Abstract]:Apple flowers are derived from the dried flowers of the Rosaceae plant apple Malus pumila Mill for.4-5 months, dry and dry, stored in the dry place. Apple flower is flat, spleen, stomach, kidney meridian. Apple flower tea has the effect of treating nerve pain, detoxification, blood supplement, eyesight, acne and whitening. No literature on apple flower is found. Apple flowers are in the market at present. Tea is sold in the form of sale, but it is not taken in any quality standard, its quality is difficult to control effectively. Therefore, this experiment has studied the characteristics of apple flower, microscopic identification, TLC identification, impurities, moisture, total ash, acid insoluble ash, alcohol soluble extract (hot leaching) and so on, and studied the method for the determination of the content of the effective component of the root bark. On this basis, the HPLC fingerprint of 12 batches of apple flowers collected in 8 provinces was studied and analyzed, which could provide a certain basis for the identification and quality standard of apple flower. Objective: To study the various items according to the requirements of quality standard formulation and establish the foundation for the quality standard of apple flower. Method: 1 to 8. The 12 batch of apple samples collected in the province were identified, microscopic identification, determination of impurities, moisture, total ash, acid insoluble ash, and the content of the extract content by.2 thin layer chromatography (1) extraction conditions in 12 batches of apple (1) extraction conditions: different extraction solvents and extraction methods were explored during the experiment, and the best extraction solvent and extraction were selected. Methods: (2) expansion conditions: according to the characteristics of the apple flower composition, determine the suitable expansion agent, fixed phase and color reagent and other.3 content determination methods (1) extraction conditions: different extraction methods, different extraction solvents and their concentration, different extraction time, different concentration of hydrochloric acid hydrolysate, different powder granularity to the extraction of apple flower exclusive. The extraction conditions of the highest extraction rate of rhizotin were selected. (2) chromatographic conditions: selecting the best detection wavelength, selecting the appropriate stationary phase, adjusting the composition of the mobile phase, the ratio of the quercetin peak, the rhizin peak, the kaempferol peak and the adjacent complex peaks. (3) the system applicability test: Under the above chromatographic conditions, the theoretical plate number and the separation degree of the chromatographic peak of the root bark were investigated. (4) the standard curve: a series of concentration of the root skin hormone control solution was prepared, and the peak area of the root skin element was recorded respectively. The root bark concentration was taken as the horizontal coordinate, the corresponding peak area was the vertical coordinate, and the standard curve was drawn. (5) the precision test: the same control respectively. Product solution and the same sample solution, continuously sample 6 times, determine the peak area of the root of the root, calculate the RSD value. (6) repeatability test: precisely called the same batch of apple samples 6, preparation of the sample solution, sample respectively, determine the peak area of the root bark, calculate the RSD value. (7) stability test: the same control solution and the same supply, respectively. Test product solution, 0,2,4,6,12,24,48h analysis, determine the area of the peak of root bark and calculate the RSD value. (8) recovery rate test: a proper amount of the apple flower, known as the known root bark content, is added to the solution of 80%, 100%, 120% of the root skin element in the sample, respectively. The content of the root skin element, calculate its recovery and RSD value. (9) determination of detection limit and quantitative limit: dilute the root bark control solution step by step, when the signal to noise ratio is greater than 3, the detection limit; when the signal to noise ratio is greater than 10, the quantitative limit. (10) durability test: using three different brands of chromatographic column, two different brands of high performance liquid color color The content of 1 batches of samples was measured by the spectrometer, and the value of RSD and RD were calculated. (11) the determination of the content of.4 fingerprints in the 12 batches of apple flowers under the above chromatographic conditions (1) the selection of the extraction conditions and chromatographic conditions except the detection wavelength was 260nm, the other conditions and the content determination. (2) precision test: the same one 6 times for the sample solution, record retention time and peak area. (3) repeatability test: 6 copies of the same batch of apple flowers were accurately weighed, and the sample solution was prepared. The retention time and peak area were recorded respectively. (4) the stability test: the same sample solution was taken in the 0,2,4,6,12,24,48h sample analysis to record the retention time and peak surface. (5) (5) establishment of fingerprints: 12 batches of apple flowers from different provinces, preparation of sample solution, fingerprint analysis and similarity evaluation. Results: 1 determine apple flower character and powder microscopic identification; impurity determination result is 