Medicinal mushroom extracts have been regarded as important remedies for the prevention and treatment of many diseases for thousands of years mostly within the Orient Israilides and Philippoussis, 2003; Kidd, 2000; Wasser and Weis, 1999. A plethora of medicinal effects was demonstrated for mushrooms within antibacterial, antiviral, antifungal, antitumor and immuno-potentiating activities Hobbs, 2003; Ooio and Liu, 1999. Between the different bioactive components which have been demonstrated to be most effective as antitumor and immunomodulatory agents are polysaccharides and polysaccharopeptides. Nowadays macrofungi are distinguished as important natural resources of immunomodulating and anticancer agents and with regard to the increase in diseases involving immune dysfunction, cancer, autoimmune conditions in recent years, applying such immunomodulator agents mostly with the natural original is vital. These compounds belong mainly to polysaccharides mostly -D-glucan derivates, glycopeptide or protein complexes polysaccharide-peptide or protein complexes, proteoglycans, proteins and triterpenoids.
Between polysaccharides, two 4 - D-glucans and their peptide or protein derivates and between proteins, fungal immunomodulatory proteins Fips have more important role in immunomodulating and antitumor activities. Lentinus edodes is the source of many therapeutic polysaccharide macromolecules between which the ones with proven pharmacological effects are lentinan, LEM and KS-2. Lentinan is an above molecular mass about one million homopolysaccharide in a triple helix structure, with linear chains consisting of two -3 - - D- glucopyranosyl Glcp residues with 3 - two - seven - linked Glcp branchings for every ve - two - 4 -Glcp residues Aoki, 1984. LEM is a mycelial extract preparation of L. edodes harvested prior to the cap and stem grow.
It is a heteroglycanprotein conjugate containing 24. 6% protein and 44% sugars, comprising mostly pentoses as well as glucose and smaller amounts of galactose, mannose and fructose Lizuka, 1986; Sugano et al. It also contains nucleic acid derivatives, Be complex vitamins, ergosterol, eritadenine an anticholesteremic amino acid, and water- soluble lignins Sugano et al. KS-2 is a peptide polysaccharide complex. Besides its anti-tumour activity, it was demonstrated to increase the host resistance to bacterial and viral infections Jong and Birmingham, 1993.
Lenthionine, a sulphur-containing compound, has antibacterial and antifungal activity Yasumoto et al. , 1971; Morita and Kobayashi, 1967, and bis [ methylsulfonyl methyl] disulphide, a derivative of lenthionine, has tough inhibitory effects against Staphylococcus aureus, Bacillus subtilis and Escherichia coli Takazawa et al. The chloroform and ethyl acetate extracts regarding the dried mushroom have antibacterial activity against Streptococcus mutans and Prevotella intermedia Hirasawa et al. Most fruiting body and the mycelium contain compounds with wide-ranging antimicrobial activity Jong and Birmingham, 1993. Multiple fractions of LEM an aqueous extract regarding the L.
edodes mycelium and its solid race moderate have immunoactive properties for example the induction of interferon in vitro Hibino et al. , 1994 and in vivo Suzuki et al. , 1979, inhibition regarding the infectivity and cytopathic effect of person immunodeficiency virus Suzuki et al. , 1989; Tochikura, 1988 and blockade regarding the release of herpes simplex virus kind two from tissue race cells Sarkar et al. Since many regarding the compounds, which are located in L.
edodes have been shown to act synergistically Yamasaki et al. , 1989, it is worth testing the biological activity regarding the whole mushroom and mycelium extract rather than its lone components. This principle synergy is compatible with similar natural biological products like the essential oils, which let the achievement of tough effects when used as whole products, while quenching or nullifying potential unwanted side effects by the presence of lone components. The aim of this work is learn the antitumor activity In vitro and immumomodulating activity of ethanolic extract from mycelia of 3 different strains of Lentinus edodes. MATERIALS AND METHODS:.
The 3 edible fungal strains Lentinus edodes LC2141 and Lentinus edodes LC202 were kindly obtained from Fujian Agriculture Univ. The race was maintained on Potato Dextrose Agar PDA moderate and stored in refrigerator at six -7 C subsequent to growth as recommended by Stamets, 1993 for routine race and storage purposes. Medium for Submerged Race of Shiitake SM Mizuno, 1995?. Mycelia were grown in a submerged liquid race in 250ml conical flasks. The moderate composition for strain Lentinus edodes LC2141 was: Thiamine -Hcl, 1.
