Antioxidant And Selective Cytotoxic Activities On Cancer Cell Lines By Bioactivatable Compounds In Extracts Of Termitomyces Schimperi (Lyophyllaceae)

Abstract

BACKGROUND: Termitomyces schimperi (TS) is an edible mushroom that is known to possess certain therapeutic properties. Anecdotal report from La Cote d’Ivoire suggests that the mushroom is traditionally used in combination with kaolin, to manage a number of cancers. There is, however, a dearth of information on its antioxidant and anticancer properties with or without kaolin. AIMS: The aims of this study were to determine the antioxidant and cytotoxic potential of aqueous and ethanolic extracts of TS, and elucidate the effect of microsome-dependent bioactivation of these extracts in combination with kaolin on cancer cell lines. METHODOLOGY: Aqueous and ethanolic (20:80, v/v) extracts of TS were prepared from the dried mushroom in combination with kaolin (TSK), and extracts of kaolin alone (K) was also prepared, giving 6 extracts in all. The six extracts were screened for secondary metabolites. Total phenolic and flavonoid content of the extracts were determined by Folin Ciocalteu and Aluminum Chloride methods, respectively. The antioxidant properties of all extracts (0 - 10 mg/ml) were evaluated, using 2, 2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reducing power assays. Cytotoxic activity of increasing concentrations of the extracts (0, 62.5, 125, 250, 500 and 1000 μg/ml) were ascertained, using 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay on a panel of six human cancer cell lines Jurkat (blood), PC-3, LNCaP (prostate), MCF-7 (breast), HMV II (vaginal malignant epithelial cells), and Hep G2 (liver), and a non-cancerous Chang liver cell line. Rat liver microsomes were prepared and used in bioactivation studies. RESULTS: Secondary metabolites screening: Both aqueous and ethanolic extracts of TS contained phenols, terpenoids, saponins and cardiac glycosides. Flavonoids were present in the ethanolic but not the aqueous extract of TS. Similar results were obtained for the aqueous and ethanolic extracts of the mixture of the TS and kaolin (TSK). None of the secondary metabolites were found in the extracts of kaolin only. Alkaloids were found to be absent in all the extracts. Antioxidant activity: The aqueous and ethanolic extracts of TS and TSK exhibited concentration-dependent scavenging activities. The ethanolic extract of TS exhibited a higher free radical scavenging potential than the aqueous extract, as shown by EC50 values; 1.285 ± 0.038 and 1.971 ± 0.231 mg/ml, respectively (p<0.001). Kaolin extracts did not exhibit any scavenging activity. The ethanolic extract of TSK also showed a higher scavenging potential than aqueous extract; EC50 of 1.964 ± 0.124 and 4.659 ± 0.050 mg/ml, respectively, (p<0.001). When compared to the aqueous and ethanolic extracts of TS, TSK showed a reduced scavenging potential; 2.36 and 1.53-fold reduction, respectively, (p<0.001). The standard, butylated hydroxytoluene (BHT), had an EC50 of 0.047 ± 0.008 mg/ml (p<0.001). Additionally, there was no significant difference between the total phenol content of both aqueous and ethanolic extracts of TS. The aqueous extract of TSK showed a higher total phenol content than its ethanolic extract (3.418 ± 0.047 g and 1.682 ± 0.436g GAE, respectively, p≤0.001). Both aqueous and ethanolic extract of all samples (TS, TSK and K) showed little or no flavonoid content. Cytotoxic activity: The aqueous and ethanolic extracts of TS, and the aqueous extract of the TSK were found to exhibit concentration-dependent cytotoxic activity on PC-3 and Jurkat. Aqueous extract of TS demonstrated significant selectivity for PC-3 (selective index = 2.65). Cytotoxic activity of aqueous extract of TS on PC-3 was significantly higher than its ethanolic extract as shown by their IC50 values of 377.42 ± 32.06 μg/ml and 862.17 ± 23.82 μg/ml, respectively, (p<0.01). The IC50 value of aqueous extract of TSK on PC-3 was found to be 1.98-fold higher than aqueous extract of TS. The IC50 value of the ethanolic extract of TS on Jurkat was 545.23 ± 3.67 μg/ml. Aqueous and ethanolic extracts of TS and the aqueous extract of TSK did not show any cytotoxic activity on normal Chang liver cell line. Increasing concentration of curcumin (0 to 36.838 μg/ml) demonstrated drastic concentration-dependent cytotoxic activity on all cancer and normal cell lines with IC50 values ranging from 3.89 ± 0.76 to 16.86 ± 0.95 μg/ml (p<0.001). Liver microsome-dependent bioactivation: In the presence of liver microsomes, the aqueous extract of TS exhibited a lower IC50 value; from >1000 μg/ml to 28.24 ± 9.53 μg/ml on Jurkat cells, representing a potentiation index (PI) of 35.41. Also, the IC50 of the ethanolic extract of TS was found to be lower after bioactivation of extract; IC50 value from 545.23 ± 3.67 μg/ml to 31.56 ± 2.98 μg/ml on Jurkat cells (PI = 17.28). This potentiation index was significantly higher than the one obtained by curcumin (PI = 3.64). There was no potentiation of the cytotoxic effect of the aqueous extract of TSK on Jurkat cell lines after bioactivation. Potentiation of the cytotoxic activity of the aqueous and ethanolic extracts of TS and curcumin were observed on PC-3 cell line, as shown by their potentiation indices of 1.83, 2.87 and 48.08, respectively. CONCLUSION: The current study shows that TS possesses antioxidant and selective cytotoxic activities on PC-3 and Jurkat cell lines. This could be attributed to its appreciable free radical scavenging potential and phenolic content. Also, kaolin did not show any potentiating effect on the antioxidant and cytotoxic activities of the extracts of TS, however, after liver microsomedependent bioactivation, there appeared to be a significant potentiation of the cytotoxic activity of TS.

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Thesis (MPhil)

Keywords

Antioxidant, Selective Cytotoxic Activities, Cancer Cell, Bioactivatable Compounds, Termitomyces Schimperi (Lyophyllaceae)

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