30年陈皮价格_陈皮的化学成份_陈皮化学成分的研究

  本文关键词：陈皮化学成分的研究，由笔耕文化传播整理发布。

        陈皮（Citri Reticulatae Pericarpium）为芸香科柑桔属植物橘（Citrus reticulataBlanco）及其栽培变种的干燥成熟果皮，是一种药食同源的中药。陈皮气香，微苦，主治脘腹胀满，食少吐泻，咳嗽痰多等症。药理学研究表明，陈皮在心血管系统、免疫、抗氧化以及抗肿瘤等方面具有良好的药用价值。陈皮的化学成分以黄酮类化合物为主，此外还含有柠檬苦素类、生物碱类、挥发油类及微量元素等。本实验对陈皮的化学成分进行了研究，从陈皮80%乙醇提取物中共分得十二个单体化合物，经结构鉴定，确认了其中十个化合物的化学结构，它们分别是：5,6,7,8,4′-五甲氧基黄酮，3,5,6,7,8,3′,4′-七甲氧基黄酮，柠檬苦素，Limonexic acid，8-羟基-3,5,6,7,3′,4′-六甲氧基黄酮，6,7,8,4′-四甲氧基黄酮，5-羟基-3,7,3′,4′-四甲氧基黄酮，3,6,7,8,2′,5′-六甲氧基黄酮，5,4′-二羟基-3,6,7,8,3′-五甲氧基黄酮，5-羟基-3,6,7,8,3′,4′-六甲氧基黄酮。其中8-羟基-3,5,6,7,3′,4′-六甲氧基黄酮，5,4′-二羟基-3,6,7,8,3′-五甲氧基黄酮为首次从陈皮中分得。本实验还建立了陈皮中3,5,6,7,8,3′,4′-七甲氧基黄酮、8-羟基-3,5,6,7,3′,4′-六甲氧基黄酮和5-羟基-3,6,7,8,3′,4′-六甲氧基黄酮RP-HPLC-DAD的含量测定方法。一、陈皮的提取、分离和结构鉴定1、提取将干燥的陈皮药材5.0kg粉碎，用80%乙醇回流提取三次，乙醇用量分别为50、40、30L，提取时间分别为1.5、1.0、1.0h，过滤后，合并提取液，减压回收溶剂得到陈皮提取物0.5kg。2、分离和纯化将0.5kg的提取物进行硅胶柱层析分离，以流动相I洗脱，得到A、B、C、D、E五个部分。C部分采用硅胶柱层析分离，，以流动相II洗脱，得到F和G两部分。F再进行硅胶柱层析，以流动相IV洗脱，得到J部分，然后再经过流动相V洗脱，得到化合物CP-4（500mg）。G部分进行硅胶柱层析，流动相VI洗脱，得到K和L部分。 L部分经硅胶柱层析分离，以流动相VIII洗脱，得到化合物CP-3（1.5g）和CP-11（12mg）。而K部分进行硅胶柱层析，以流动相VII洗脱得到N和O部分，再分别经过ODS柱层析分离，流动相分别为IX和X，得到CP-1（2.0g）、CP-2（1.8g）、CP-5（15mg）和CP-8（20mg）四个化合物。D部分进行硅胶柱层析分离，以流动相III洗脱，得到H和I两个部分。其中I部分经过ODS柱层析，以流动相XI洗脱，得到化合物CP-12（50mg）。H部分利用ODS柱层析，流动相XI洗脱得到化合物CP-6（14mg）、M部分和P部分， M部分再通过ODS柱层析，流动相X洗脱后得到化合物CP-9（16mg）和CP-7（26mg），P部分经过重结晶得到CP-10（15mg）。3、结构鉴定根据各化合物的理化性质，经核磁共振和质谱等波谱数据分析并与文献对照，鉴定了十个化合物的化学结构，结果如下：化合物CP-1为5,6,7,8,4′-五甲氧基黄酮，化合物CP-2为3,5,6,7,8,3′,4′-七甲氧基黄酮，化合物CP-3为柠檬苦素，化合物CP-4为8-羟基-3,5,6,7,3′,4′-六甲氧基黄酮，化合物CP-5为6,7,8,4′-四甲氧基黄酮，化合物CP-6为5-羟基-3,7,3′,4′-四甲氧基黄酮，化合物CP-8为3,6,7,8,2′,5′-六甲氧基黄酮，化合物CP-9为5,4′-二羟基-3,6,7,8,3′-五甲氧基黄酮，化合物CP-11为limonexic acid，化合物CP-12为5-羟基-3,6,7,8,3′,4′-六甲氧基黄酮。二、RP-HPLC-DAD法测定陈皮中三种多甲氧基黄酮的含量1、仪器和色谱条件高效液相色谱仪：Agilent1200HPLC-DAD检测器天平：METTLER TOLEDO AB135-S色谱柱：Cosmosil packed column C18-Ar-II （4.6×250mm，5μm）色谱条件：流动相：乙腈-水（0.2%醋酸）（梯度：0-30min，39%乙腈；30-33min，39-52%乙腈；33-45min，52%乙腈）流速：1.0mL/min；柱温：25℃；检测波长：258nm；进样量：20μL。2、方法学考察2.1线性关系化合物CP-2、CP-4和CP-12分别在0.01~10.0μg、0.05~10.0μg和0.025~5.0μg的范围内峰面积（Y）和进样浓度（X）均呈现良好的线性关系。它们的线性回归方程和相关系数分别为：CP-2：Y=23606X+40.533，r=0.9996；CP-4：Y=26996X-31.177，r=0.9997；CP-12：Y=17519X+0.8356，r=0.9999。2.2检测限和定量限当化合物CP-2的浓度分别降至7.5×10-6mg/mL和1.0×10-4mg/mL时，信噪比S/N分别为3.8和10.0，因此CP-2的检测限为1.5×10-4μg，定量限为2.0×10-3μg。当化合物CP-4的浓度分别降至5.0×10-4mg/mL和1.25×10-3mg/mL时，信噪比S/N分别为3.2和11.6，因此CP-4的检测限为1.0×10-2μg，定量限2.5×10-2μg。当化合物CP-12的浓度分别降至5.0×10-5mg/mL和2.5×10-4mg/mL时，信噪比S/N分别为3.2和10.9，因此CP-12的检测限为1.0×10-3μg，定量限5.0×10-3μg。2.3精密度将化合物CP-2、CP-4和CP-12混合对照品溶液按上述色谱条件进行测定，结果显示化合物CP-2峰面积的日内和日间精密度的RSD值分别为0.69%和0.63%，化合物CP-4峰面积的日内和日间精密度的RSD值分别为1.29%和1.18%，化合物CP-12峰面积的日内和日间精密度的RSD值分别为1.05%和1.42%。RSD值均小于2%，表明仪器精密度良好。2.4重复性测定六份供试品溶液，结果显示化合物CP-2、CP-4和CP-12含量的RSD值分别为0.89%、1.21%和0.96%，均小于2%，表明重复性良好。2.5稳定性将供试品溶液每间隔2h测定一次，连续12h，结果显示这三个化合物峰面积的RSD值分别为0.49%、0.71%和1.13%，保留时间的RSD值分别为0.09%、0.16%和0.11%，各RSD值均小于2%，表明供试品溶液在12h内稳定性良好。2.6加样回收率化合物CP-2、CP-4和CP-12的平均加样回收率分别为99.72%、101.78%和100.22%，回收率的RSD值依次分别为0.50%、1.97%和1.75%，其RSD值均小于2%，表明回收率良好。3、含量测定陈皮药材中化合物CP-2、CP-4和CP-12的平均含量分别为0.08%、0.01%和0.0025%。4、总结本论文在国内外学者研究的基础上，对陈皮药材的化学成分进行了深入研究，共分离十二个化合物，鉴定了十个化合物，其中两个为首次从陈皮中分得。此外，本文还建立了陈皮中三种多甲氧基黄酮含量的RP-HPLC-DAD测定方法，为完善陈皮药材的质量标准，以及陈皮的开发利用和药理活性的进一步研究提供新的科学依据。

