| Cancer is the disease that humen still has not overcome, which seriously threaten human health. Lung cancer attracted much attention for high its incidence and mortality. At present, surgery, radiotherapy and chemotherapy are the main means for tumor treatment. Among them, chemotherapy is currently the most active research area. Some anticancer drugs currently used in the clinic more than seventy, but still can not effectively control the occurrence and development of tumors. Although many anticancer drugs enter clinical studies, few drugs are efficiency and low toxicity. Natural products are an important source of anticancer drugs. Traditional Chinese medicine has used medicinal plant for more than 2500 years. Thus, looking for antitumor drugs from traditional Chinese medicine is a speedy way. Euphorbia lunulata Bge is one of the herbal medicines in traditional Chinese medicine, some studies reported that its extract has anti-tumor effect, but the material basis and the detailed mechanism were still unkown. In our study, the active ingredients of Euphorbia lunulata Bge were isolated, and its anti-cancer effects were explored.1. The extraction techniques of flavonoids from Euphorbia lunulata BgeUsing uniform design to extract the total flavonoids from Euphorbia lunulata Bge, ethanol as solvent, four factors were investigated to determine the optimal extraction conditions, including ethanol concentration (30% -90%), ethanol volume (W / V: 9 ~ 15 times), reflux time (0.5 to 3.5 hours) and returning the number (1 to 3 times). The results showed that total flavonoid extraction from the Euphorbia lunulata Bge could increasethe (3.23%) under the condition: 50% ethanol solution at 14 times of the original ingredients (W / V), reflux extract 2 times, each time the conditions for 1 hour.2. Determination of anti-tumor effect for Euphorbia lunulata Bge extract by serum pharmacologyTo further determin the anti-tumor effects, serum pharmacology and Western Blot were used. The results showed that rabbit serum containing Euphorbia lunulata Bge extract could inhibit the proliferation of A549 lung cancer cells in a concentration- and time-dependencemanner. At the concentration of 20% for 72h, the A549 proliferation inhibiting rate was 39.08%. Flow cytometry assay showed that A549 cells were arrested in G1 phase by Euphorbia lunulata Bge extract rabbit serum, and showed significant apoptosis. Western Blot analysis exhibited that the rabbit serum containing Euphorbia lunulata Bge extract could promote the apoptosis-related protein Caspase-9 and Caspase-3 protein cutting, thereby causing activation of PARP protein, increase the expression of Bax, and decrease Bcl-2 expression and Protein phosphorylation of Bad.The results above mentioned suggested that Euphorbia lunulata Bge could induce apoptosis of A549 cells.3. Determination of the anti-tumor effect for Euphorbia lunulata Bge extractTo evaluate the effects of Euphorbia lunulata Bge extracts on tumor growth, Lewis lung tumor mouse models were used, and the tumor weight, the general performance of animals, body weight and thymus and spleen weight were observed. The results showed that after continuously administrated for 19 days, Euphorbia lunulata Bge extract at concentration of 1 and 0.5g/kg could significantly inhibit the Lewis lung cancer growth in mice (compared with model group, p <0.01 and 0.05), and inhibiting rates were 41.14% and 40.40%. Euphorbia lunulata Bge extract at 0.25g/kg also showed inhibitive effects on Lewis lung tumor in mice, and inhibiting rate was 21.31%. But not statistically significant was showed. Euphorbia lunulata Bge extract at 1 and 0.5g/kg concentrations reduced the weight of the thymus and spleen, but only 1g/kg for spleen showed statistical significance (compared with model group, p <0.01). The corresponding calculation of the spleen index and thymus index also showed the same trend, which suggests the influence on immune function. But it also might be the indirect effects after suppression of tumor growth. For general observation during the experiment, only cyclophosphamide and high dose of extract of Euphorbia lunulata Bge (1g/kg) group showed slightly body weight loss. The mental state of each group of animals, including food, water, feces and urine showed no abnormals.4. Extraction of Euphorbia lunulata Bge active ingredients and activity trackingThe major flavonoids component of Euphorbia lunulata Bge 50% alcohol extract were identified as quercetin-3-O-β-D-glucuronide and kaempferol ( 1 ) and 3-O-β-d-glucuronic acid(2)by structure guided fractionation. The subfractions together with the pure compounds were also test for their antitumor activity against A-549 and HL-60 cell lines, which lead to the results that compounds 1 and 2 were not show antitumor activities directely, while the organic soluable components were active.5. The anti-tumor effects and mechanisms of Euphorbia lunulata Bge active ingredients in molecular and cellular levelsUsing kinase screening platform and related technologies, combined with Western Blot, the mechanism and anti-tumor effect of Euphorbia lunulata Bge active ingredients were explored. Results showed that the Euphorbia lunulata Bge active ingredient could specifically inhibit the phosphorylation of EGFR, and the IC50 value was 0.32μg/ml. Using human umbilical vein endothelial cells to simulate blood vessels formation, Euphorbia lunulata Bge active ingredients showed the inhibitive effect on the tube formation, suggesting that inhibition of angiogenesis. Next, we observed the effects of Euphorbia lunulata Bge active components on cell migration. The results showed that the Euphorbia lunulata Bge active ingredient could significantly inhibit tumor cell migration. Related signaling pathway analysis showed that Euphorbia lunulata Bge active ingredients could inhibit the phosphorylation of EGFR and downstream AKT and ERK activation.The active ingredient in Euphorbia lunulata Bge was extracted in this study. The anti-tumor effects and mechanism were first explored, which provide theoretical basis for the development and utilization of Chinese medicine Euphorbia lunulata Bge... |
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