Research On Material Base Of Anticancer Formula “Shenqi Powder”

Chinese medicine formula, anticancer of “Shenqi San”, which is a hospital preparations from Kede hospital of wuhan, the prescription from professor chang tet jong ancestral secret recipe, composed of ten flavors traditional Chinese medicine formula, such as American ginseng, notoginseng, spreading hedyotis herb and so on, which can beneficial blood, soft firm fights, analgesic efficacy of detoxification and so on. Professor Chang tet jong experienced for more than 40 years, used in tumor patients treated cases for thousands in clinical prescriptions, which can improve symptoms, control tumor development, reduce patient pain, improve the quality of survival, welcomed by the masses of patients. In this paper, on the basis of clinical curative effect, studied its anticancer active substance, aims to interpret the hospital preparations efficacy material base, provide scientific basis for the clinical curative effect.Review of relevant literature of chemical constituents of single flaver in formula of anticancer “Shenqi San”. Retrieval chemical constituents and pharmacological action of each flaver in this formula, and has carried on the square to the traditional Chinese medicine compound solution. At the same time, retrieval large number literature of previous studies in traditional Chinese medicine formula on antitumor and traditional Chinese medicine formula material substance basic research, learn other people’s thinking, draw lessons from the research method of others. On the basis of literature review, the completion of this paper were reviewed, for the study of the paper will provide referential basis for the design and the subject.As anticancer formula “Shenqi San” as a whole, using modern supercritical CO2 extraction technology and the traditional solvent extraction, got seven different chemical parts, the separation of supercritical CO2 extract A, total polysaccharides B, petroleum ether extract C, chloroform extract D, ethyl acetate extract E, n-butyl alcohol extract F and water soluble substances G; Establishe lung adenocarcinoma A549 cell model, determined the most anti-cancer activity in these chemical parts by MTT method. The results show that the chloroform extraction material parts has significant antitumor activity, other material parts antitumor activity is not obvious, therefore determine chloroform extraction material parts as anticancer active material parts in Chinese medicine formula.Using HPLC – MS modern chromatographic techniques, analyzes the chemical composition of chloroform extraction material parts, identified eight compounds, such as 3- pyridine carboxylic acid, barbatin C, asperulosidic acid, deacetylasperuloside, 2- cyclohexene- 1- ketone, scutebarbatine F, barbatine D and scutebarbatine G. Through literature retrieval, the above are bioactive compounds for a variety of cancer cells.We use the chromatographic separation methods such as high performance liquid chromatography(HPLC) instrument, and eight monomer composition were isolated from effective parts of the communist party, we adopted the method of experiment in vitro activity tracking filter,and by 1H-NMR、13C-NMR、MS and other modern spectral methods to analyze these structures, identified five compounds, respectively: Prosaikogenin D 、 Prosaikogenin F 、β-sitosterol、SS-6、ursolic acid. Five compounds which have identified, the IC50 were 65.4, 57.2, 58.9, 4.9, 88.4μg/m L respectly. Because SS-6(3β,16β,23-3hydroxy-13,28-dimethyl-11-ene-3-O-β-D-pyran methyl glucoside) IC50 is ideal, and did not see related anti-tumor research through the literature,so we select its effective concentration(1, 2, 4 and 6μg/m L) lays the foundation for the research of the mechanism of the subsequent.On the basis of determining characteristics of anticancer active material parts and anticancer active ingredients, use MTT method determined the cell vitality, use fluorescent staining method determination of cell morphology, use flow cytometry instrument to detect cell cycle and cell apoptosis changes, methods of protein activity assay and western blot experiments, discusses the mechanism of anticancer active substance. The results show that the chloroform extract has obvious inhibitory effect on proliferation of A549 cells, and the dose and time dependent; Cell morphology changes obviously, presents the typical apoptosis, and with the increase of drug concentration in the apoptosis rate increased; Western blot test the effect of anticancer effective material for protein expression, can be activated Caspase 3, 8, 9, promote enhanced Bax apoptosis gene expression, cut the Bcl-2 expression suppression of apoptosis gene, Bax/Bcl-2 ratio increased significantly. Effective substances show that anticancer formula “Shenqi San” induced A549 lung cancer cell apoptosis through death receptor pathway and mitochondrial pathway, and related to increase Bax and cut the Bcl-2 expression.Using Western blot method to study the mechanism of compound SS-6 action on human lung adenocarcinoma A549 cells, tested the PI3K/Akt/m TOR pathway and protein expression in autophagy related. Research results show that the compound SS-6 can obviously inhibit proliferation of A549 cells, in a dose-response relationship, which can cut the expression of Bcl- 2, raised the expression of Bax, via increased Bax/Bcl- 2 activated Caspase protein family, prompted PARP shear, mitochondria induced by apoptosis of A549 cells. We detect PI3K/AKT/m TOR signaling pathways. Autophagy as another way of programmed cell death,we also detected the expression of autophagy related proteins by western blot method, results show that the LC3Ⅱ/LC3Ⅰ, beclinl, Atg5, Atg7 and Atg12 protein expression were significantly increased, the results can explain SS-6 on A549 cell PI3K/AKT/m TOR signaling pathway and its downstream target protein phosphorylation has inhibitory effect, suggests that SS-6 resistance mechanism of action of human lung adenocarcinoma A549 cells is inhibition of PI3K/AKT/m TOR signaling pathways, cause cell autophagy and apoptosis.This paper by the method of interdisciplinary research, screening the anticancer effective material part of hospital preparations anticancer formula “Shenqi San”, analysis and identify the main anticancer active ingredients of effective material part, studied the mechanism of the anticancer active substance. Results explain the efficacy material base of the hospital preparations, provides the scientific basis for its clinical curative effect, and laid a certain foundation for its further development and innovation of TCM varieties.