| Oroxyli semen is a traditional Chinese medicine, which is the dry seeds of Oroxylum indicum (L.) Vent. and clinically used in the treatment of acute and chronic bronchitis,tuberculosis,cough,sore throat and tonsillitis etc. A series of chemical investigations revealed that flavonoid aglycones and flavonoid glycosides were the major compositions of Oroxylum indicum. In addition,there are phenylethanoids and cycolhexylethanoids as well as volatile oil in it.In this research, ten compounds were isolated from the seeds of Oroxylum indicum (L.) Vent. Various spectral methods, such as NMR, MS, IR and UV, were used to identified their structures,which were testified as Oroxin B (M1), Oroxin A (M2), Chrysin (M3), Chrysin-7-O-β-diglucoside (M4), Chrysin-7-O-β-galactopyranuronoside. (M5), Apigenin (M6), Lupeol (M7),2α,3β-dihydroxyllupeol, Stigmasterol (M9), Oleic acid (M10). In which, Chrysin-7-O-β-galactopyranuronoside (M5), Lupeol (M7),2 aα,3β-dihydroxyllupeol (M8) and Stigmasterol (M9) were first isolated from the seeds of Oroxylum indicum (L.) Vent.In this paper, the quantification analytical method of two flavonoid glycosides (M1 and M2) using HPLC were also investigated in order to improve the quality standards for the seeds of Oroxylum indicum (L.) Vent. The contents of Oroxin A and Oroxin B in the seeds of were found absolutely high by detecting three batches of samples.Extraction, separation and identificationDried seeds of Oroxylum indicum (L.) Vent. in 3 kilograms were crushed and extracted by heat reflux in 85% ethanol (the volume fraction) twice (1.0h and 0.5h respectively).The combined extract was evaporated to dryness under reduced pressure and 185g of extracts were obtained.By using silica gel,ODS and polyamide column repeatedly.ten compounds previously described were isolated. The mobile phase was made of petroleum ether, methanol,water and so on in various ratio.According to their physical and chemical properties, UV,IR,MS and NMR were used to identify the structures of the compounds isolated after comparing with the literature reports.All the isolated compounds were identified as Oroxin B(M1), Oroxin A(M2), Chrysin(M3), Chrysin-7-O-β-diglucoside(M4), Chrysin-7-O-β-galactopyranuronoside(M5), Apigenin(M6), Lupeol(M7),2,3-dihydroxyllupeol(M8), Stigmasterol(M9), Oleic oil(M10).Contents determination of Oroxin B and Oroxin AThe research was performed on Agilent 1200 HPLC instrument and COSMOSIL-MS-II C18 coloum (4.6 X 250mm,5 u m), using a mixture of methanol-water-phosphate in ratio of 40:60:0.2 as the mobile phase. The flow rate was 1.0 mL.min-1 at 25℃, the wavelength of VWD detector was set at 278 nm.ResultsI Theoretical plate numner and ResolutionThe Theoretical plate numner of M1 and M2 were 3800 and 6500 respectively; The Resolution of Ml and M2 were 6.43 and 2.91 respectively.II Precision testSolution of compounds M1 and M2 was injected into HPLC for six times in one day, the RSD of peak area and retention time for M1 were 0.76% and 0.09%,for M2 were 1.47% and 0.10%,respectively;The same solution was injected into HPLC once a day for six days, the RSD of peak area and retention time for M1 were 0.98% and 0.28% for M2 were 0.85% and 0.33%.ⅢThe standard curveThe linearity was in the range of 0.05-2.0μg for both compounds M1 and M2,and the correlation coefficients were both 0.9999. The regression equations were Y=2371.9X-10.88(M1) and Y=2978.8X-21.93 (M2).IV RepeatabilitySix copies of the same compound were injected into HPLC, the RSD of peak area for M1 and M2 were 0.76% and 0.42% respectively. V Stability testThe same solution of sample was injected into HPLC in 0h,2h,4h,8h,16h and 24h, the RSD of peak area and retention time for Ml were 0.28% and 0.34% respectively, the RSD of peak area and retention time for M2 were 0.43% and 0.48% respectively.ⅥLOD and LOQFor both compound compound M1 and M2, the experiment gave their detection limit (LOD) in 2ng and quantification limit (LOQ) in 5ng.ⅦRecovriesThe average recovery for compound M1 was 100.41% and the RSD (n=6) was 2.26%; the average recovery for compound M2 was 101.95% and the RSD (n=6) was 1.07%.ⅧContents determination of samplesThree batches of samples of the seeds of Oroxylum indicum (L.) Vent. were determined,and the results showed that the average content of compound Ml was 3.58%,3.85% and 3.12 the average content of compound M2 was 4.16%,4.41% and 3.80%,respectively.In this paper, the chemical constituents of Oroxylum indicum was furtherly studied, and new index of content determination was offered to improve quality standard of the seeds of Oroxylum indicum (L.) Vent..The results of this paper provided a theoretical support for further study and a scientific basis for development and utilization of Oroxylum indicum. |
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