University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
Number
Pl/13
Active substance methanol mushroom extracts
Ganoderma lucidum
Radoja Domanovića 12, 34000 Kragujevac, Serbia http://cpctas.pmf.kg.ac.rs

e-mail: cpctas@kg.ac.rs
User request Material reception /
Start of testing
Responsible person
Dejan Poslon
Medical Herbs, Novi Sad
June, 2011. Dragana Đačić
Model system
HCT-116 Cell viability assay (MTT assay)
Analysis
Letinus edodes
Phellinus linteus
UM.01, UM.02, UM.03,
UM.04, UM.05,
Coprinus comatus
Cordiceps sinensis
Title
Objective
Material, methods, patients
CYTOTOXIC ACTIVITIES OF MUSHROOM METHANOL EXTRACTS
The aim of this study was to determinate the antprolifertive effects of methanol extracts of mushroom on colon cancer adenocarcinoma cell line HCT-116.
Cell preparation and culturing(UM.01, UM.02, UM.03, UM.04)
The colon cancer adenocarcinoma cell line HCT-116 was obtained from the
American Tissue Culture Collection (Manassas, VA, USA). These cells were propagated and maintained in DMEM (Dulbecco’s Modified Eagle Medium),
(Gibco, USA) and supplemented with 10% fetal bovine serum (PAA), antibiotics
100 IU/mL penicillin and 100 μg/mL streptomycin.
Cells were growth in 75 cm 2 culture bottles supplied with 15 ml DMEM, and after a few passages cells were seeded in 96-well plate. Cells were cultured in a humidified atmosphere of 5%
CO
2
at 37 °C.
Cell viability assay (MTT assay) (UM.05)
After 24 and 72 h of treatment the cell viability was determined by MTT assay.
The proliferation test is based on the color reaction of mitochondrial dehydrogenase from living cells with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide, Sigma, USA). HCT-116 cells was seeded in a 96well plate (10 4 cells per well). After 24 h incubation cells were treated with 100
µL of each concentrations of mushrom methanol extracts (in concentration of 10-
500 µg/ml) for 24 and 72 h. Untreated cells served as a control. At the end of the tretmant period, MTT (final concentration 5 mg/ml PBS) was added to each well, wich was then incubated at 37 °C in 5% CO
2
for 2 h. The colored crystals of produced formazan were dissolved in DMSO (dimethyl sulfoxide, Sigma, USA).
The absorbance was measured at 550 nm. Cell proliferation was calculated as a ratio of the apsorbance of the treated group divided by the apsorbance of control group, multiplied by 100 to give percentage proliferation (Mosmann, 1983).
Statistical analysis
The data are expressed as the means
± standard errors (SE). Biological activity is result of three individual experiments, performed in triplicate for each dose. The magnitude of correlation between variables was done using a SPSS (Chicago, IL)
Dragana Đačić, MSci Snežana Marković, Ph.D.

Results
Discussion
Conclusion
References statistical software package (SPSS for Windows, ver. 17, 2008). The effect of each extract were expressed by IC
50
(inhibitory dose which inhibit 50% growth cells) and by the magnitude of maximal effect in exposed cells. The IC
50
values were calculated from the dose curves by a computer program (CalcuSyn).
1.
Antiproliferative activity
After cell seeding in standard DMEM medium cells were exposed to different drugs concentrations for 24 and 72 h at 37 °C. Percent cell survival, evaluated by
MTT assay (see Materials and methods), was calculated as the ratio between absorbance at each dose of the drugs and absorbance of untreated control x 100.
The results obtained with antiproliferative assays are represented in Fig. 1-5 .
According to the American National Cancer Institute (NCI), the criteria of cytotoxic activity for the crude extracts is an IC
50
< 30 μg/ml (Itharat et al. 2004).
Cell viability was reduced in dose-dependent manner for Ganoderma lucidum and Phellinus linteus methanol extracts ( Figure 1 and Figure 3 ). The longer time exposure induced higer cell sensitivity for methanolic ecstrat of Phellinis linteus, except in lower concentrations after 72 h ( Figure 3 ).
The shape of dose response curves indicates a not significant inhibition of cell growth in dose-dependent and time-dependent manner for exstrcts of Letinus edodes, Coprinus comatus and Cordiceps sinensis ( Figure 2 , Figure 4 and
Figure 5 ). Our results show better activity in higer concentration. The longer time exposure induced low cell sensitivity ( Figure 2, Figure 4 and Figure 5 ).
The methanol fraction of Phellinus linteus which inhibited the highest antiproliferative potential among the five tested mushroom.
According to the American National Cancer Institute (NCI), the criteria of cytotoxic activity for the active substances extracted from Ganoderma lucidum,
Letinus edodes, Coprinus comatus and Cordiceps sinensis were completely unable to inhibit HCT-116 cell growth at the used concentrations (IC
50
>30
μg/ml) ( Table 1 ) and this extracts not demonstrated antiproliferative activity after
24 and 72 h.
The data resulting from three different experiments are summarized in Table 1.
All of tested drugs show inhibition of cell proliferation. The maximal effects to inhibit proliferated had extract from Phellinus linteus for 72 h. The methanol fraction of Phellinus linteus have the highest antiproliferative potential among the five active mushroom with IC
50 values of 200.58 μg/ml fall within the NCI criteria, thus are considered as of promising anticancer potential. The active substances extracted from other lichens was completely unable to inhibit HCT-
116 cell growth at the used concentrations (IC
50
>30 μg/ml), and this extracts demonstrated an intermediate activity after 24 and 72 h.
To further determine the activity and mechanism of action of the methanol extract, or adders different extracts, we would like to isolate and identify the active principle and investigate the effects in more detail.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 1983, 65:
55-63
Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A. In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer.
J Ethnopharmacol. 2004; 90: 33–38.
Dragana Đačić, MSci Snežana Marković, Ph.D.

