| Murraya paniculata (L.) Jack is a traditional Chinese medicine in China. The plant is widely used in the treatment of stomachache, toothache, rheumatism and paralysis. Modern research found that Murraya paniculata had obvious medicinal value in the anti-fertility, treatment of diabetes and stomach .In this study, the chemical constituents of Murraya paniculata (L.) Jack have been researched futherly, and the quantification method of two flavanones using HPLC were investigated in order to develop a new drug with higher medicinal and social value .The test results obtained as follows:1 Chemical constituents of the extraction, separation and structural identificationSeven compounds have been isolated from the leaves of Murraya paniculata (L.) Jack. They were identified by various spectral methods as 2'-hydroxy- 3,4,5,4',6'- pentamethoxychalcone(S-1),5-hydroxy-6,7,8,3',4'-pentamethoxyflavone(S-2),6,7,8,3',4'-pentamethoxyflavanone(S-3),Spathulenol(S-4),2'-hydroxy-3,4,5,3',4',6'-hexametho-xychalcone(S-5),5,7,3',4',5'-pentamethoxyflavanone(S-6),2'-hydroxy-3,4,4',6'tetramet-hoxychalcone(S-7). Compounds S-3 and S-5 were isolated from the natural plant for the first time, while compound S-7 was isolated from Murraya paniculata (L.) Jack. for the first time.1.1 Extraction5kg of dry grinding leaves of Murraya paniculata (L.) Jack.mixed with 85% (the volume fraction) were extracted by heat reflux three times .The volume of ethanol used were 40L, 38L and 36L. The extraction time was 3h, 2h and 1h.Then the extract was combined and concentrated before it was diluted with water and flowed through the AB-8 macroporous resin column. After the resin was eluted by the volume fraction of 95% ethanol and the solvent was recovered, 300g of extract was obtained1.2 Isolation and purificationThe extraction obtained was subjected to silica gel column and ODS column eluted repeatedly to afford compounds S-1,S-2,S-3,S-4,S-5,S-6,S-7. The mobile phase were Petroleum ether, Ethyl acetate, Chloroform, Methanol and water with several proportions.1.3 IdentificationAccording to the physical and chemical properties and their characters of UV, MS, one-dimensional NMR and two-dimensional NMR, comparing to the literatures, the chemical structures of all isolated compounds were identified as follow.S-1 was identified as 2'-hydroxy-3,4,5,4',6'-pent- amethoxychalcone,S-2 was identified as 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, S-3 was identified as 6,7,8,3',4'-pentamethoxyflavanone, S-4 was identified as Spath- ulenol, S-5 was identified as 2'-hydroxy-3,4,5,4',6'-hexameth-oxy-chalcone, S-6 was identified as 5,7,3',4',5'-pentamethoxyflavanone, S-7 was identified as 2'-hydroxy- 3,4, 4',6'tetramethoxychalcone.2 The Contents determination of 6,7,8,3',4'-pentamethoxyflavanone and 5,7,3',4',5'-pentamethoxyflavanone2.1 Equipment, reagents and reference substanceChromatograph: Agilent 1200 HPLC Detector: PDWD variable wavelength UV detector Column: Agilent HC-C8 4.6×150mm 5μm column Balance: Mettlertoledo AG135 electronic balance Reagents: Chromatography of methanol,acetic acid,distilled water twice Reference substance:6,7,8,3',4'-pentamethoxyflavanone(compound S-3,97.8% ) and5,7,3',4',5'-pentamethoxyflavanone (compound S-6,99.2%).2 .2 Methodology validation1) Chromatographic conditions Column: Agilent HC-C8 4.6×150mm 5μm Mobile phase: methanol-0.1% acetic acid Detection wavelength: 286nm 2) The standard curveIn the range of 0.1～5.0μg ,the linear relationships of the sample peak area (Y) and mass(X,μg) of compound S-3 and S-6 were in high linearity. The regression equations of S-3 and S-6 were: Y = 3.398×103X +61.70 r = 0.9999 Y = 3.605×103X + 2.94 r = 1.00003) Detection LimitsWhen the concentration was reduced to 1×10-7mg.mL-1, the S/N of the compound S-3 was 3.75. So the detection limit of S-3 was 1×10-7×10 =1×10-6μg; When the concentration was reduced to 1×10-5mg.mL-1, the S/N of the compound S-6 was 3.57. So the detection limit of S-6 was 1×10-5×10 =1×10-4μg .4) Precision testThe same sample was tested 6 times in parallel. The RSD of peak area was 1.34% for compound S-3 and was 1.35% for compound S-6. The RSD of retention time was 0.10% for compound S-3 and was 0.04% for compound S-6. Both of them were lower than 2%, indicated that they are in good precision.5) repeatabilityWith 6 copies of the same compounds, the RSD of the content of compound S-3 was 1.28%; the RSD of the content of compound S-6 was 0.93%. They all less than 2%.6) Stability testingThe same sample was tested once every 2 hours within 12 hours. The RSD of the peak area of compound S-3 was 1.35%; The RSD of the peak area of compound S-6 was 1.26%. The RSD of retention time were in same value of 0.04% for both compounds. The results showed that compound S-3 and S-6 was stable within 12 hours and the ready sample solution can be determined within 12 hours.7) RecoveriesThe average recovery of compound S-3 was 99.9% and the RSD was 1.20%;The average recovery of compound S-6 was 101.7% and the RSD was 1.25% respectively.2.3 The Contents determinationThe compounds of Murraya paniculata (L.) Jack.were determined. The average content of the compound S-3 is 0.104% and the average content of the compound S-6 is 0.212%.This paper , we have studied on Chemical Constituentsof Murraya paniculata(L.) Jack.furtherly, has also established the quality standards of two flavanones and provides a scientific theoretical data for further research. |
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