| Piper laetispicum C.DC. (Piperaceae), which enjoys vast uses for invigorating circulation and reducing stasis, detumescence and analgesic in the southern part of China, is popularly known in folk as a folk medicine, Present studies show that extracts of P. laetispicum C.DC. have distinctly antidepressant activity. In addition, its activity relates with a serial of amides.To further study on the antidepressant bioactive constituents and analytical method of Piper laetispicum, which can serve research and development of new drug, a new model called Reverse R&D was used. Based on the absorbed components analysis, preparation, structure identifying and bioactivity screening of absorbed components of Piper laetispicum C.DC., the antidepressant bioactive constituents were confirmed. Finally, a method for the simultaneous determination of multicomponent was developed, it is useful as a technical support for further study on the antidepressant bioactive constituents and a measure method for intermediate and preparation. The main contents were as follows:1.Analysis of components migrating into bloodAt the beginning, in vivo animal model of depression was used to verify antidepressant activity of Piper laetispicum C.DC..The results showed that it has distinctly anti-depressant activity. Then everted gut scas were used to analysis the intestinal components of the extractive Piper laetispicum C.DC. absorbed by the different intestinal section in vitro. It showed that most of compounds which can be detected by the HPLC were absorbed in the intestine.16 compounds in the duodenum, 15 compounds in the colon and jejunum respectively,10 compounds in the rectum were detected considering 19 compounds as total in solution. This indicates that most of the compounds of Piper laetispicum C.DC. can be absorbed into the blood.2.preparation and structure identifying of components migrating into bloodTake the HPLC chromatogram of constituents migrating to blood as the reference, 14 compounds including 12 amide alkaloids were separated and purified by the means of Macroporous resin, silica gel and flash rapid preparative chromatography. G13-1(1-[1-oxo-9(3,4-methylenedioxy-phenyl)-2E,4E,7E-nonatrienyl] pyrrolidine) and G13-2(1-[1-oxo-9(3,4-methylenedioxy-phenyl)-2E,7E-nonadienyl] pyrrolidine) are new compounds, G9(N-isobutyl-7(3,4-methylenedioxy-phenyl)-2E-4E-HeptaDie-namide),G12 (1-[1-oxo-3(3,4-methylenedioxy-5-methoxy-phenyl)-2E-propenyl] pyrrolidine) and G14 (1-[1-oxo-7(3,4-methylenedioxy-phenyl)-2E,4E-heptadienyl] pyrrolidine) are separated initially from Piper laetispicum C.DC. The purity determined by HPLC-PDA of 12 amides alkaloids is more than 98% indicating these amides alkaloids can be used as reference substances in the following study.3.Study on antidepressant activityForced swimming test and tail suspension test ha been used in this part. The results demonstrated that compound G5, G6 and Gll have distinctly anti-depressant activity. In the one single dose test, the activity of G5 and G6, which was stronger than the same dose of positive drug fluoxetine group was significantly different from the control group, G6 is the laetispieine and its activity is consistent with the reported, which shows that amide alkaloids are material basis of antidepressant activity of Piper laetispicum C.DC.4.Study on Analysis MethodA simple, rapid, precise and accurate reversed-phase HPLC method had been developed and validated for the simultaneous determination of the 12 amide alkaloids in Piper laetispicum. The method had shown adequate separation for separating these 12 compounds from their associated main impurities. Separation was achieved on a 5μm,250mm×4.6mm i.d. column using a mobile phase consisting of acetonitrile-water (0 min,55:45;20 min,60:40;30min,90:10; 32 min,55:45;37 min,55:45,) at a flow rate of 1 ml/min and UV detection at 240 nm. Good linearity over the range 0.8-300μg/ml was observed, and the regression coefficient for the linear equations for the 12 compounds lay between 0.9995 and 1. The intra-day and inter-day precis-ions (CV) of analysis lay in the range of 0.22-1.86 and 0.55-1.90%, respectively, and the accuracy ranged from 85.46-116.36%. This method had been successfully applied to the analysis of 12 amide alkaloids in Piper laetispicum. |
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