本文關(guān)鍵詞：陳皮化學成分的研究，由筆耕文化傳播整理發(fā)布。

        陳皮（Citri Reticulatae Pericarpium）為蕓香科柑桔屬植物橘（Citrus reticulataBlanco）及其栽培變種的干燥成熟果皮，是一種藥食同源的中藥。陳皮氣香，微苦，主治脘腹脹滿，食少吐瀉，咳嗽痰多等癥。藥理學研究表明，陳皮在心血管系統(tǒng)、免疫、抗氧化以及抗腫瘤等方面具有良好的藥用價值。陳皮的化學成分以黃酮類化合物為主，此外還含有檸檬苦素類、生物堿類、揮發(fā)油類及微量元素等。本實驗對陳皮的化學成分進行了研究，從陳皮80%乙醇提取物中共分得十二個單體化合物，經(jīng)結(jié)構(gòu)鑒定，確認了其中十個化合物的化學結(jié)構(gòu)，它們分別是：5,6,7,8,4′-五甲氧基黃酮，3,5,6,7,8,3′,4′-七甲氧基黃酮，檸檬苦素，Limonexic acid，8-羥基-3,5,6,7,3′,4′-六甲氧基黃酮，6,7,8,4′-四甲氧基黃酮，5-羥基-3,7,3′,4′-四甲氧基黃酮，3,6,7,8,2′,5′-六甲氧基黃酮，5,4′-二羥基-3,6,7,8,3′-五甲氧基黃酮，5-羥基-3,6,7,8,3′,4′-六甲氧基黃酮。其中8-羥基-3,5,6,7,3′,4′-六甲氧基黃酮，5,4′-二羥基-3,6,7,8,3′-五甲氧基黃酮為首次從陳皮中分得。本實驗還建立了陳皮中3,5,6,7,8,3′,4′-七甲氧基黃酮、8-羥基-3,5,6,7,3′,4′-六甲氧基黃酮和5-羥基-3,6,7,8,3′,4′-六甲氧基黃酮RP-HPLC-DAD的含量測定方法。一、陳皮的提取、分離和結(jié)構(gòu)鑒定1、提取將干燥的陳皮藥材5.0kg粉碎，用80%乙醇回流提取三次，乙醇用量分別為50、40、30L，提取時間分別為1.5、1.0、1.0h，過濾后，合并提取液，減壓回收溶劑得到陳皮提取物0.5kg。2、分離和純化將0.5kg的提取物進行硅膠柱層析分離，以流動相I洗脫，得到A、B、C、D、E五個部分。C部分采用硅膠柱層析分離，，以流動相II洗脫，得到F和G兩部分。F再進行硅膠柱層析，以流動相IV洗脫，得到J部分，然后再經(jīng)過流動相V洗脫，得到化合物CP-4（500mg）。G部分進行硅膠柱層析，流動相VI洗脫，得到K和L部分。 L部分經(jīng)硅膠柱層析分離，以流動相VIII洗脫，得到化合物CP-3（1.5g）和CP-11（12mg）。而K部分進行硅膠柱層析，以流動相VII洗脫得到N和O部分，再分別經(jīng)過ODS柱層析分離，流動相分別為IX和X，得到CP-1（2.0g）、CP-2（1.8g）、CP-5（15mg）和CP-8（20mg）四個化合物。D部分進行硅膠柱層析分離，以流動相III洗脫，得到H和I兩個部分。其中I部分經(jīng)過ODS柱層析，以流動相XI洗脫，得到化合物CP-12（50mg）。H部分利用ODS柱層析，流動相XI洗脫得到化合物CP-6（14mg）、M部分和P部分， M部分再通過ODS柱層析，流動相X洗脫后得到化合物CP-9（16mg）和CP-7（26mg），P部分經(jīng)過重結(jié)晶得到CP-10（15mg）。3、結(jié)構(gòu)鑒定根據(jù)各化合物的理化性質(zhì)，經(jīng)核磁共振和質(zhì)譜等波譜數(shù)據(jù)分析并與文獻對照，鑒定了十個化合物的化學結(jié)構(gòu)，結(jié)果如下：化合物CP-1為5,6,7,8,4′-五甲氧基黃酮，化合物CP-2為3,5,6,7,8,3′,4′-七甲氧基黃酮，化合物CP-3為檸檬苦素，化合物CP-4為8-羥基-3,5,6,7,3′,4′-六甲氧基黃酮，化合物CP-5為6,7,8,4′-四甲氧基黃酮，化合物CP-6為5-羥基-3,7,3′,4′-四甲氧基黃酮，化合物CP-8為3,6,7,8,2′,5′-六甲氧基黃酮，化合物CP-9為5,4′-二羥基-3,6,7,8,3′-五甲氧基黃酮，化合物CP-11為limonexic acid，化合物CP-12為5-羥基-3,6,7,8,3′,4′-六甲氧基黃酮。