A Experimental Study On The Antineoplastic,Antimutagenic And Immunoregulatory Activity Of Rhizoma Menipermi Extracts

Objective: To study the antineoplastic,antimutagenic and immunoregulatory activity and their mechanisms of Chinese medicinal herb Rhizoma Menipermi extracts. To compare the antineoplastic activity of Rhizoma Menispermi extract with water (RMW) and Rhizoma Menispermi extract with ethanol (RME). To isolate and purify active component and find out the active part and the active component. To produce the scientific evidence for developing and manufacturing new antitumor drugs.Methods: 1. The suppressive effect of Rhizoma Menipermi extracts on the proliferation of K562,BGC823,TE13,U937,HGC27,KATO-Ⅲ,TE1,SK-OV3,ASPC-1,MCF-7,QG-56 and SMMC-7721 tumor cells and 9 tumor cells coming from tumor tissues of patients by primary culture were assayed in vitro using cell culture technique and MTT colorimetric method. 2. Rhizoma Menipermi extract with ethanol was purified preliminariy using three steps method. PE2 was isolated and purified farther by Pole Chromatogram. The components were identified by Thin Layer Chromatogram,ESI-MS and Ultraviolet Spectro photometer. 3. The apoptosis of human gastric carcinma cell BGC823 induced by Rhizoma Menipermi extract PE2 was studied by fluorescent staining, flow-cytometry (FCM), electric microscope and DNA agarose gel electrophoresis technique.4. The effect of Rhizoma Menipermi extracts on lymphocytes and the effect on the metabolism and phagocytosis of mouse peritoneal macrophages were studied using MTT colorimetric method and neutrol red method.5. The modles bearing tumor mouse were constructed by injecting subcutaneously tumor cells, then were treated with Rhizoma Menipermi extract PE2 to observe its antitumor activity on tumor cells in vivo.6. The antimutagenicity and mutagenicity of Rhizoma Menipermi extracts were investigated using the antimutagenicity and mutagenicity synchronous test by bacterium culture technique.Results: 1. Rhizoma Menipermi extracts RMW and RME inhibited markedly the proliferation of tumor cells such as K562,BGC823 and TE13. Purified RME-PE2 had stronger inhibitory. effect than RME. PE2 also inhibited very markedly the proliferation of U937,HGC27,KATO-Ⅲ,TE1,SK-OV3,ASPC-1,MCF-7,QG-56 and SMMC-7721 tumor cells with dose-dependent relationship. PE2 also inhibited the proliferation of tumor cells coming from tumor tissues of patients by primary culture. The two monomers PF1 and PF2 also had very strong inhibitory effect. But Rhizoma Menipermi extract RMP had not inhibitory effect on K562,BGC823 and TE13 tumor cells. 2. Rhizoma Menipermi extract with ethanol RME was purified preliminarily and PE2 was isolated from RME. PE2 was testified to be alkloid by dragendorff staining. PE2 is the active part of RME by antitumor test in vitro. PE2 was purified farther and then PF1 and PF2 were isolated from PE2. Their molecular weight are 624 and 610 respectively by ESI-MS and accord with the molecular weight of Dauricine and Daurisoline. PF1 and PF2 were testified to be the active components of PE2 by antitumor test in vitro. Rhizoma Menipermi extract RMP was testified to be comparatively pure amylose by Ultraviolet Spectro photometer.3. After treatment with PE2, the BGC823 cells presented some typical morphologic features and super-microstructural changes of apoptosis, including cell shrinkage, cytoplasm concentration, nclius forming outwardly acute angle promineney, chromatin concentrating on karyotheca or forming meniscus, nuclear condensation,electric density increasing, nuclear fragmentation and formation of apoptotic bodies. Some typical subdiploid peaks before G0/G1 phase were observed. The apoptotic rate of BGC823 was 22.9% after 50μg/ml PE2 treating for 72h. DNA agarose gel electrophoresis showed characteristic "DNA ladder" pattern. After treatment with 5μg/ml PE2, the cells in G0/G1 phase were blocked and increased remarkably. But the cells in S phase were blocked after treatment with 50μg/ml PE2. And the expression of bcl-2 was inhibited whereas the expression of bax was up-regulated.4. RMP can directly st...