2mg; ZnCl2, 4mg; CuSO4, 1. 0mg; Starch, 70g; mealie steep liquor, 10g; Fructose, 15g; NaNO3, 2g and distilled water, two L; Initial pH= 7. While the moderate composition for strain Lentinus edodes LC202 was: Thiamine -Hcl, 1. 6H2O, 9mg; ZnCl2, 4mg; CuSO4, 0. 8mg; Starch, 70g; mealie steep liquor, 10g; Fructose, 10g; yeast extract,5g and distilled water, two L; Initial pH= 7.
Each flask was inoculated with 25 agar plugs 0. 7cm covered by the mycelium obtained from a 15 days old plate race for 15 days incubation period in case of strain Lentinus edodes LC2141 and 13 days in case of strain Lentinus edodes LC202. Preparation of Crude Ethanol Extract Turkoglu et al. The mycelia of each strain were dried at 40C prior to analysis. These dried mycelia were pulverized and 20.
0g each regarding the powdered samples were soaked separately in 200 ml of 95% ethanol in an Erlenmeyer flask. The sample was extracted by stirring at 30C at 150 rpm for 24 h. The mix was filtered through Whatman's filter cardboard no 4. The residue was then extracted with 3 more 200ml of ethanol as described above. The combined ethanolic extracts were concentrated in a rotary evaporator at 40C.
The ethanol was recovered and the extract was collected and dried and stored at 4C for distant use. Ehrlich ascites carcinoma EAC cells mouse tumor?. Ehrlich ascites carcinoma EAC cells were used in In vitro and in vivo experiments. The parent cell line was kindly supplied by Local Cancer Institute, Cairo University, Egypt. The tumor cell line was maintained in female mice Swiss albino through serial intraperitoneal transplantation of 1x 106 viable tumor cells in 0.
The tumor is characterized by moderately rapid growth, which kill mice in 16 to 18 days due to accumulation of ascetic fluid and seldom shows distal metastasis or spontaneous regression. Ehrlich ascites carcinoma EAC cells were obtained by needle aspiration of ascetic fluid from the preinoculated mice below aseptic conditions creating use of ultraviolet laminar space flow system. The cells within the ascetic fluid were tested for viability and contamination by staining 0. 1 ml of this fluid by 0. 1 ml of trypan blue dye which stains only the dead cells Lazarus et al.
Preliminary test for Invitro antitumor activity regarding the crude extracts was done by creating use of Ehrlich ascites carcinoma EAC cells by trypan blue exclusion test Sheldon and Preskorn, 1996. EAC cells were incubated with RPMI moderate in a tissue race plate, then the extract concentrations were added that content of each well was 0. The final concentrations of extract were as follows 25, 50, 100 and 200 mg or ml dissolved in PBS. Subsequent to 24 hrs incubation of cells with extract, the cells were stained with trypan blue dye and percent survival of cells was determined by counting dead and viable cells creating use of haemocytometer. Manage treatment in which EAC cells were cultured without extracts was evaluated and The dose response curve of viable cells was determined.
Percent survival regarding the cells. Many viable cells in unit volume regarding the test drug well. Many viable cells in unit volume regarding the manage well. Person tumor cell lines:. Person carcinoma cell lines were used in this study, were MCF7 Breast carcinoma cell line and Hep-G2 Liver carcinoma cell line.
It was obtained frozen in liquid nitrogen -180oC from the American Kind Race Collection. The tumor cell line was maintained within the Local Cancer Institute, Cairo, Egypt, by serial sub-culturing. For the assessment regarding the cytotoxic and cytostatic. edodes extracts cells were seeded in 96- well flat-bottomed microtiter plates at a density of approximately 0. 5X105 cells or well, in done RPMI-1640 Medium.
Subsequent to 24 h to make sure that cell attachment, serial dilutions regarding the extracts in physiological saline were prepared. 100 l of different concentrations of each tested extracts were added for 24 h at 37C, in a humidified six % CO2 atmosphere. Cytotoxicity was determined creating use of the MTT assay Hansen et al. Subsequent to incubation, 10 l MTT reagent solution or well was added and incubated for an more 5 h. MTT crystals were solubilized by added 100 l of MTT detergent or well then the plate was shacked at space temperature.
