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Studies Of Ethanolic Extract from Lentinus edodes On Different Cell Lines And Lymphocytes Separated From Cancer Breast Patients

introducion

Medicinal mushroom extracts have been considered as important remedies for the prevention and treatment of many diseases for thousands of years especially in the Orient (Israilides and Philippoussis, 2003; Kidd, 2000; Wasser and Weis, 1999). A plethora of medicinal effects has been demonstrated for mushrooms including antibacterial, antiviral, antifungal, antitumor and immuno-potentiating activities (Hobbs, 2003; Ooio and Liu, 1999). Among the various 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 especially with the natural original is vital. These compounds belong mainly to polysaccharides especially β-D-glucan derivates, glycopeptide/protein complexes (polysaccharide-peptide/protein complexes), proteoglycans, proteins and triterpenoids. Among polysaccharides, β (1 →3) - D-glucans and their peptide/protein derivates and among proteins, fungal immunomodulatory proteins (Fips) have more important role in immunomodulating and antitumor activities.

     Lentinus edodes is the source of many therapeutic polysaccharide macromolecules among which the ones with proven pharmacological effects are lentinan, LEM and KS-2. Lentinan is a high molecular weight (about one million) homopolysaccharide in a triple helix structure, with linear chains consisting of (1 -3)- β - D- glucopyranosyl (Glcp) residues with two β-(1 - 6)- linked Glcp branchings for every five β -(1 - 3)-Glcp residues (Aoki, 1984 ). LEM is a mycelial extract preparation of L. edodes harvested before the cap and stem grow. It is a heteroglycan–protein 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., 1982). It also contains nucleic acid derivatives, B complex vitamins, ergosterol, eritadenine (an anticholesteremic amino acid), and water- soluble lignins (Sugano et al., 1985). KS-2 is a peptide –polysaccharide complex. Besides its anti-tumour activity, it has been 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 strong inhibitory effects against Staphylococcus aureus, Bacillus subtilis and Escherichia coli (Takazawa et al.,1982). The chloroform and ethyl acetate extracts of the dried mushroom have antibacterial activity against Streptococcus mutans and Prevotella intermedia (Hirasawa et al., 1999). Both fruiting body and the mycelium contain compounds with wide-ranging antimicrobial activity (Jong and Birmingham, 1993). Several fractions of LEM (an aqueous extract of the L. edodes mycelium and its solid culture medium) have immunoactive properties such as the induction of interferon in vitro (Hibino  et al., 1994) and in vivo (Suzuki et al., 1979), inhibition of the infectivity and cytopathic effect of human immunodeficiency virus (Suzuki et al.,  1989; Tochikura, 1988) and blockade of the release of herpes simplex virus type 1 from tissue culture cells (Sarkar et al., 1993).

     Since many of the compounds, which are found in L. edodes have been shown to act synergistically (Yamasaki et al., 1989), it is worth testing the biological activity of the whole mushroom and mycelium extract rather than its individual components. This principle (synergy) is compatible with similar natural biological products like the essential oils, which allow the achievement of strong effects when used as whole products, while quenching or nullifying potential unwanted side effects by the presence of individual components.

     The aim of this work is study the antitumor activity ( In vitro) and immumomodulating activity of ethanolic extract from mycelia of two different strains of Lentinus edodes.

 

MATERIALS AND METHODS:

Fungal Strains:

     The two edible fungal strains Lentinus edodes LC2141 and Lentinus edodes LC202 were kindly obtained from Fujian Agriculture Univ., China. The culture was maintained on Potato Dextrose Agar (PDA) medium and stored in refrigerator at 5 -7 °C after growth as recommended by Stamets, (1993) for routine culture and storage purposes. 

 

Culture Media:

Medium for Submerged Culture of Shiitake(SM) (Mizuno, 1995):

     Mycelia were grown in a submerged liquid culture in 250ml conical flasks. The medium composition for strain Lentinus edodes LC2141 was: Thiamine -Hcl, 1.0 mg; KH2 PO4 , 1.0g; Cacl2 .2H2O, 0.5g; MgSO4 . 7H2O, 0.5g; FeSO4. 7H2O, 10mg; MnSO4 .6H2O, 7.2mg; ZnCl2 , 4mg; CuSO4, 1.0mg; Starch, 70g; corn steep liquor, 10g; Fructose, 15g; NaNO3 , 2g and distilled water, 1 L; Initial pH= 7). While the medium composition for strain Lentinus edodes LC202  was: Thiamine -Hcl, 1.0 mg; KH2PO4 , 1.0g; Cacl2 .2H2O, 0.5g; MgSO4 . 7H2O, 0.5g; FeSO4. 7H2O, 10mg; MnSO4 .6H2O, 9mg; ZnCl2 , 4mg; CuSO4 , 0.8mg; Starch, 70g; corn steep liquor, 10g; Fructose, 10g; yeast extract,5g and distilled water, 1 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 culture 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., 2007):