二、RP-HPLC-DAD法測定陳皮中三種多甲氧基黃酮的含量1、儀器和色譜條件高效液相色譜儀：Agilent1200HPLC-DAD檢測器天平：METTLER TOLEDO AB135-S色譜柱：Cosmosil packed column C18-Ar-II （4.6×250mm，5μm）色譜條件：流動相：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；柱溫：25℃；檢測波長：258nm；進樣量：20μL。2、方法學考察2.1線性關(guān)系化合物CP-2、CP-4和CP-12分別在0.01~10.0μg、0.05~10.0μg和0.025~5.0μg的范圍內(nèi)峰面積（Y）和進樣濃度（X）均呈現(xiàn)良好的線性關(guān)系。它們的線性回歸方程和相關(guān)系數(shù)分別為：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檢測限和定量限當化合物CP-2的濃度分別降至7.5×10-6mg/mL和1.0×10-4mg/mL時，信噪比S/N分別為3.8和10.0，因此CP-2的檢測限為1.5×10-4μg，定量限為2.0×10-3μg。當化合物CP-4的濃度分別降至5.0×10-4mg/mL和1.25×10-3mg/mL時，信噪比S/N分別為3.2和11.6，因此CP-4的檢測限為1.0×10-2μg，定量限2.5×10-2μg。當化合物CP-12的濃度分別降至5.0×10-5mg/mL和2.5×10-4mg/mL時，信噪比S/N分別為3.2和10.9，因此CP-12的檢測限為1.0×10-3μg，定量限5.0×10-3μg。2.3精密度將化合物CP-2、CP-4和CP-12混合對照品溶液按上述色譜條件進行測定，結(jié)果顯示化合物CP-2峰面積的日內(nèi)和日間精密度的RSD值分別為0.69%和0.63%，化合物CP-4峰面積的日內(nèi)和日間精密度的RSD值分別為1.29%和1.18%，化合物CP-12峰面積的日內(nèi)和日間精密度的RSD值分別為1.05%和1.42%。RSD值均小于2%，表明儀器精密度良好。2.4重復(fù)性測定六份供試品溶液，結(jié)果顯示化合物CP-2、CP-4和CP-12含量的RSD值分別為0.89%、1.21%和0.96%，均小于2%，表明重復(fù)性良好。2.5穩(wěn)定性將供試品溶液每間隔2h測定一次，連續(xù)12h，結(jié)果顯示這三個化合物峰面積的RSD值分別為0.49%、0.71%和1.13%，保留時間的RSD值分別為0.09%、0.16%和0.11%，各RSD值均小于2%，表明供試品溶液在12h內(nèi)穩(wěn)定性良好。2.6加樣回收率化合物CP-2、CP-4和CP-12的平均加樣回收率分別為99.72%、101.78%和100.22%，回收率的RSD值依次分別為0.50%、1.97%和1.75%，其RSD值均小于2%，表明回收率良好。3、含量測定陳皮藥材中化合物CP-2、CP-4和CP-12的平均含量分別為0.08%、0.01%和0.0025%。4、總結(jié)本論文在國內(nèi)外學者研究的基礎(chǔ)上，對陳皮藥材的化學成分進行了深入研究，共分離十二個化合物，鑒定了十個化合物，其中兩個為首次從陳皮中分得。此外，本文還建立了陳皮中三種多甲氧基黃酮含量的RP-HPLC-DAD測定方法，為完善陳皮藥材的質(zhì)量標準，以及陳皮的開發(fā)利用和藥理活性的進一步研究提供新的科學依據(jù)。