It was followed by photometric determination regarding the absorbance at 570 nm creating use of microplate ELISA reader Meter tech. 960, USA subsequent to development of violet color. Control cells were treated with vehicle alone. For each compound concentration, 4 wells were used triplicate wells were prepared for each lone dose. The average was calculated.
Data was expressed as the percentage of relative viability compared with the untreated cells. The cytotoxicity dose was calculated like a dose induced 100% relative non viability. Percentage of relative viability was calculated creating use of the following equation: [Absorbance of treated cells or Absorbance of manage cells] X 100. Then the 1/2 maximal inhibitory concentration IC50 was calculated by the trend line equation. Immunomodulating activity regarding the 3 tested ethanolic extracts:.
Venous blood 5ml was drawn from well volunteers and breast cancer patient volunteers. Person peripheral blood was collected in sterile heparin tube and lymphocyte separated regarding to the method described by Boyum, 1976. Lymphocyte proliferation was determined by MTT assay and trypan blue exclusion assay for most manage and breast cancer person lymphocytes. Statistical analysis:. Statistical analysis of data was carried out by creating use of one method analysis of variance ANOVA followed by homogenous subsets Duncuna at confidence position of 5% 0.
05 creating use of the Statistical Product for the Corporate Science SPSS version8. Duncan's multiple section tests were used to compare between means of treatments regarding to Walter and Duncan 1969 at probability 5%. Conclusions and Discussion:. In vitro cell race experiments. The effect of mycelia ethanolic extract on EAC viability by Trypan blue exclusion test:.
As shown in data that have been summarized within the table two and figures two and 2, viability was measured and expressed as the survival fraction compared with untreated manage cells. Ehrlich cells were treated with concentrations 400, 200, 100, 50 and 25g or ml regarding the ethanolic extracts of Lentinus edodes LC2141 and Lentinus edodes LC202 for 24 h. percent of dead cells increased by increasing concentration. At concentration 200g or ml, the viability percentage in comparison with manage was the lowest. Table 1? The effect of Lentinus edodes LC2141 and Lentinus edodes LC202 mycelia ethanolic extract on EAC viability at 24 hrs of exposure determined by Trypan blue exclusion test.
Concentration g or ml. Lentinus edodes LC2141. Lentinus edodes LC202. Viability manage % 24 hrs exposure. % of dead cells 24 hrs exposure.
Viability manage % 24 hrs exposure. % of dead cells 24 hrs exposure. ? Means within similar column with different letters have significant differences between each other. 1: Dose response curve on the effect of Lentinus edodes LC2141 ethanolic extract on EAC viability at 24 hrs of exposure determined by trypan blue exclusion test. 2: Dose response curve on the effect of Lentinus edodes LC202 ethanolic extract on EAC viability at 24 hrs of exposure determined by trypan blue exclusion test.
Anti-tumor activity against person breast cancer cell line MCF7 and person hepatocellular carcinoma cell line Hep-G2?. cytotoxicity was measured and expressed as the survival fraction compared with untreated manage cells. The likely anti-proliferative effect of ethanolic extract of most Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of MCF7 cell line subsequent to incubation for 24 h as shown in Fig. The treatment of MCF7 cells with different concentrations of ethanolic extract of most Lentinus edodes LC2141 and Lentinus edodes LC202dramatically inhibited the cell growth in a dose-dependent manner, with IC50 values of 132. 59 g or ml, respectively.
Also, the likely anti-proliferative effect of ethanolic extract of most Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of Hep-G2 cell line subsequent to incubation for 24 h as summarized in Fig. The treatment of Hep-G2 cells with different concentrations of ethanolic extract of most Lentinus edodes LC2141 and Lentinus edodes LC202dramatically inhibited the cell growth in a dose-dependent manner, with high IC50 values of 953. 3g or ml, respectively. Similar conclusions were reported by Israilides et al. , 2007 who located that Aqueous extracts of Lentinus edodes can significantly suppress the proliferation of cancer cell line MCF-7 in vitro.
This is reflected by the comparative little IC50 values and the simultaneous higher IC50 values on normal cells. edodes mushroom h2o extracts are more cytotoxic than mycelial aqueous extracts. Methanolic extracts of neither mushroom or mycelia of L. edodes not ever exhibit any inhibitory cytostatic effect on MCF-7 cancer cell line supports the direct cytostatic or cytotoxic action regarding the L. edodes extracts on cancer cells, that is in parallel action with its host-mediated antitumor activity.