1%~3%; water determination result is 5.9%~7.9%; total ash determination result is 6.2%~8.5%; acid not The result of soluble ash determination is 0.6%~2.0%, and the result of alcohol soluble extract (hot leaching) is 34.9%~41.7%.2 thin layer chromatography (1) extraction method: take the powder 0.5g, insert the plug triangle flask, add ethanol 40m L, the dense plug, the ultrasonic treatment for 20 minutes, put cold, shake well, filter through 20 m L, set up 250 m L triangle flask, and add ethanol 20 m L, 25% Hydrochloric acid solution 10 m L, shake well, heat reflux for 30 minutes in 85 centigrade water bath, evaporate dry. Residue plus ethanol 4m L to dissolve. (2) expansion condition: thin plate is silica gel G plate, toluene ethyl acetate formic acid (7: 2: 1) supersolution is expanded, spread, remove, dry, and spray with 3% aluminum chloride ethanol solution at 105 C About 10 minutes, under the ultraviolet light (365nm) inspection, in the sample chromatography, in the corresponding position of the control sample chromatography and the control product chromatography, the same color fluorescence spot.3 content determination method (1) extraction conditions: the powder (over four sieves) is about 0.2g, the fine density is called, the plug triangle flask, precision adding ethanol 20m L, dense plug, called Set weight, ultrasonic treatment for 20 minutes, put cold, then weigh the weight, use ethanol to reduce the weight of lost, shake well, filter, and take the continuous filtrate 10m L, set 100m L triangle flask, add ethanol 10m L, 25% hydrochloric acid 5m L, shake well, heat back for 1 hours at 85 C water bath, cool to room temperature, transfer to 50m L measuring bottle, and shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake and shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake to scale, shake with ethanol to scale, shake Uniform, filtration, that is, (2) chromatographic conditions: using YMC ODS-A C18 (250 mm*4.6 mm, 5 m) column; mobile phase: methanol -0.4% phosphoric acid solution (50:50); detection wavelength 286nm; flow rate 1.0m L min-1; column temperature 30 C, sample quantity is 10 micron (3) system applicability test: under this chromatographic condition, the theoretical plate number should not be less than 5000 according to the root pepein peak calculation. Draw the standard curve more than 1.5. (4) standard curve: the linear relationship is good in the range of 1.23137~246.274 g/m L, the regression equation is y=40466x-6026, r=1.0000 (n=6). (5) precision test: the precision of the instrument and method is good, RSD is 0.1%. (6) repeatability test: the sample reproducibility is good, RSD is 0.2%. (7) stability test: the control solution solution The stability of the test solution in 48h was good, the RSD value was 0.2% and 0.1%. (8) recovery test respectively: the average recovery rate of the root skin pigment was 104%, the RSD was 1.7%. (9), the limit of 1.7%. (9) was 1.2ng. (10) durability test: the chromatographic column and the instrument had good durability. (11) the result of the content of the root bark in the apple flower was 2.3%~4.2%.4 finger. The pattern method (1) the selection of the extraction conditions and the chromatographic conditions except the detection wavelength is 260nm, the other conditions and the content determination items. (2) the precision test: the root bark peak is the reference peak, the relative peak area of the total peak relative retention time and the total peak area over 2% of the total peak is calculated, and the RSD value is 0.03%~0.11% and 0.25%~0.98%, respectively. (3) repeatability test: the relative peak area of the 12 main peak relative retention time and the total peak area above 2% was no obvious change, and the RSD value was 0.02%~0.10% and 0.19%~1.36%, and the repeatability was good. (4) the stability test: the sample was in the 48h, the relative retention time of the 12 common peaks and the total peak area above 2% chromatography The relative peak area of the peak was no obvious change, and its RSD value was 0.04%~0.19% and 0.10%~1.04%, and the stability was good. (5) fingerprint establishment: fingerprint and 12 common peaks of apple flower from different habitats. (6) data analysis: application of "chromatographic fingerprint similarity evaluation system software of traditional Chinese Medicine" (National Pharmacopoeia Committee) 2004 (National Pharmacopoeia Committee) Analysis of the obtained data, the results can provide the basis for the identification and quality standard of apple flower. Conclusion: 1 character identification, microscopic identification, inspection item, alcohol soluble extract method stable.2 thin layer chromatography, the spot is clearly visible, the degree of separation, reproducibility, stability are good, and it can be used as apple flower well. The quantitative analysis method of.3 was established by qualitative identification method. The quantitative analysis method of apple flower was established. This method is precise, repeatable and stable. It can be used to evaluate the quality of apple flower, and lay the foundation of.4 fingerprint for the quality standard of apple flower to establish the height of apple flower. The HPLC fingerprint was used to generate the control map and calculate the similarity. The precision and repeatability of the method were good. It could control the quality of apple flower and provide data support for the authenticity identification of apple flower.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284.1

【参考文献】