BÁO CÁO KHOA HỌC: "Nghiên cứu độc tính và tác dụng kìm hãm tăng sinh dịch chiết nước và cồn của nấm đa niên lên tế bào invitro của người và khỉ" ppt
Nghiên cứu độc tính và tác dụng kìm hãm tăng sinh dịch chiết nước và cồn của nấm đa niên lên tế bào invitro của người và khỉ
Dịch chiết nước va cồn của một số loai nấm lỗ đa nien thuộc các chi Ganoderma ( duối chi Elfwingia),Phellinus, Nigrofomes, Perenniporia cũng như hỗn hợp của chúng không thể hiện ảnh hưởng độc tế bào đối với các dòng tế bào đã được thử nghiệm bao gồm Hella( tế bào ung thư cổ tử cung),Vero-B4 ( tế bào thận của loài khỉ xanh Châu Phi), Hep-G2 (Tế bào ung thư gan), CLS-354 (tế bào ung thư hình vảy trong miệng) và MDA-MB 436 ( tế bào ung thư vú của người). Sau khi sử lýyý với dịch chiết nước và cồn, chúng ta quan sát thấy sự ức chế phụ thuộc nồng độ dịch chiết lên quá trình tăng sinh của tế bào ở nồng độ cao; một số dịch chiết biểu hiện tác dụng kìm hãm quá trình tăng sinh tế bào. Các tế bào thể hiện nhiều đặc tính quan trọng của quá trình appoptosis. INTRODUCTION
For millennia, mushrooms have been valued by humankind
as an edible and medical resource. A number of bioactive molecules, including antitumor substances, have been identified in many mushroom species. Polysaccharides are the best known and most potent mushroom-derived substances with antitumor and immunomodulateing properties (Wasser, 2002). Moreover, the small-weighed molecules such as terpens are also proved that having pharmaceutical properties.
In recent decades, people have studied much about fungi classification, biology, cultivation techniques and about bioactive compounds, medical properties, etc. of some important annual fungal species. However, the perennial polypore species are still not studied carefully.
In the present study, we investigated the effects ethanolic and aqueous extracts of some wood-inhabiting polypores o- n the growth of human cervix carcinoma (Hela), African green monkey kidney (Vero-B4), human hepatocellular carcinoma (Hep-G2), squamous carcinoma (mouth) (CLS- 354) and human breast carcinoma (MDA-MB 436) cells. (Strains are signed E1, H1, H3, N1, and P1).
1. Preparation the polypore extractions
We have 5 samples of 5 Vietnamese fungal species, they are: Phellinus sps (H3, H1), Nigrofomes sp (N1), Perenniporia sp (P1), Ganoderma sp, sub genus Elfwingia (E1). To extraction, we take 300g each sample. With each sample, we boil it with water 3 times, each in 2 hours. Then, mix 3 solutions and boil down until we have about 25-30ml. The solutions are lyophilized. Then we dissolve them again with water and store in the refrigerator.
The rest materials are dried by sunlight. All 5 samples are again extracted in Ethanol 96%, 98% in 24 hours. After 24
hours, we distill them and then dissolve again with EtOH and store in the refrigerator for later experiments.
2. Cells and culture conditions
Cells of adherent cell lines were cultured in RPMI 1640 medium (GIBCO BRL g/ml42402-016), supplemented with 100 U/ml penicillin G-sodium salt/100 streptomycin- sulfate (GIBCO BRL 15140-114), 10% heat-inactivated fetal bovine serum (GIBCO BRL 10500-064), and L- glutamine (GIBCO BRL 25030-024) at 37ºC in high density polyethylene flasks (NUNC 156340). Hela cells were grown in RPMI 1640 culture medium (GIBCO BRL 21875-034) g/mlsupplemented with 100 U/ml penicillin G-sodium salt/100 streptomycin-sulfate (GIBCO BRL 15140-114), 10% heat-inactivated fetal bovine serum (GIBCO BRL 10500-064), NS 10 ml/l non-essential amino-acid (GIBCO BRL 1140-035) at 37ºC in high density polyethylene flasks (NUNC 156340). The cells were harvested at the logarithmic growth phase after soft trypsinization, using 0,25% trypsin in HBSS containing 0,038% EDTA (GIBCO BRL 25200-056).
Antiproliferative and cytotoxic assays
The cells were incubated with ten concentrations of the test compounds. For eacg assay 10000 cells were seeded with 0,1 ml RPMI 1640 culture medium (GIBCO BRL 21875- 034), containing 10% heat-inactivated fetal bovine g/mlserum (GIBCO BRL 10500-064), 100 U/ml penicillin G-sodium salt/100 streptomycin-sulfate (GIBCO BRL 15140-114) into 96-well microplates. The plates were previously prepared with ten dilutions of test substances in 0,1 ml RPMI 1640 medium. Hela cells were preincubated for 48 h without the test substances. Cells were incubated for 72h at 37ºC in a humidified atmosphere and 5% CO 2 .
The monolayer of the cells were fixed by glutaraldehyde and stained with a 0,05% solution of methylene blue for 15 min. After gently washing the stain was eluted by 0,2 ml of 0,33 N HCl in the wells. The optical densities were measured at 630 nm in a Dynatech MR 7000 microplate reader. Comparisons of the different values were performed with Microsoft Excel. From the dose response curves the IC50 values (concentration which inhibited cell growth by 50%) were calculated with CASYSTAT (Schọfe), software for data evaluation. The IC50 was defined as being where the concentration-response curve intersected the 50% line, determined by means of the cell counts/ml, compared to control.
RESULTS AND DISCUSSION
The reseach data are demonstrated in table 1 and differend figures:
The IC50 values were found and shown in the table above. The IC50 values of the perenial polypores extracts in these cell lines are completely different. In some cases, the IC50 values are higher than even the first concentrations used, such as in Hela with all aqueous extracts or in Vero-B4 with aqueous extracts of N1, E1, P1 and mixture. However, in g/ml.some other cases, the IC50 values are smaller than 10
The dose-resonse curves of the perenial polypore extracts on cell proliferation after 72 hours of incubation are depicted in the following figures.
All extracts and their mixtures do not exhibit cytotoxic effect against the tested cell lines. Upon treatment with ethanolic and aqueous extracts, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis. At high concentration, some of them express antiproliferative effect.
Some authors confirm that many, if not all, Basidiomycetes contain biologically active polysaccharides in fruit bodies, cultured mycelium, culture broth. Their polysaccharides are of different chemical composition, most of which belong to the group of ò-glucans, these have ò-(1 >3) linkages in the main chain of the glucan and additional ò-(1 >6) branch points that are needed for antitumor actions. Mushroom polysaccharides prevent oncogenesis, show direct antitumor activity against various allogeneic and syngeneic tumors, and prevent tumor metastasis. Polysaccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antiumor action of polysaccharides requires a T-cell component, their activity is mediated through a thymus-dependent immune mechanism. These conclusions are given after many satisfactory results obtained by many research groups on Grifolia frondosa, Trametes versicolor, Flamulina velutipes, Lentinula edodes, Wolfiporia cocos, Agaricus blazei …. Ganoderma lucidum is the most concerned and studied fungi among medicinal mushrooms and its commercial products are common all over the world. So that we can completely trust that its related species, the perennial species of subgenus Elfvingia and some other perennial polypore groups, also have pharmaceutical properties and can be used to treat many diseases, even cancers.
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