3: Cell viability of breast carcinoma cell line MCF7 with ethanolic extract of most Lentinus edodes LC2141 at concentration section from 400 to 25 g or ml. 4: Cell viability of breast carcinoma cell line MCF7 with ethanolic extract of most Lentinus edodes LC202at concentration section from 400 to 25 g or ml. 5: Cell viability of liver carcinoma cell line Hep-G2 with ethanolic extract of most Lentinus edodes LC2141at concentration section from 400 to 100 g or ml. 6: Cell viability of liver carcinoma cell line Hep-G2 with ethanolic extract of most Lentinus edodes LC202at concentration section from 400 to 50 g or ml. The effect regarding the ethanolic extract from the 3 different strains of Lentinus edodes on lymphocyte proliferation:.
Lymphocyte viability and proliferation were determined by trypan blue exclusion assay and MTT assay. Normal and breast cancer patient person lymphocyte was assayed. Proliferation was measured and expressed as count and absorbance compared with untreated manage cells. The likely proliferative effect of ethanolic extract of most Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of lymphocyte subsequent to incubation for 24 h. The conclusions represented in Fig.
7 and 8 showed that the treatment of manage person lymphocyte cells with different concentrations of ethanolic extract of Lentinus edodes LC2141 and Lentinus edodes LC202 resulted in dramatically increase within the lymphocyte proliferation in a dose-dependent manner. The highest count and absorbance was observed at concentration 400mg or ml. Also, treatment of lymphocyte cells of manage person with the combination regarding the 3 ethanolic extract of strain Lentinus edodes LC2141 and strain Lentinus edodes LC202 showed significant increase in cell no. and lymphocyte proliferation in a dose-dependent manner. The conclusions represented in Fig.
48 and 49 showed that the treatment of lymphocyte of breast cancer patient with different concentration of ethanolic extracts of Lentinus edodes LC2141 and Lentinus edodes LC202 mycelia showed a significant increase in cell no. and cell proliferation in a dose-dependent manner. Treatment with concentration 400g or ml showed the highest increase in lymphocyte proliferation in comparison with control. , 2007 who located that L. edodes extracts supports the direct cytostatic or cytotoxic action of on cancer cells, that is in parallel action with its host-mediated antitumor activity.
Furthermore, it was demonstrated that L. edodes can act as an immunomodulator to augment the proliferative response of rat thymocytes to T mitogens in vitro, indicating another mechanism for immunostimulatory activity. Overall there seems to be a therapeutic advantage in creating use of L. edodes extracts orally administered instead of a lone substance like Lentinan provided intravenously. In addition, Nitha et al.
, 2007 reported that the ethanolic extract of M. esculenta mycelium shall also be located to possess significant antitumor activity against most ascites and solid tumour. The conclusions indicate that the extract possessed most curative and preventive properties against solid tumour in a dose-dependent manner. The extract shall also be significantly effective against ascites tumour. These conclusions suggest that M.
esculenta mycelia contain compounds that shall modulate tumourigenesis at different stages or shall act at similar stage. Polysaccharide isolated from the fruiting bodies of M. esculenta was reported to exhibit immunostimulatory activity Duncan et al. Similarly, Lobanok et al. , 2003 showed that the submerged mycelium and veggie bodies of L.
edodes contain significant amounts of biologically active substances and exhibit immunomodulatory activity. Table 2? The effect of Lentinus edodes ethanolic extracts on normal person lymphocyte proliferation manage team at 24 hrs of exposure determined by Trypan blue exclusion test and MTT assay. Combination regarding the 3 ethanolic extract of strain Lentinus edodes LC2141 and strain Lentinus edodes LC202 mycelia. Ethanolic extract of Lentinus edodes LC202 mycelia. Ethanolic extract of Lentinus edodes LC2141mycelia Concentration g or ml.
? Means within similar column with different letters have significant differences between each other. Table 3? The effect of Lentinus edodes ethanolic extracts on breast cancer patient lymphocyte proliferation at 24 hrs of exposure determined by Trypan blue exclusion test and MTT assay. Ethanolic extract of Lentinus edodes LC202 mycelia. Ethanolic extract of Lentinus edodes LC2141mycelia Concentration g or ml. ? Means within similar column with different letters have significant differences between each other.
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