| Background:Cancer is a major cause of death in the world. Comprehensive treatments with primary surgery integrated with chemotherapy or radiotherapy is the main therapeutic method currently, but the recurrence and metastasis of tumor after operation is one of the most difficult issues in clinical. In particular, there is lack of effective therapeutic method to prevent or cure advanced tumors. Therefore, it is urgently and necessary to find an effective, low cost and innocuous drug for preventing and curing cancer diseases. E. helioscopia (Family Euphorbiaceae) is widely distributed in the most parts of China. It has been traditionally used for the prevention or cures of various diseases such as liver cancer, esophagus cancer, nasopharyngeal carcinoma, bronchitis, acute glomerulonephritis, and epidemic parotitis in China for centuries. Modern researches reveal that the water extraction of E. helioscopia (EWE) has anti-tumor activity in vitro and in vivo. Quercetin, Lupeol, gallic acid, and hyperoside, which are extracted from E. helioscopia, are proved to have anti-tumor activities. Therefore, it is necessary and urgent to develop and utilize of E. helioscopia. But there are only a few studies on anti-tumor effects of E. helioscopia. Which part is the active site of anti-tumor effects? Which component plays an important and key role in its pharmacologic actions? What is the role of trace elements? Are trace heavery metals the source of toxicity? There are no answers about all of these issues.Objectives:To study the anti-tumor activity and the mechanism of the E. helioscopia; To standardized a reversed-phase high-performance liquid chromatography (RP-HPLC) assay for active component detection; To determine the contents of Ferrum, Zinc, Cuprum, Manganese Plumbum, Cadmium, Arsenic, and Mercury in E. helioscopia.Methods:Systemic research ways of phytochemistry were adopted for extraction and separation active principles; Human gastric carcinoma SGC-7901, hepatic carcinoma HepG2, SMMC7721, and lung cancer A549 were treated respectively with different extraction of E. helioscopia, and then MTT assay was used to observe the inhibition rate of carcinoma cell lines; The morphologic examination and the ultrastructure of tumor cells was conducted through inverted microscope and TEM, respectively; The apoptosis and cell cycle were examined through FCM; The invasiveness was examined in Transwell chamber; Relative concentrations of MMP-9 in the culture medium were measured with ELISA. RT-PCR was used to detect the expressions of Caspase3, Bcl-2, Bax, Fas, Cytochrome C, and P53 mRNA in the cell lines. The main active principles were separated and analyzed by RP-HPLC using Agilent 1120 HPLC TC-C18 column (250 mm x 4.6 mm; 5μm) with UV-detector system. The mobile phase of methanol-0.2% phosphoric acid (65:35) solution was used with the flow rate of 1.0 mL-min-1 at 30℃and the detection was performed at 360 nm wavelength. The contents of Ferrum, Zinc, Cuprum, and Manganese were determined by flame atomic absorption spectrometry; The Plumbum, and Cadmium were determined by graphite furnace atomic absorption spectrometry; The Arsenic, and Mercury were determined by atomic fluorescence spectrometry.Results:The inhibition rate of chloroform extraction in 400μg-mL-1 on human hepatic carcinoma HepG2, SMMC7721, gastric carcinoma SGC-7901, and lung cancer A549 were 48.3%,86.4%,48.3% and 25.8%, respectively. The inhibition rate of petroleum ether extraction in 400μg·mL-1 on human hepatic carcinoma HepG2, SMMC7721, gastric carcinoma SGC-7901, and lung cancer A549 were all less than 30%. Round or irregular in shape, the decrease of intercellular junction and many deposits of brown granular materials in cytoplasm could be observed through the common inverted microscope and the shrinking of cell volume, the decrease of microvillui and mitochondria, and the increase of nuclear heterochromatin could be observed through electron microscope in hepatic carcinoma HepG2, SMMC7721, gastric carcinoma SGC-7901, and lung cancer A549 cells in both chloroform extraction group and petroleum ether extraction group when the concentration was 400μg·mL-1. The analysis of cell cycle indicated that chloroform extraction blocked hepatic carcinoma HepG2, SMMC7721, and gastric carcinoma SGC-7901cells at G2/M phases and cells of G0/G1 and S phase reduced both in the 300μg·mL-1 group and 400μg·mL-1 group. The apoptosis rate of SMMC7721,SGC-7901, HepG2 cells in 400μg·mL-1 group were 1.518%,11.361%,18.693% at 24 hours and the apoptosis rate of SMMC7721, SGC-7901 cells were 26.738% and 18.28% at 48 hours. The invasive inhibiton rate of chloroform extraction on gastric carcinoma SGC-7901, hepatic carcinoma HepG2, and SMMC7721 cells were 25.5%,20.4%,21.0% in the 300μg·mL-1 group and 46.5%,40.7%,35.6% in the 400μg·mL-1 group; The concentrations of MMP-9 in the culture medium of gastric carcinoma SGC-7901, hepatic carcinoma HepG2, and SMMC7721 cells were 46.5±2.14 ng·mL-1,29.3±3.01 ng·mL-1,31.8±195ng·mL-1 in the 300μg·mL-1 group and 33.1±233 ng·mL-1 2.98 ng·mL-1,26.8±2.29 ng·mL-1 in the 400μg·mL-1 group. Compared to the control group, the expression of P53, Bax, Cytochrome C, Caspase3, Fas mRNA were increased and Bcl-2 mRNA was decreased in experimental group. The ratio of Bax/bcl-2 was raised. The highest quercetin content (average 1.42 mg·g-1 and 3.57 mg·g-1 dry-weight) were present in leaves and in June, respectively. The contents of Ferrum, Zinc, Cuprum, and Manganese is 713.4677μg·g-1,23.9684μg·g-1,18.4504μg·g-1,257.4068μg·g-1, respectively; The contents of Plumbum, Cadmium, Arsenic, and Mercury is 2666.8877 ng·g-1,127.1414 ng·g-1,0.4918μg·g-1,0.2098μg·g-1, relatively.Conclusions:(1) Both petroleum ether extraction and chloroform extraction had significant effects on the morphology and ultrastructure of SMMC7721, HepG2, SGC-7901 and A549 cells; (2) Hepatic carcinoma SMMC7721 was inhibited most significantly by chloroform extraction, the order of inhibitory effect was SMMC7721 > SGC-7901> HepG2> A549. (3)The chloroform extraction could block SMMC7721, HepG2, and SGC-7901cells at G2/M phases and cells of G0/G1 and S phase reduced. (4)The chloroform extraction could inhibit the proliferation and induce SMMC7721, HepG2, and SGC-7901 cells apoptosis time-dependently and dose-dependently. (5) The chloroform extraction could inhibit the inverse ability of SMMC7721, HepG2, and SGC-7901cells through decrease the concentrations of MMP-9 in the culture medium. (6) The chloroform extraction could induce apoptosis through signal transduction of mitochondrial and caspase pathways or fas and caspase pathways. (7) Quercetin was one of the main active components. HPLC could be used as quality control for content determination of quercetin. The leaves of E. helioscopia are the best raw material of quercetin and the best acquisition time is June. (8) The contents of Ferrum, Zinc, Cuprum, and Manganese are relatively lower in the roots, stems and leaves of E. helioscopia. The contents of Plumbum, Cadmium, Arsenic, and Mercury are below the limit reference to import and export of medicinal plants and preparation of green industry standards, they may not be the toxic principles of E. helioscopia. |
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