      The mycelia of each strain were dried at 40°C before analysis. These dried mycelia were pulverized and 20.0g each of the powdered samples were soaked separately in 200 ml of 95% ethanol in an Erlenmeyer flask. The sample was extracted by stirring at 30°C at 150 rpm for 24 h. The mixture was filtered through Whatman's filter paper no 4. The residue was then extracted with two additional 200ml of ethanol as described above. The combined ethanolic extracts were concentrated in a rotary evaporator at 40°C. The ethanol was recovered and the extract was collected and dried and stored at 4°C for further use. 

 

Tumor cell lines: 

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 National 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 0.2 ml of saline. 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 under aseptic conditions using ultraviolet laminar air flow system. The cells in 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., 1966). Preliminary test for Invitro antitumor activity of the crude extracts was done by using Ehrlich ascites carcinoma (EAC) cells by trypan blue exclusion test (Sheldon and Preskorn, 1996). EAC cells were incubated with RPMI medium in a tissue culture plate, then the extract concentrations were added that content of each well was (0.8 ml medium + 0.1 ml cells + 0.1 ml extracts). The final concentrations of extract were as follows (25, 50, 100 and 200 mg/ml) dissolved in PBS. After 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 using haemocytometer. Control treatment in which EAC cells were cultured without extracts was evaluated and The dose response curve of viable cells was determined.

 

*Percent survival of the cells

= T/C x 100

      Where:

T …. Number of viable cells in unit volume of the test drug well.

C …. Number of viable cells in unit volume of the control well.

 

Human tumor cell lines:

     Human 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 Type Culture Collection. The tumor cell line was maintained in the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. For the assessment of the cytotoxic and cytostatic

activities of L. edodes extracts cells were seeded in 96- well flat-bottomed microtiter plates at a density of approximately (0.5X105 cells/well), in complete RPMI-1640 Medium. After 24 h to ensure cell attachment, serial dilutions of the extracts in physiological saline were prepared. 100 µl of different concentrations of each tested extracts were added for 24 h at 37ºC, in a humidified 5 % CO2 atmosphere. Cytotoxicity was determined using the MTT assay (Hansen et al.,1989). After incubation, 10 µl MTT reagent solution/well was added and incubated for an additional 4 h. MTT crystals were solubilized by added 100 µl of MTT detergent/well then the plate was shacked at room temperature. It was followed by photometric determination of the absorbance at 570 nm using microplate ELISA reader (Meter tech. Σ 960, USA) after development of violet color.Control cells were treated with vehicle alone. For each compound concentration, 3 wells were used (triplicate wells were prepared for each individual dose). The average was calculated. Data was expressed as the percentage of relative viability compared with the untreated cells. The cytotoxicity dose was calculated as a dose induced ≈100% relative non viability.

Calculation:

      Percentage of relative viability was calculated using the following equation:

 

[Absorbance of treated cells / Absorbance of control cells] X   100

    

      Then the half maximal inhibitory concentration IC50 was calculated by the trend line equation.

.

 

Immunomodulating activity of the two tested ethanolic extracts:

Collection of blood: 

     Venous blood (5ml) was drawn from healthy volunteers and breast cancer patient volunteers. Human peripheral blood was collected in sterile heparin tube and lymphocyte separated according to the method described by (Boyum, 1976).Lymphocyte proliferation was determined by MTT assay and trypan blue exclusion assay for both control and breast cancer human lymphocytes.

 

Statistical analysis:

Statistical analysis of data was carried out by using one way analysis of variance (ANOVA) followed by homogenous subsets (Duncuna) at confidence level of 5% (0.05) using the Statistical Package for the Social Science (SPSS) version8. Duncan's multiple range tests were used to compare between means of treatments according to Walter and Duncan (1969) at probability 5%.