    Citri Reticulatae Pericarpium is dried fruit peel of Citrus reticulata Blanco andits cultivars. It is a widely used traditional Chinese herbal medicine which is fragrantand slightly bitter, with stimulating the appetite, regulating the spleen and stomach,treating the act of vomiting, etc. Pharmacological studies have shown that CitriReticulatae Pericarpium has good activities such as cardiovascular system, immune,antioxidant, and anti-tumor.Chemical constituents of Citri Reticulatae Pericarpium are mainly flavones, inaddition to containing limonoids, alkaloids, volatile oils and trace elements, etc.Twelve compounds were isolated and purified from80%ethanol extract of CitriReticulatae Pericarpi，and ten of them were identified as5,6,7,8,4′-pentamethoxyflavo-ne,3,5,6,7,8,3′,4′-heptamethoxyflavone, Limonin, Limonexic acid,8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone,6,7,8,4′-tetramethoxyflavone,5-hydroxy-3,7,3′,4′-tetra-methoxyflavone,3,6,7,8,2′,5′-hexamethoxyflavone,5,4′-dihydroxy-3,6,7,8,3′-penta-methoxyflavone and5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone.8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone and5,4′-dihydroxy-3,6,7,8,3′-pentamethoxyflavonewere obtained from this medical material for the first time.The experiment also established a RP-HPLC-DAD method to determine thecontents of3,5,6,7,8,3′,4′-heptamethoxyflavone,8-hydroxy-3,5,6,7,3′,4′-hexamethox-yflavone and5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone.1Extraction, isolation and structural identification of the ChemicalConstituents1.1Extraction5.0kg of dry Citri Reticulatae Pericarpium were crushed and then extracted byheat reflux for three times in80%ethanol. The volume of80%ethanol were50L(1.5h),40L(1.0h),30L(1.0h) in turn. The extracting solution was combined after filerted. After the solvent was evaporated by rotary vaporization under reducedpressure,0.5kg crude extract of Citri Reticulata Pericarpium was obtained.1.2IsolationIsolation methods mainly used recrystallization and column chromatographywhich included silica gel column and ODS column, and isolation process needed to berepeated continuously. Reagents used as eluents included methanol, ethanol, ethylacetate, chloroform, methylene chloride, acetone, cyclohexane, petroleum ether anddistilled water, etc. Finally twelve compounds in total were isolated from0.5kg crudeextract of Citri Reticulata Pericarpium. They were named CP-1, CP-2, CP-3, CP-4,CP-5, CP-6, CP-7, CP-8, CP-9, CP-10, CP-11, CP-12.1.3Structural