Notes
Date
Dragana Đačić, MSci
This report applies only to the tested substances. Responsible for the report,
(accuracy and technical explanations of results) are researcher and manager and are considered the report's authors, which they have confirmed with their signature. The report should not be used or reproduced partially, except in its entirety in form of the publication of results as an integral part of the report.
Publication of results based on this report must be approved by the authors.
06.07.2011.
Snežana Marković, Ph.D.

Figure 1. The dose responses curve of effects of methanol extract Ganoderma lucidum on cell growth in
HCT-116 cells after 24 and 72 h exposure . The cells were treated with methanol extract Ganoderma lucidum in concentration range from 10-500 µg/ml. The antiproliferative effects was measured by MTT assay after 24 and 72 h exposure. Results were expressed as the means ± SE from three independent determinations.
Figure 2. The dose responses curve of effects of methanol extract Letinus edodes on cell growth in HCT-
116 cells after 24 and 72 h exposure . The cells were treated with methanol extract Letinus edodes in concentration range from 10-500 µg/ml.. The antiproliferative effects was measured by MTT assay.
Result were expressed as the means ± SE from three independent determinations.
Figure 3. The dose responses curve of effects of methanol extracts Phellinus linteus on cell growth in
HCT-116 cells after 24 and 72 h exposure . The cells were treated with methanol extract Phellinus liteus in concentration range from 10-500 µg/ml.The antiproliferative effects was measured by MTT assay.
Result were expressed as the means ± SE from three independent determinations.
Figure 4. The dose responses curve of effects of methanol extracts Coprinus comatus on cell growth in
HCT-116 cells after 24 and 72 h exposure . The cells were treated with methanol extract Coprinus comatus in concentration range from 10-500 µg/ml. The antiproliferative effects was measured by MTT assay. Result were expressed as the means ± SE from three independent determinations.
Figure 5. The dose responses curve of effects of methanol extracts Cordiceps sinensis on cell growth in
HCT-116 cells after 24 and 72 h exposure . The cells were treated with methanol extract Cordiceps sinensis in concentration range from 10-500 µg/ml. The antiproliferative effects was measured by MTT assay. Result were expressed as the means ± SE from three independent determinations.
Table 1. Growth inhibitory effects - IC
50
values (μg/ml) of methanolic extracts on HCT-116 cell line after
24 and 72 h exposure.
Dragana Đačić, MSci Snežana Marković, Ph.D.

Figure 1.
Figure 2.
Dragana Đačić, MSci Snežana Marković, Ph.D.

Figure 3.
Figure 4.
.
Dragana Đačić, MSci Snežana Marković, Ph.D.

Figure 5.
Table 1.
Tested extracts mushroom
Ganoderma lucidum
Letinus edodes
Phellinus linteus
Coprinus comatus
Cordiceps sinensis
24 h
>500
>500
261.1±11.21
>500
>500
IC 50 (µg/ml)
72 h
>500
>500
200.58±6.85
>500
>500
Dragana Đačić, MSci Snežana Marković, Ph.D.