 

Results and Discussion:

In vitro "cell culture" experiments

The effect of mycelia ethanolic extract on EAC viability by Trypan blue exclusion test:

      As shown in data that have been summarized in the table (1) and figures (1 and 2), viability was measured and expressed as the survival fraction compared with untreated control cells. Ehrlich cells were treated with concentrations (400, 200, 100, 50 and 25μg/ml) of the ethanolic extracts of Lentinus edodes LC2141 and Lentinus edodes LC202 for 24 h. percent of dead cells increased by increasing concentration. At concentration 200μg/ml, the viability percentage in comparison with control 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 (μg/ml)

Lentinus edodes LC2141

Lentinus edodes LC202

Viability (control %) (24 hrs exposure)

% of dead cells (24 hrs exposure)*

Viability (control %)(24 hrs exposure)

% of dead cells (24 hrs exposure)*

25

94.60

5.40e

96.80

3.20e

50

72.00

28.00d

64.50

35.50d

100

36.60

63.40c

38.70

61.30c

200

15.20

84.80a

20.50

79.50a

400

22.60

77.40b

26.90

73.10b

*: Means in the same column with different letters have significant differences between each other.

Fig. 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.

Fig. 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 human breast cancer cell line (MCF7) and human hepatocellular carcinoma cell line (Hep-G2):

      cytotoxicity was measured and expressed as the survival fraction compared with untreated control cells. The possible anti-proliferative effect of ethanolic extract of both Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of MCF7 cell line after incubation for 24 h as shown in Fig. (3 and 4). The treatment of MCF7 cells with different concentrations of ethanolic extract of both Lentinus edodes LC2141 and Lentinus edodes LC202dramatically inhibited the cell growth in a dose-dependent manner, with IC50 values of 132.05μg /ml and 153.59 μg/ml, respectively. Also, the possible anti-proliferative effect of ethanolic extract of both Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of Hep-G2 cell line after incubation for 24 h as summarized in Fig. (5 and 6). The treatment of Hep-G2 cells with different concentrations of ethanolic extract of both Lentinus edodes LC2141 and Lentinus edodes LC202dramatically inhibited the cell growth in a dose-dependent manner, with high IC50 values of 953.1μg/ml and 1868.3μg/ml, respectively. Similar results were reported by Israilides et al., 2007 who found 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 low IC50 values and the simultaneous higher IC50 values on normal cells. L. edodes mushroom water extracts are more cytotoxic than mycelial aqueous extracts. Methanolic extracts of either mushroom or mycelia of L. edodes do not exhibit any inhibitory (cytostatic) effect on MCF-7 cancer cell line supports the direct cytostatic/cytotoxic action of the L. edodes extracts on cancer cells, which is in parallel action with its host-mediated antitumor activity.

 

 

 

Fig. 3: Cell viability of breast carcinoma cell line MCF7 with ethanolic extract of both Lentinus edodes LC2141 at concentration range from 400 to 25 μg/ml.

 

Fig. 4: Cell viability of breast carcinoma cell line MCF7 with ethanolic extract of both Lentinus edodes LC202at concentration range from 400 to 25 μg/ml.

 

 

 

Fig. 5: Cell viability of liver carcinoma cell line Hep-G2 with ethanolic extract of both Lentinus edodes LC2141at concentration range from 400 to 100 μg/ml.

Fig. 6: Cell viability of liver carcinoma cell line Hep-G2 with ethanolic extract of both Lentinus edodes LC202at concentration range from 400 to 50 μg/ml.

 

 The effect of the ethanolic extract from the two 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 human lymphocyte was assayed. Proliferation was measured and expressed as count and absorbance compared with untreated control cells. The possible proliferative effect of ethanolic extract of both Lentinus edodes LC2141 and Lentinus edodes LC202wasstudied on the growth of lymphocyte after incubation for 24 h. The results represented in Fig. (7 and showed that the treatment of control human lymphocyte cells with different concentrations of ethanolic extract of Lentinus edodes LC2141 and Lentinus edodes LC202 resulted in dramatically increase in the lymphocyte proliferation  in a dose-dependent manner. The highest count and absorbance was observed at concentration 400mg/ml. Also, treatment of lymphocyte cells of control human with the combination of the two ethanolic extract of strain Lentinus edodes LC2141 and strain Lentinus edodes LC202 showed significant increase in cell number and lymphocyte proliferation in a dose-dependent manner. The results 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 number and cell proliferation in a dose-dependent manner. Treatment with concentration 400μg/ml showed the highest increase in lymphocyte proliferation in comparison with control. Israilides et al., 2007 who found that L. edodes extracts supports the direct cytostatic/cytotoxic action of on cancer cells, which 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 using L. edodes extracts orally administered instead of a single substance like Lentinan given intravenously. In addition, Nitha et al., 2007 reported that the ethanolic extract of M. esculenta mycelium is also found to possess significant antitumor activity against both ascites and solid tumour. The results indicate that the extract possessed both curative and preventive properties against solid tumour in a dose-dependent manner. The extract is also significantly effective against ascites tumour. These results suggest that M. esculenta mycelia contain compounds that may modulate tumourigenesis at different stages or may act at the same stage. Polysaccharide isolated from the fruiting bodies of M. esculenta has been reported to exhibit immunostimulatory activity (Duncan et al., 2001).Similarly, Lobanok et al., 2003 showed that the submerged mycelium and fruit 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 human lymphocyte proliferation "control group" at 24 hrs of exposure determined by Trypan blue exclusion test and MTT assay.

