Anti Lung Cancer Effect Of Alkaloidal Compounds Extracted From Houttuynia Cordata Thunb

Lung cancer is the fastest-growing cancer with the highest morbidity and mortality at present, which has become the biggest threat of malignant tumor for human life and health. Studies on natural anti-cancer drugs are more and more popular at present,especially the study on the bioactive components from higher plant. Houttuynia cordata Thunb(HCT)is a medicinal plant of the Saururaceae family which features antiviral and antimutagenic properties. HCT has a good therapeutic effect for lung carbuncle which is closed related to lung cancer. The bioactive components of HCT are mainly volatile oil, flavonoids and alkaloids. The studies of HCT are focused on volatile oil and flavonoids for the antiviral and antimutagenic properties. The anti-cancer therapeutic effect of alkaloids in HCT is less reported. The issue of this topic is to observe the effect of HCT-A to lung cancer cells and try to explain the mechanism. The topic consists of three sections which are respectively the extraction and purification of HCT-A, apoptosis assay induced by HCT-A and the study of apoptosis mechanism.Firstly, the extraction and purification of HCT-A. Refluxing extraction method was used. Then optimized procedure of purification was conducted. Berberine hydrochloride、piperolactam and aristololactam B were used as indexes. The result was that the loading concentration regulated as 20 mg/ml, p H regulated approximately 3, the maximal loading volume up to 40 ml, absorbing flow velocity reached to 0.5 ml/min, stewing 2 h, purified water of 60 ml used to rinse impurities, 1% ammonia ethanol(0.5N ammonium hydroxide, 80% ethyl alcohol) used as elution solvent, the volume of elution solvent up to 70 ml, the elution rate setted as 3.0ml/min. Finally, 11.375 g alkaloidal extractum was harvested from 4 kg hay and the yield of alkaloids extracted from Houttuynia cordata Thunb was measured as 0.2759%.Secondly, cell prolifation inhibition and the apoptosis assay induced by HCT-A. Five lung cancer cells(95D、A549、SPC-A-1、H1975、H460) are chosen as the experimental subject. MTS assay was conducted to observe the proliferation effect in vitro. Data showed that HCT-A exerted markedly inhibition effect to the five lung cancer and the effect increase with the increase of dose. The ICS50 of 95D、A549、SPC-A-1、H1975、H460 were respectively 29.51 μg/ml、72.04 μg/ml、67.94 μg/ml、54.00 μg/ml、130.17 μg/ml. Then observe the cell morphology after the incubation of a series of concentrations(0、1/2IC50、IC50、2IC50). Cells begun to shrink and became small 48 h later. With the increasing concentration, cells partly fall off with the increasing black spot. In order to find out whether apoptosis happened, Hoechst 33258 and Annexin V/PI assay were designed.Pictures of the cells stained by Hoechst 33258 under the microscope showed that the blue apoptosis body appeared. The result of Annexin V/PI assay showed that the percentage of early apoptosis cells increase with the dosage increasing and the difference is statistically significant.Thirdly, the study of the cell prolifation inhibition and the apoptosis mechanism induced by HCT-A. The effect of HCT-A to cell cycle was conducted. 95 D, A549 and H460 were chosen as the assay subject and the concentration was set as 0,1/2IC50, IC50. The result showed that HCT-A can arrest cell cycle at G0/G1 phase. Next, western blot was conducted in A549 and the concentration was set as 0,40, 70, 140 μg/ml. Total protein was extracted after a incubation of 48 h. The result showed that expression quantity of p53 and Bax increased and Bcl-2 decreased. So the ratio of Bax/Bcl-2 increased. It showted that the apoptosis induced by HCT-A is related to p53 and Bcl-2 family. At last,the Caspase activities were measured after a incubation of 48 h. The result showed that activity of Caspase-3/-9 increased and exerted no obvious impact on Caspase-8. It could be concluded that the apoptosis has a close relation with the increasing activities of Caspase-3/-9.This topic studied the effect of HCT-A on lung cells from several aspects. The mechanism of apoptosis induced by HCT-A was preliminary explained. All of this work can provide theoretical basis for the exploitation of anti-lung cancer drugs from HCT.