identificationBased on the physical and chemical properties of the compounds, nuclearmagnetic resonance spectroscopy and mass spectrometry data, the chemical structuresof ten compounds were identified after compared with the literatures. They were5,6,7,8,4′-pentamethoxyflavone (CP-1),3,5,6,7,8,3′,4′-heptamethoxyflavone(CP-2),Limonin(CP-3),8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone(CP-4),6,7,8,4′-tetrame-thoxyflavone(CP-5),5-hydroxy-3,7,3′,4′-tetramethoxyflavone(CP-6),3,6,7,8,2′,5′-hexamethoxyflavone(CP-8),5,4′-dihydroxy-3,6,7,8,3′-pentamethoxyflavone(CP-9),Limonexic acid(CP-11),5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone(CP-12).2HPLC determination of3,5,6,7,8,3′,4′-heptamethoxyflavone,8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone and5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone2.1Equipment and Chromatographic conditionsChromatograph-Detector: Agilent1200HPLC-DAD DetectorScale: METTLER TOLEDO AB135-SColumn: cosmosil C18-Ar-II (4.6×250mm,5μm)Conditions: Mobile phase: Acetonitrile-water(0.2%HAc) gradient elution; Flow rate:1.0mL/min; Column temperature:25℃;Injection volume:20μL; Detection wavelength:258nm.2.2Methodology Investigation2.2.1linear relationWithin the range of0.01~10.0μg,0.05~10.0μg and0.025~5.0μg, the peakareas(Y) of Compound CP-2, CP-4, CP-12and the sample concentration (X) was ina good linear relationship respectively. Linear regression equations and correlationcoefficients are showed as follows:CP-2: Y=23606X+40.533, r=0.9996;CP-4: Y=26996X-31.177, r=0.9997;CP-12: Y=17519X+0.8356, r=0.9999.2.2.2Limit of detection and quantificationWhen the concentration of CP-2diluted to7.5×10-6mg/mL and1.0×10-4mg/mL,the corresponding S/N ratio were3.8and10.0respectively. Therefore, detection limitof CP-2was1.5×10-4μg and quantification limit was2.0×10-3μg. When theconcentration of CP-4diluted to5.0×10-4mg/mL and1.25×10-3mg/mL, thecorresponding S/N ratio were3.2and11.6respectively. Therefore, detection limit ofCP-4was1.0×10-2μg and quantification limit was2.5×10-2μg. When theconcentration of CP-12diluted to5.0×10-5mg/mL and2.5×10-4mg/mL, thecorresponding S/N ratio were3.2and10.9respectively. Therefore, detection limit ofCP-12was1.0×10-3μg and quantification limit was5.0×10-3μg.2.2.3PrecisionA mixed solution of the reference substances was measured in accordance withthe corresponding requirements, and recorded peak areas of each compound. Intradayand inter-day RSD of peak area were0.69%and0.63%for compound CP-2,1.29%and1.18%for compound CP-4,1.05%and1.42%for compound CP-12respectively.It indicated the equipment was in good precision that all data were less than2%.2.2.4Repeatability Six copies of the test solution was measured and the RSD of the contents ofcompound CP-2, CP-4and CP-12were1.11%,1.21%and1.30%respectively. Thesedata