Combination of the two 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 (μg/ml)

Absorbance*

 

Count*

Absorbance*

 

Count*

Absorbance*

 

Count*

0.55d

5050d

0.55d

5050d

0.55c

5050c

Control

0.83c

5425c

0.65c

6633c

0.53d

4166d

100

0.88b

6966b

0.89b

8410b

0.62b

6516b

200

0.95a

8600a

0.97a

16466a

0.74a

7523a

400

*: Means in the same 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 (μg/ml)

Absorbance*

Count*

Absorbance*

Count*

0.02e

8500e

0.02e

8500e

Control

0.16d

9250d

0.31d

9900d

50

0.21c

10120c

0.43c

10450c

100

0.23b

12650b

0.50b

14750b

200

0.26a

16850a

0.59a

22275a

400

*: Means in the same column with different letters have significant differences between each other.

REFERENCES:

Aoki, T. (1984): Lentinan. In: Fenichel, R.L., Chigris, M.A. (Eds.), Immune Modulation Agents and Their Mechanism. Marcel Dekker, Inc., New York and Basel, pp. 63–77.

 

Boyum, A. (1976): Separation of white blood cells. Nature 204: 793-794.

 

Chihara, G.; Hamuro, J.; Maeda, Y.Y.; Arai, Y. and Fukuoka, F. (1970): Fractionation and purification of the polysaccharides with marked antitumor activity, especially Lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom). Cancer research 30: 2776-2781.

 

Duncan, C.J.; Pugh, N.; Pasco, D.S. and Ross, S.A. (2001): Isolation of a galactomannan that enhances macrophage activation from the edible fungus Morchella esculenta. J. Agric. Food Chem.( 50): 5681–5685.

 

Hansen,  M.B; Nielsen,  S.E.  and  Berg,  K. (1989): Method.   J. Immunol.119: 203-210.

 

Hibino, Y.; Konishi, Y.; Koike, J.; Tabata, T.; Ohashi, Y. and Sugano, N. (1994): Productions of interferon-g and nitrite are induced in mouse splenic cells by a heteroglycan-protein fraction from culture medium of Lentinus edodes mycelia. Immunopharmacol. 28:77–85.

 

Hirasawa, M.; Shouji, N.; Neta, T.; Fukushima, K. and Takada, K. (1999): Three kinds of antibacterial substances from Lentinus edodes (Berk.) Sing. (Shiitake, an edible mushroom). Int. J. Antimicrob. Agents 11:151–157.

 

Hobbs, Ch. (2003). In: Miovic, Michael (Ed.), Medicinal Mushrooms: An Exploration Of Tradition, Healing and Culture. Botanica Press, Williams, OR.

 

Ikekawa,T.; Uehara, N.; Maeda, Y.Y.; Nakanishi, M. and Fukuoka, F. (1969): Antitumor activity of aqueous extracts of edible mushrooms. Cancer research 29: 734-735.

 

Israilides, C. and Philippoussis, A.(2003): Bio-technologies of recycling agro-industrial wastes for the production of commercially important fungal polysaccharides and mushrooms. Biotechnol. Genet. Eng. Rev. 20:  247–259.

 

Israilides, C.; Kletsas, D.; Arapoglou, D.; Philippoussis, A.; Pratsinis, A.; Ebringerova, H.; Hrˇı'balova, V. and Harding, S.E. (2007): In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes. Phymed. 1-8.

 

Ivankovic, S.; Hirsl, N.; Jakopovic, I. and Jurin, M. (2004): the influence of medicinal mushroom preparations on mouse tumors. International journal of medicinal mushrooms 6: 107-116.

 

Jong, S.C. and Birmingham, J. M. (1993): Medicinaland therapeutic value of the Shiitake mushroom. Adv.Appl. Microbiol. 39:153-184.

