| Traditional mongolian medicine Pipper longum L. is one of research topics in our laboratory. Piperine and piperlonguminin are two of the major alkaloids in Pipper longum L. Piperine (GBO) exhibits a wide variety of biological effects, including antioxidant properties, anti-platelet and hepatoprotective effect, etc. Piperlonguminin (GBN) was separated from Piper Longum L. and had been found to have a remarkable Hypolipidemic activity with low toxicity at the first time in our team. First, the components of Mongolian medicine Pipper longum L. were analyzed in vitro.We got the HPLC-UV fingerprints of Pipper longum L. and did in vivo detection. Then, three kinds of sample of Pipper longum L.(75% ethanol extract of Piper longum L., water extract of Piper longum L., the crude drug of Piper longum L.) were administered to rats. The blank rat serum and rat serum sample after an oral administration of drug are treated by acetonitrile precipitation, methanol precipitation, ethyl acetate extraction and solid-phase extraction and determined by UPLC-UV. The result shows that he optimal analytical conditions were using ACQUITY UPLC(?) HSS T3 column (2.1 mmx 100mm,1.8μm, Waters Corporation, USA), column temperature being set at 30℃, sample cell temperature was set at 10℃, with solvent of acetonitrile-water-formic acid (50:50:0.01%, v/v) at a flow rate of 0.2mL/min, samples were monitored at 338 nm, when choosing the solid phase extraction method as a pretreatment method for serum samples and the extract of Pipper longum L. by 75% ethanol as the administration drug. On this basis, the method was established for simultaneous SPE-UPLC determination of GBN and GBO in rat serum after oral administration of extract of Pipper longum L.The calibration curves showed good linearity of GBN and GBO in rat serum in the concentration range from 1-100μg/ml and 0.6～12.8μg/ml; linear regression equations are y=61916.46x+13473.46 (r= 0.9990) and y=61457x+5725.385 (r= 0.9989); the recoveries for method are 101.69%～108.71%(n=5) and 100.61%～111.53% (n=5); the limit of detection values are both 0.15～0.2μg/ml; the limit of quantitation are both 0.6-0.7μg/ml; the relative standard deviation (RSD) between intra-day and inter-day is less than 10% (n=5). Therefore this method is suitable for simultaneous determination of GBN and GBO in rat serum. |