    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.

陳皮化學成分的研究

摘要4-8Abstract8-11第一章 緒論15-32    1.1 資源分布15    1.2 性狀15    1.3 化學成分15-25        1.3.1 黃酮類16-22        1.3.2 檸檬苦素類22-23        1.3.3 生物堿類23-24        1.3.4 揮發(fā)油24-25        1.3.5 微量元素25        1.3.6 其它成分25    1.4 含量測定25-27    1.5 藥理作用27-31        1.5.1 抗氧化作用27-28        1.5.2 對胃腸道的作用28        1.5.3 對呼吸系統(tǒng)的作用28-29        1.5.4 抗癌抗腫瘤的作用29        1.5.5 抗炎殺菌29-30        1.5.6 心血管的作用30        1.5.7 其他作用30-31    1.6 研究目的與研究內(nèi)容31-32第二章 陳皮的化學成分研究32-55    2.1 陳皮的化學成分結(jié)構(gòu)分析32-50        2.1.1 化合物 CP-1 的結(jié)構(gòu)分析32-34        2.1.2 化合物 CP-2 的結(jié)構(gòu)分析34-35        2.1.3 化合物 CP-3 的結(jié)構(gòu)分析35-37        2.1.4 化合物 CP-4 的結(jié)構(gòu)分析37-39        2.1.5 化合物 CP-5 的結(jié)構(gòu)分析39-41        2.1.6 化合物 CP-6 的結(jié)構(gòu)分析41-42        2.1.7 化合物 CP-8 的結(jié)構(gòu)分析42-44        2.1.8 化合物 CP-9 的結(jié)構(gòu)分析44-46        2.1.9 化合物 CP-11 的結(jié)構(gòu)分析46-48        2.1.10 化合物 CP-12 的結(jié)構(gòu)分析48-50    2.2 實驗部分50-54        2.2.1 實驗材料、儀器、試劑及層析條件50-51        2.2.2 化學成分的提取和分離51-53        2.2.3 結(jié)構(gòu)鑒定數(shù)據(jù)53-54    2.3 小結(jié)54-55第三章 RP-HPLC-DAD 法測定陳皮中三種多甲氧基黃酮的含量55-66    3.1 實驗儀器、試劑和對照品55    3.2 色譜條件55-57        3.2.1 檢測波長的選擇55-56        3.2.2 流動相的選擇56-57    3.3 提取條件的選擇57-59        3.3.1 提取溶劑的選擇57        3.3.2 提取溶劑用量的選擇57-58        3.3.3 提取方法的選擇58-59        3.3.4 提取次數(shù)的選擇59        3.3.5 供試品溶液的制備59    3.4 方法學考察59-64        3.4.1 對照品溶液的制備59        3.4.2 線性關(guān)系59-61        3.4.3 檢測限和定量限61        3.4.4 精密度61-62        3.4.5 重復(fù)性62        3.4.6 穩(wěn)定性62-63        3.4.7 加樣回收率63-64    3.5 含量測定64-66        3.5.1 供試品溶液的制備64        3.5.2 對照品溶液的制備64-65        3.5.3 樣品的測定65-66第四章 結(jié)果與討論66-67參考文獻67-74附圖74-99作者簡介99-100致謝100

  本文關(guān)鍵詞：陳皮化學成分的研究，由筆耕文化傳播整理發(fā)布。