 

Kidd, P.M. (2000): The use of mushroom glucans and proteoglycans in cancer treatment. Altern. Med. Rev. 5: 4–27.

 

Lazarus, H.; Tegeler, M.; Mazzone, H.; Leroy, J.; Boone, B. and Foley, G. (1966): Determination of sensitivity of individual Biopsy specimens to potential inhibitory agents. Evaluation of some explants cultures as assay systems. Cancer chemother. Rep. 50: 543-554.

 

Lizuka, C. (1986): Antiviral Substance and the Manufacturing Method Thereof. United States Patent 4: 627-629.

 

Lobanok, A.G.; Babitskaya, V.G.; Plenina, L.V.; Puchkova, T.A. and Osadchaya, O.V. (2003): Composition and biological activity of submerged mycelium of the xylotrophic Basidiomycete Lentinus edodes. Appl. Biochem. Microbiol. 39: 60–64.

 

Mizuno, T. (1995): Shiitake, Lentinus edodes: functional properties for medicinal and food purposes. Food Rev. Int. 11: 7–21.

 

Mizuno, T. (1999): The extraction and development of antitumor-active polysaccharides from medicinal mushrooms in Japan. International Journal of Medicinal Mushrooms 1: 9-29.

 

Morita, K. and Kobayashi, S. (1967): Isolation, structure, and synthesis of lenthionine and its analogs. Chem. Pharm. Bull 15:988–93.

 

Nitha, B.; Meere, C.R. and Janardhanan, K.K. (2007): Anti-inflammatory and antitumor activities of cultured mycelium of morel mushroom, Morchella esculenta. Current science 92(2): 235-239.

 

Ooio, V.E. and Liu, F. (1999): A review of pharmacology activities of mushroom polysaccharides. Int. J. Med. Mushrooms 1: 195–206.

 

Sheldon, H. and Preskorn,  M.D. (1996):  Why Did Terry Fall Off the Dose-Response Curve?  Journal of Practical Psychiatry and Behavioral Health; 39-43.

 

Stamets, P. (1993): Growing gourmet and Medicinal mushrooms. Ten Speed Press, Berkeley, CA 94707.

 

Sugano, N.; Choji, Y.; Hibino, Y.; Yasumura, S. and Maeda, H. (1985): Anticarcinogenic actions of alcohol-insoluble fractions (LAP) from culture medium of Lentinus edodes mycelia. Cancer Lett., 1–6.

 

Suzuki, F.; Suzuki, C.; Shimomura, E.; Maeda, H.; Fujii, T. and Ishida N. (1979): Antiviral and interferon-inducing activities of a new peptidomannan, KS-2, extracted from culture mycelia of Lentinus edodes. J. Antibiot. 32:1336–45.

 

Suzuki, H.; Okubo, A.; Yamazaki, S.; Suzuki, K.; Mitsuya, H. and Toda, S. (1989): Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus by water-soluble lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM). Biochem. Biophys. Res. Commun. 160:367–73.

 

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Tochikura, T.S. (1988): Inhibition (in vitro) of replication and of the cytopathic effect of human immunodeficiency virus by an extract of the culture medium of Lentinus edodes mycelia. Med. Microbiol. Immunol. 177: 235–244.

 

Turkoglu, A.; Duru, M.E. and Mercan, N. (2007): Antioxidant and antimicrobial activity of Russula delica Fr. An edible wild mushroom. Eurasian journal of analytical chemistry 2(1): 54-67.

 

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About the Author

Blue Veronica

What are a good name for triplets?

Ok i sad they are twins on http://answers.yahoo.com/question/index?qid=20100502171224AAb50tN
they are actually triplets
1. Dark brown hair, blue eyes : Chrissa or Veronica ? I love Veronica and want one of the triplets to be named Veronica.
2.Light Brown hair brown eyes: maybe Jessica or Kendall? Which one sounds good with Veronica
3.Caramel Hair Blue eyes: Emily or Paige? Which ones sound good

I don't want them to all start the same and you can suggest names
and the format is
1 first and middle name(dark brown hair blue eyes)
2 first and middle name (brown hair brown eyes)
3 first and middle name (caramel hair blue eyes)

If you cant think of a middle name let me know and you can suggest names as well

LAST NAME IS MAXWELL

Veronica Michelle- The sassy older triplet with the sarcastic personality. Nick name Roni.

Kendall Elise- The middle triplet with superb athletic skill. Nick name Keni.

Paige Camille- The youngest of the three with the sweet personality who is very smart and sometimes witty. Nick name Millie.

