| 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. |
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