Separation,Purification And Activity Analysis Of The Active Ingredient From Pegasus Laternarius Cuvier

Pegasus laternarius Cuvier is also known as the Sea Sparrow or Swallow.It distributes widely in tropical and subtropical waters of the western Pacific and Indian Ocean,the southern East China Sea,the Taiwan Strait and the South China Sea.In history,Pegasus laternarius Cuvier was seen as one of folk medicinal herbs for Relieving cough and sputum,dispersing lung qi and promoting eruption,tonifing the kidney yang and promoting bone xiaoyin sanjie,damping diarrhea and so on in China's coastal areas.On the basis of previous research results,We employed High Performance Liquid Chromatography(HPLC)to separate and purify chemical constituents from ethyl acetate crude extract and petroleum ether crude extract and methanol crude extract of Pegasus laternarius Cuvier,The Study showed that The optimal conditions for ethyl acetate crude extract and petroleum ether crude extract were as follows:column:SinoChromODS-BP;UV detection wavelength:200nm.mobile phase:methanol-water by gradient elution(VMethanol/Vaqueous=60/40-100/0),And the optimal mobile phase condition for methanol extract is water.It was shown that the optimal conditions were a successful method for separating ethyl acetate,petroleum ether extract and methanol extract.We separated six high-purity chemical compounds(A1,A2,A3S A4,A5,A6)from ethyl acetate and petroleum ether extract,four high-purity chemical compounds(J1,J2,J3,J4)from methanol extract with corresponding preparative column(20.0mm×250mm).Further research on the inhibition effect of compounds with variety of tumor cells:A1,A3 have no significant inhibition on proliferation of HeLa;component A2 had an inhibitory effect on human cancers,such as cell line MCF-7,cell line HepG-2,cell line 293T;component A4 had an inhibitory effect on cell line MCF-7,cell line A549,cell line 293T,it had weaker cytotoxicity for HepG-2 and had the weakest Cytotoxicity for HeLa,A5?A6 had inhibitory effect on growth of humans cell line HeLa.They are displayed higher activities with inhibitory concentration 50%(IC50)of 302.4±2.33?g/mL and 50.98±3.43?g/mL than the positive control 5-fluorouracil(IC50 = 415.410.82(?g/mL),and have no significantly inhibit the proliferation of 293T cells,and is thus of enormously potential application values.We analyzed the function of cell apoptosis with components A5 and A6 induced through flow cytometry,with the increase of drug concentration,more and more cells turned into the stage of late apoptosis or death directly,which showed that little drug could suppress cell growth obviously,a great proportion of those cells entered into the early period of apoptosis with a lower drug concentration induced,it had a limited impact on those early apoptotic cells with raised concentrations,and then they turned to the late apoptosis or death eventually.Samples were analyzed by mass spectrometry and nuclear magnetic resonance,it was difficult to determine the results accurately on account of the purity and the sample size,we concluded that A5 might be an aromatic compound,A6 might contain a steroid or terpene compounds due to its high field of hydrogen.