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Dark Blue ; Patrick/Veronica. (PS there's violence and blood)

Free Seeds Too

my downloading speed got really slow suddenly?

it was 20ks 2 days ago then it went down to .5 now?
what do i do?
there is nothing wrong with my internet though and there are enough seeds and peers too and the swarm average is about 15..
i have already done a defragmentation and my computer has more than half of its memory free
what can i do ppls help

it sounds like your ISP is doing a FAP (fair access Policy) so you would need to check this out with them.

A FAP is where your only allowed to download so many megabytes a day.. like I am on 200 meg a day.. after that the download speed drops to nothing.

if I download 200 meg in a day then I have to wait 24 hours before it gets back up to speed..
tough I know but there is nothing you can do about it...

so get on to your ISP and find out if they are using the fair access policy

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Growing Heirloom Seeds & Seed Saving : Collecting Seeds from Tomatoes

Any one know where I can get gluten and dairy free basics at a reasonable price in the UK?

I ma gluten and dairy free and on income support, I need to stick to the diet but it is very expensive. I find it really hard to get hold of gram flour, soya flour, and cheap nuts and seeds. Are there any websites out there that have this stuff if not cheap then not too expensive. I cant go between lots of different shops as I have mobility problems, I could pick this stuff up quite cheap but I would have to make about 5 trips and I cant get there.

I live in CHicago Illinois so I cant help you I wish I could. If you want to talk ever about your situation Im here.

Seeds Real Pretty

My boyfried's vegan Help?

I'm the total opposite of my bf, especially when it comes to diet. He's a vegan and pretty adamant about it. All his kitchen has his nuts and seeds, fruit, veggies, potatoes, soy products, rices, noodles and vegan pasta, tofu, vegan sushi, soup, wholemeal bread, and mock meats. Mine is stocked full of steaks, hamburgers, hotdogs, pork, chicken, turkey, eggs, fish and seafood, milk, all that good stuff.

We're talking about moving in with each other, but I'm sure he won't let any meat into where we would live. And I can't survive of those faux meats, I LOVE the real thing. What should I do/say to him?

It depends. To you, it might seem like he's asking you to give up something that you greatly enjoy. To him, it probably seems like like you're asking him to abandon his ethics.

If you so greatly enjoy animal products (to the exclusion of eating plant-based foods), this relationship likely has no long-term potential.

If he knows that you eat meat and dairy and hasn't tried to get you to stop, then he probably won't ask you to give it up outside of the home. For example, he'd probably be okay with you eating whatever the heck you wanted outside of the house. To him, meat and animal by-products don't even seem like food. He likely is as upset by cow meat or pig meat as he would be if you stuck a dead, skinned cat or puppy in the fridge.

Take this opportunity to discuss the importance of meat with him. If it is merely a question of pleasure, and you're not willing to go without when you are at home, then you might need to re-think this relationship.

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Strain Hunters Africa Expedition - Part 1 - Green House Seed Co

Successful Methods Of Real Estate Lead Generation

Acquiring leads is the single most important and difficult
aspect of the real estate business. Without a lead, there is no
client, so all realtors dedicate a significant amount of time to
collecting contact information and establishing relationships
with potential prospects.

There are many ways to generate leads, all of them with their
respective advantages and disadvantages. Whether traditional or
modern, it's well worth your time and effort to try each one
until you find the best combination that is expense-effective,
time-saving and successful.

This articles aims to discuss the most common methods, the
decision ultimately belonging to you.

For the sake of organization, we have split lead generation
methods into two large groups: traditional methods (printing
materials, phone and mail advertising, networking and others)
and modern methods, that take advantage of the continuous growth
of the importance of internet for home buyers and sellers.

Traditional methods

Use your friends

If your friends don't trust you enough to do business with you,
then who will? So talk to them about their real estate plans,
get their contact information and ask for referrals and
testimonials. Your friends have other friends and family as
well, so this circle will keep expanding with a domino-like
effect. You might even get 100 names or so, at the very least
and they will come with a trust factor already in place.

The disadvantage? There are many people that believe in keeping
business and personal life separate, so they might refuse
dealing with you professionally for various reasons. However,
you can still ask them for information and feed-back, even help
if the situation occurs.

Face-to-Face contact

This classic method involves basically going around the
neighborhood and knocking on doors. Stay for a short chat, let
the people know you and in the same time, you will get to know
them. Don't use the aggressive approach ("Hello, do you want to
sell/buy a house? Then come to me, the greatest realtor on this
side of the Atlantic."). You're not a door-to-door salesman.
Farming a community is a long-time process.