were less than2%, which showed the experiment had good repeatability.2.2.5StabilityMeasured the test solution by interval of2h and continued12h. The RSD ofpeak areas of compound CP-2, CP-4and CP-12were0.49%,0.71%and1.13%respectively, all data less than2%. Therefore the test solution was stable within12h.2.2.6RecoveriesThe average recovery of CP-2was99.72%and the RSD was0.50%; Theaverage recovery of CP-4was101.78%and the RSD was1.97%; The averagerecovery of CP-12was100.22%and the RSD was1.75%. All data were less than2%,so the recoveries were good.3Content determinationAfter measured contents of compound CP-2,CP-4and CP-12in Citri ReticulataePericarpium medicinal materials, the average contents were0.08%,0.01%and0.0025%respectively.4SummaryOn the basis of the domestic and foreign scholars studies, the chemicalconstitution of Citri Reticulatae Pericarpium was studied deeply in this thesis. Twelvecompounds were isolated and ten of them were identified. Two polymethoxylatedflavonoids were obtained for the first time. Three compounds were analyzedquantitatively by RP-HPLC-DAD method. The study further improved theestablishment of quality standards, and provided favorable scientific basis for research,development and utilization of Citri Reticulatae Pericarpium.

陈皮化学成分的研究

摘要4-8Abstract8-11第一章 绪论15-32    1.1 资源分布15    1.2 性状15    1.3 化学成分15-25        1.3.1 黄酮类16-22        1.3.2 柠檬苦素类22-23        1.3.3 生物碱类23-24        1.3.4 挥发油24-25        1.3.5 微量元素25        1.3.6 其它成分25    1.4 含量测定25-27    1.5 药理作用27-31        1.5.1 抗氧化作用27-28        1.5.2 对胃肠道的作用28        1.5.3 对呼吸系统的作用28-29        1.5.4 抗癌抗肿瘤的作用29        1.5.5 抗炎杀菌29-30        1.5.6 心血管的作用30        1.5.7 其他作用30-31    1.6 研究目的与研究内容31-32第二章 陈皮的化学成分研究32-55    2.1 陈皮的化学成分结构分析32-50        2.1.1 化合物 CP-1 的结构分析32-34        2.1.2 化合物 CP-2 的结构分析34-35        2.1.3 化合物 CP-3 的结构分析35-37        2.1.4 化合物 CP-4 的结构分析37-39        2.1.5 化合物 CP-5 的结构分析39-41        2.1.6 化合物 CP-6 的结构分析41-42        2.1.7 化合物 CP-8 的结构分析42-44        2.1.8 化合物 CP-9 的结构分析44-46        2.1.9 化合物 CP-11 的结构分析46-48        2.1.10 化合物 CP-12 的结构分析48-50    2.2 实验部分50-54        2.2.1 实验材料、仪器、试剂及层析条件50-51        2.2.2 化学成分的提取和分离51-53        2.2.3 结构鉴定数据53-54    2.3 小结54-55第三章 RP-HPLC-DAD 法测定陈皮中三种多甲氧基黄酮的含量55-66    3.1 实验仪器、试剂和对照品55    3.2 色谱条件55-57        3.2.1 检测波长的选择55-56        3.2.2 流动相的选择56-57    3.3 提取条件的选择57-59        3.3.1 提取溶剂的选择57        3.3.2 提取溶剂用量的选择57-58        3.3.3 提取方法的选择58-59        3.3.4 提取次数的选择59        3.3.5 供试品溶液的制备59    3.4 方法学考察59-64        3.4.1 对照品溶液的制备59        3.4.2 线性关系59-61        3.4.3 检测限和定量限61        3.4.4 精密度61-62        3.4.5 重复性62        3.4.6 稳定性62-63        3.4.7 加样回收率63-64    3.5 含量测定64-66        3.5.1 供试品溶液的制备64        3.5.2 对照品溶液的制备64-65        3.5.3 样品的测定65-66第四章 结果与讨论66-67参考文献67-74附图74-99作者简介99-100致谢100

  本文关键词：陈皮化学成分的研究，由笔耕文化传播整理发布。