Study On Chemical Constituents From Aurantli Fructus

Aurantii Fructus is the dried and immature fruit of Citrus aurantium L.and itscultivars. It has the traits of fragrance, taste bitter and slightly acidic etc. As a widelyused traditional Chinese herbal medicine,Aurantii Fructus is used in the treatment ofmany diseases, such as dyspepsia,prolapse of visceral organ, etc.The chemical constituents of Aurantii Fructus consist of flavones,alkaloids,coumarins,volatile oils and trace elements,etc. This experiment had gained tencompounds from80%ethanol extract of Aurantii Fructus by using the main methodsof column chromatography, preparative chromatograph and recrystallization,etc. Allthe chemical structures of ten compounds had been elucidated by means of variousspectroscopy methods combining with the physical and chemical properties. The tencompounds were identified as limonin(Lm1), β-sitosterol(Lm2), nobiletin(Lm3),nomilin(Lm4),sinensetin(Lm5),naringenin(Lm6),5,7,8,4′-tetramethoxyflavone(Lm7),3,5,7,8,3′,4′-hexamethoxyflavone(Lm8),5,7,8,3′,4′-pentamethoxyflavone(Lm9) anddaucosterol(Lm10). Among the ten compounds, nomilin(Lm4),3,5,7,8,3′,4′-hexamethoxyflavone (Lm8) and5,7,8,3′,4′-pentamethoxyflavone(Lm9)were gained from Aurantii Fructus for the first time.In this experiment, the content determination of sinensetin (Lm5) and5,7,8,3′,4′-pentamethoxyflavone (Lm9) in Aurantii Fructus using HPLC wereestablished.1.The extraction, isolation and structure identification of thechemical constituents in Aurantii Fructus1.1ExtractionThe20.0kg dried Aurantii Fructus medicinal materials were crushed firstly, thenwere extracted using the method of heating reflux extraction.The extraction solventwas80%ethanol, the extraction times was three, the dosage of the solvent were ten, eight and eight times (V/W) and the extracting time were1.5h,1.0h,1.0h respectively.The extracted solution were put together, filtered and then processed using rotaryevaporators, the3.0kg crude extracts were gained finally.1.2Isolation and purificationThe3.0kg crude extracts were subjected to silica gel column chromatographyand eluted by mobile phase1, then eight parts A、B、C、D、E、F、G、H were obtained.(1)The five parts (C1,C6,D1,D3and D4) were separated repeatly using themethod of silica gel column chromatography and ODS column chromatography, thensix compounds were gained and marked as Lm1, Lm2, Lm3, Lm4, Lm5and Lm6.(2)The two parts(D5and D6) were separated repeatly using preparative liquidchromatograph, then received three compounds Lm7, Lm8and Lm9.(3)The part D7were isolated repeatly using recrystallization. Then gained thecompound Lm10.In summary, ten compounds were gained from Aurantii Fructus.1.3Structure identificationBy means of various spectroscopy methods,combining with the physical andchemical properties and compared with the relevant literatures, the structures of theten compounds were identified as limonin(Lm1), β-sitosterol(Lm2), nobiletin(Lm3),nomilin(Lm4),sinensetin(Lm5),naringenin(Lm6),5,7,8,4′-tetramethoxyflavone(Lm7),3,5,7,8,3′,4′-hexamethoxyflavone(Lm8),5,7,8,3′,4′-pentamethoxyflavone(Lm9),dau-costerol(Lm10).2.The contents determination of sinensetin(Lm5) and5,7,8,3′,4′-pentamethoxyflavone(Lm9) in Aurantii Fructus usingHPLC2.1Instruments and chromatographic conditionsChromatography: Agilent1200HPLC; Diode array detector(DAD);Chromatographic column: Acchrom Unitary C18Column(4.6×250mm,5μm); Chromatographic conditions: Mobile phase was methanol-water(52:48); Flowrate was1.0mL·min-1; Column temperature was25.0℃; Wave length of DAD was setat310nm; Injection volume was20μL.2.2Methodology Investigation2.2.1Linear relationThe two compounds sinensetin(Lm5) and5,7,8,3′,4′-pentamethoxyflavone (Lm9)had a good linear relationship with the ranges of0.011~5.3μg and0.010~5.1μgrespectively. The linear equations and linear coefficients were as follows respectively:Lm5: y=53411x+56.071r=0.99995Lm9: y=22869x+21.006r=0.999952.2.2LOD and LOQAs shown in the result, the LODs of Lm5and Lm9were1.3×10-3μg and2.7×10-3μg respectively; the LOQs of Lm5and Lm9were4.6×10-3μg and1.1×10-2μgrespectively.2.2.3PrecisionAs shown in the result, the intraday RSDs of peak areas for Lm5and Lm9were1.6%and0.58%respectively; the interday RSDs of peak areas for Lm5and Lm9were1.1%and1.5%respectively.That above datas were less than2%,it illustratedthat the method had a good precision.2.2.4RepeatabilityAs shown in the result,the RSDs of the contents were0.16%and1.5%respectively, the RSDs data were less than2%, it illustrated that the method had agood repeatability.2.2.5StabilityAs shown in the result, the RSDs of peak areas for Lm5and Lm9were0.53%and0.49%respectively; the RSDs of retention time of Lm5and Lm9were0.35%and0.29%respectively. All the RSDs were less than2%, so the sample solution had agood stability within12h.2.2.6Recoveries As shown in the result, the average recoveries of Lm5and Lm9were101.4%and100.7%respectively, and the RSDs were1.22%and1.51%respectively.3. Contents determination of Lm5and Lm9in Aurantii FructusThe experiment had determined two kinds of Aurantii Fructus purchased fromdifferent regions.The average contents of Lm5and Lm9were0.16mg·g-1and0.024mg·g-1respectively.4. SummaryOn the basis of the domestic and foreign scholars studies, the chemicalconstitution of Aurantii Fructus was studied deeply in this expriment. We gained tencompounds and established the contents determination methods of sinensetin(Lm5)and5,7,8,3′,4′-pentamethoxyflavone (Lm9) in Aurantii Fructus using HPLC. Thestudy further improved the establishment of quality standards, and provided favorablescientific basis for research, development and utilization of Aurantii Fructus.