So start slow, explain that you wish to become the trusted
neighborhood realtor. Ask for information, let them tell about
their experience with other realtors, their plans for the
future. The idea is to introduce yourself first and foremost.
Then ask if they wouldn't be interested in getting a monthly
newsletter or printed material with useful information of
general interest or specific information about buying or
selling. If they say yes, then your effort paid off. Take their
contact information (email, phone number) and continue to keep
in touch, coming back to chat every two months or so and
regularly sending them the promised leaflets or emails.

Disadvantage? It's time consuming most of all.

Networking

First of all, what is networking? Networking is the process of
finding out about potential clients from friends, business
contacts, co-workers, acquaintances, and fellow members in
professional and civic organizations. Sounds simple enough. But
where do you go to practice your networking skills?

Well, there are various opportunities from family gatherings, to
business cocktails, to seminars, conventions or presentations.
The important factor is to determine whether you will find
possible leads at these events. For example, you are sure to
find people interested in getting in touch with a realtors at a
neighborhood barbecue or an open house but less so at a medical
congress.

Be sure to bring plenty of business cards and brochures.

And remember, networking is about planting seeds. Do not expect
to find the next big deal, instead nurture a possibility of a
collaboration at some point in time.

Cold calling

Cold calling involves basically plowing through a list of phone
numbers and hoping that the people that don't slam the receiver
right away might be willing to meet with you. It's not a method
we recommend. It might have been efficient in the past, but
today's consumers have no wish to waste their time listening to
someone blabbering advertising text on the phone. This is
especially true where real estate is concerned. Who wants to
entrust their home over the phone?

Still, there might be some benefit to it, if you carefully
choose your target. So, if you decide to do it, we recommend you
treat it like a real conversation. Instead of reciting memorized
scripts, recognize that there's a person at the other end of the
line and talk to them normally. Sell yourself and try to get an
appointment that way, don't sell your services. Or give them the
URL of your website or article page and then let them decide if
they want to get in touch.

Direct mail

Direct mail is also know as mass mailing and involves sending
your sales letter or brochure to a targeted mailing list. It's
usually pretty efficient in getting your name out there, though
it can cost quite a lot, since the best response rate is around
1%.

The key is to make your letter stand out against all the other
junk mail your targets receive. Be sure to write your sales
letter with the client in mind. Instead of detailing your great
skills, try to address their concerns and give them a possible
solution. This will give them the reason to contact you, which
is your primary goal.

Take great care of the appearance of the envelope and the
contents. They need to look as professional and as official as
possible. You need to create a serious, trustworthy image.

You can always try postcards instead of letters. One of the most
important advantages of this method is the direct impact. Your
targets will see the attractive design immediately. Use creative
and compelling copy on the back and include testimonials if you
have the space. The text, combined with the appropriate visuals
will increase the possibility of your postcard being read.

Don't forget to ask your recipients to take action. It's a
detail many forget. Ask them to contact you or to visit your
website.

Direct mail can be also used to keep in touch with your
prospects and the neighborhood you are farming. Remember,
constancy is tantamount in building and keeping trust. Also, you
need to contact your target as many as eight times before they
will consider doing business with you.

Business cards

Your business cards, often overlooked, can be a very effective
means of collecting leads. Beside your contact information, you
can also include on the back: pitch phrases, your website's URL,
a call to action, a freebie offer and many more.

Try to make your card stand out. That way, you will ensure it
does not end up in the trash.

Modern methods

These modern methods have appeared with the advance of internet.
More and more homebuyers and sellers use the web to acquire
information.

Direct marketing (email)

Email marketing is very similar to classical direct marketing,
except instead of paper you use electronic text. The greatest
difference? It's FREE. And you can change the design and content
as often as you like.

Email can also be used with a feed-back form. It's much likelier
to have your targets whip up a quick email with a couple of
questions than write a letter, or even give a call.

You can use it to offer a free gift such as an e-book, or free
courses or a chance to subscribe to your newsletter.

Newsletters

The newsletter is an excellent way to get and keep good leads.
Since your recipients need to subscribe first, they will be
already interested in your and your services, for the future at
least.

But put together a real newsletter. That means to include
quality, informative articles, not self-promotion. Your
recipients don't like to be cheated. When they sign for a
newsletter, they will expect a newsletter, not a sales letter.

Don't worry if you've never written an article in your life.
It's easier than you might think. Draw from your experience and
you'll be sure to obtain a good result. You can also take
articles from free-for-reprint resources.

