| Apoptosis is referred to as cell-programmed suicide. An abnormality in apoptosis causes neurodegeneration and malignancy, which may lead to uncontrolled cell growth. Therefore, the induction of cancer cell apoptosis is one of useful methods of cancer therapy. Natural products provide rich resources for induction apoptosis agents. Lycoris aurea and Nothapodytes foetida show anticancer activity, and the effective compounds have only recently been tested as clinical anticancer drugs. It is likely that they contain other anticancer compounds. In present study, in order to screen more power anticancer compounds, extract of L. aurea (DELA) and 6 compounds were isolated from L. aurea and N. foetida, Annexin V-FITC/PI, MTT assay, Real time PCR and Western bloting analysis were employed to investigate induced cancer cells apoptosis effect and mechanism. Furthermore, active compounds in N. foetida were prepared by optimization of extraction process and preparation technologys determined by HPLC. Moreover, the anticancer activity of DELA was tested on murine sarcoma 180 (S180) cells in vivo. The main results as follows,(1) Homolycorine, galanthamine, lycoramine, and DELA were isolated from L. aurea. Camptothecin,9-methoxycamptothecin, and 5-hydroxycamptothecin (diastereoisomer) were isolated from N. foetida. Furthermore, LC-MS found DELA contained a series of lycorenine-type alkaloids such as homolycorine,2a-hydroxyoduline, oduline, hippeastrine, 2a-hydroxy-6-O-methyloduline, and 2a-methoxy-6-O-methyloduline.(2) The optimized extraction technology for 9-methoxycamptothecin and camptothecin was 0.35% cellulase, pH 6.5, extracted 10 h at 45℃. Using this technology, the 0.151% content of 9-methoxycamptothecin and the 0.613% content of camptothecin were determined simultaneously in 1 kg of N. foetida, and 85.60 mg 9-methoxycamptothecin and 132.45 mg camptothecin with purity over 98% were also obtained by further purification. The results indicated N. foetida was super camptothecin resource than C. acuminata, and will be industrialization in future years.(3) Annexin V-FITC/PI was employed to determine the apoptosis induced by the components isolated from L. aurea and N. foetida.9-methoxycamptothecin and DELA were active components with induction apoptosis rate 66.46% and 30.71%, respectively. 9-Methoxycamptothecin was showed significant inhibitory effects on murine sarcoma S180 cells with IC50 0.385μM. DELA exerted significant inhibitory effects on murine sarcoma S180 cells in a dose-response manner.(4) Quantitative Real-time PCR data showed the RNA levels of Bcl-2 in the treatment groups were lower than those in the control groups. The RNA levels of Bax increased from 1 to 1.13,1.82, and 3.79 after treatment with 0,0.19,0.38, and 0.95μM 9-methoxycamptothecin, resulting in dose-depndent Bax/Bcl-2 ratios of 1,1.61,2.43, and 4.57, respectively. The results suggest that apoptotic cell death pathway is involved in 9-methoxycamptothecin anticancer effect, and was a potential anticancer drug.(5) Western bloting data showed the DELA-evoked increase in apoptotic cell death was accompanied by decreases in the ratio of Bcl-2/Bax, and increases in cleaved caspase-3. The results indicated DELA contained lycorerine-type Amaryllidaceae alkaloids, induced murine sarcoma 180 cells apoptosis. Furthermore, the high anticancer effect of DELA was proved by in vivo test on murine sarcoma S180 cell with 53.49% inhibitory tumor rate, and higher thymus index (2.98±0.61) mg/g and spleen index (9.95±1.87) mg/g than CTX group and model control group. And these results indicate that DELA exerted an anticancer effect with low toxic effects on the immune system of mice, at least in part, by activating the apoptotic cell death pathway, and was a potential anticancer drug.
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