Website

Having a website will ensure you have a 24 hour connection with
your prospects. Include information about yourself, showcase
your listings, put up testimonials and of course, lots of
quality information with your special insider's tips.

Don't forget to add a subscription box for your newsletter.

No matter which methods you will end up using, remember these
three important aspects:

1. Even if it's ultimately about you, always develop an approach
that is about them. In other words, your targets do not care how
great you are, they only want to know what's in it for them.

2. Your primary goal is to acquire contact information (name,
address, phone number, email address) and the permission to use
them. That way, you will ensure your leads are actually
valuable, being people genuinely interested in doing business
with you.

3. Always follow up. The results of an one-time campaign of any
kind are very low and the impact decreases exponentially

About the Author

NetRealinTouch.net
is a complete email
marketing system for real estate professionals, offering quality
prospecting and farming newsletters at an affordable price. articles@netreal.net

Daisy Tree

The great gatsby daisy and myrtle question?

The character "Daisy" is named after a beautiful flower. Myrtle's name is quite different. A myrtle is a shrub that is used as a headge (a border) in a garden. However, the myrtle was also considered to be the sacred tree of Aphrodite, the goddess of love and beauty. How might these facts add meaning to author's choice of these names? Include character traits and facts about each character.

Well daisy would be quite beautiful of course, and I can see her as quite ecstatic and carefree almost.
Myrtle would be mysterious. She'd have a hidden dark side: maybe a shadowed past.

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The Daisy Tree Shelf....

Creating A Versatile Garden

There is no hard and firmly pronounce as to what a summer plot should be. This means that your special sight for your summer backyard is vacant to be fairly achievable if you take the time to do the modest seek and proposal the planting for your summer farming shrewdly and with great concern. One thing is certain, planting wisely can generate a summer patch that is stunning and maintains some point of genuine annoyance oppress. Your summer plotted can produce fruit, vegetables, flora, and even be planned to draw certain birds or butterflies. Of course, your summer backyard may also be planned to accomplish more than one of these goals as well. That's the beauty of the versatility of your ordinary summer plot.

Here are a few equipment to believe. Versatility in a summer plot means that you aren't committed to one prevailing tenacity or theme. This means that it is not at all inappropriate to place a few acme plants or herbs as mosquito dictate along with your desired summertime vegetable offerings. At the same time it is completely acceptable to add a few vegetables that you just can't appear to live lacking in your flower plot or among a, the plants in a backyard of green.

When it comes to insignia, the possibilities of the ordinary summer backyard are almost unbounded. There are ample of brightly dyed flowers that token the summer spell and beget a little dash of incline to a world that is often drained of shade by the sometimes blinding summer sunshine. By brining a few well sited ensign into your plot as a highlight or border you are adding dramatic demand. If you select to make your patch a sea of brightly colored flowers, plants, trees, and vines you can also achieve great concern that may be breathtaking in beauty. Be arranged to spend a great treaty of time tending a plot of this makeup however, as many of the brightly colored peak plants are awareness hogs to some extent.

If you live in a dryer climate you also have the decision of with low water landscaping or creative conservation techniques to construct a plot that is lovely in its own right. A garden doesn't have to be excessively green or occupied of happy brightly colored flowers to be wonderful. By using landscaping techniques that are conservative in character you are making your own conservation labors and the balance of the world should appreciate not only the exertion but also the beauty of the universe that was untaught of that crack.

If you want fussy and abundant greenery and live in a climate that will accommodate this particular design of a summer garden there is no purpose to evade this either. Be confident to use some conservation pains while because you never know when dearth conditions may offer themselves in climates that are typically rather moderate as we are learning in the Midwest this summer.

Vegetable gardens are yet another decisions when creating your summer garden and another tenuous example of just how versatile these gardens may be. Keep in wits that many people are discovering the beauty of vertical farming and killing plants for a few favorite vegetables and this may be a choice if you wish to use imperfect room wisely and to greater provoke.

Another choice when it comes to screen the incredible versatility of the summer garden is raised farming. This is fully exactly planting your garden above the ground. Some people start elaborate 'smooth boxes' of sorts in which the garden is planted. This allows great versatility for those with lesser seats in which to bury their gardens as well as those who have landscaping issues that are inhospitable for gardening.

These are but a few examples of just how versatile a summer garden may be but I think they afford brilliant insight and food for thought for those who are looking for a little something new and different for their summer gardens.

About the Author

For tips on shasta daisies and daisy plant, visit the Facts About Daisies website.