MATERIALS AND METHODS

Duration of study: This work was done between the space of five months which commenced in May, 2010 and ended in August, 2010.

Plant collection: Fruiting body of G. lucidum (Olu-iju) and the rhizome of Curculigo pilosa (Epakun) were purchased from the local herb market in Mushin Area of Lagos State, Nigeria. Sample specimens were identified at the Department of Botany, University of Lagos. The plant materials were washed and oven dried at 40°C to remove moisture.

Aqueous and organic extraction: Five hundred grams of dried samples were pulverized and 100 g of powdered samples were dissolved in 300 mL of an organic mixture of ethanol and ethylacetate (50:50) in a 2 L volumetric flask. The flask was agitated for 18-22 h and the solutions were poured through low ash filter paper into round bottom flasks for rotary evaporation at 40°C which was labelled organic extract. The insoluble material in the low ash filter paper and the funnel was returned into a 2 L volumetric flask containing a mixture of methanol and distilled water (70:30). The volumetric flasks were then agitated for 22 h after which the solutions were poured through low ash filter papers and funnels into appropriately labeled round bottom evaporator flasks. The extract was concentrated to 20 mL and subsequently lyophilized (Eldridge et al., 2008).

Tissue culture and extracts testing: HeLa cell line was cultured in DMEM (Dulbecco’s Modified Eagles Medium) with 10% Fetal Calf Serum (FCS) at 37°C and 95% O₂/5% CO₂. At 90% confluence, 0.05x10⁶ cells was seeded into each well of 24 well plates and 35000 cells was seeded in each well of 96 well plates for cytotoxicity and apoptosis assays. A stock of 40 mg mL^-1 of the individual aqueous and organic extracts of G. lucidum and C. pilosa were prepared in 0.5% ethylacetate. The stock was then used to prepare test concentrations of 8-0.5 mg mL^-1 in DMEM.

Cytotoxicity assay (neutral red assay): Cells were seeded in 96 well tissue culture plates and tested for 6 h with varying concentrations (8-0.5 mg mL^-1) of the extracts in triplicate. The supernatant was removed from the wells, and cells washed with 2 mL PBS and 100 μL of neutral red (NR) solution (100 μg mL^-1, in serum free media) was added to each well. The plates were incubated for 2 h at 37°C. Wells were washed rapidly with 100 uL, 1% paraformaldehyde. The NR dye was extracted from the cells by adding 100 uL of NR desorb (1% Glacial acetic acid, 50% ethanol and 49% dH₂O) to each well, plates were incubated for 15 min, then placed on a shaker for an additional 30 min. before determining the optical density at 540 nm (OD₅₄₀) on a MultiWell spectrophotometer (Borenfreund and Puerner, 1984; Fotakis and Timbrell, 2006). Cytotoxicity is calculated as follows:

Apoptosis assays
Caspase 3 Assay: Caspase 3 enzymatic activity was quantified by the spectrophotometric detection of p-nitroanilide (pNA) chromophore after cleavage from the labelled substrate DEVD-pNA at 405 nm. HeLa cells were plated into 24 well plates and tested for 6 hrs with varying concentrations (8-0.5 mg mL^-1) of the extracts in triplicates. Total protein content was determined using Bradford method. One hundred and fifty microgram protein was used for the determination of the relative increase of caspase-3 activity as described in the assay kit (Genscript^® Caspase-3 Colorimetric Assay Kit).

DNA fragmentation assay: Testing was carried out with 8 mg mL^-1 of the extracts. After treatment, cells were washed with PBS then lysed with cold lysis buffer TE (10 mM Tris, pH 7.4, 5 mM EDTA, 0.2% Triton X). The lysates were centrifuged at 27000 g for 25 min, the supernatant was treated with phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v) containing 0.1% (w/v) hydroxyquinoline and DNA was extracted from the aqueous phase of the mixture. DNA was precipitated with cold 70% (v/v) ethanol and dried. Precipitates were dissolved in TE buffer containing 10 mg mL^-1 RNAse and incubated at 37°C for 30 min. The DNA pellet was washed with 3 M sodium acetate and cold 70% ethanol and kept at -20°C for 1 h. The pellet was rinsed with 70% ethanol and air dried for 10 min. Washed pellet was dissolved in 30 μL TE buffer; 10 μL of the sample was loaded unto 1.2% agarose gel containing 0.05 μg mL^-1 ethidium bromide and ran at 5 V for 5 min before increasing to 100 V. Image was analysed with BioDoc image analyzer (Khalafalla et al., 2009).

Calculations and statistical analyses: All calculations and statistical analyses were done using the computer software GraphPad Prism^® 5. Values from cytotoxity and apoptotic assays were expressed as relative cell death in percent. Two-way analysis of variance (ANOVA) using Bonferroni post test to compare apoptosis and cytotoxicity in HeLa cells at different extract concentrations (N = 3) was done. A p-value of less than 0.05 was considered significant.

RESULTS AND DISCUSSION

The inhibition of neoplasia without toxicity to normal cells has attracted attention as an important target in cancer therapy (Li et al., 2009). More than half of the new chemicals approved between 1982 and 2002 were derived from medicinal plants (Newman et al., 2003; Tan et al., 2011). Tan et al. (2011) reported several anti-cancer natural products isolated from Chinese medicinal herbs. This initiative in Nigeria would also be of immense benefit to further isolate and purify new lead compounds or bioactives with efficacious anti-cancer property. Literature is yet to report on the anti-neoplastic effect of C. pilosa but in our findings, the organic extract of C. pilosa in the study caused a concentration dependent increase in cytotoxicity and apoptosis (Fig. 1a) which both shows a relative cell death (%) that is lower than 50%. Nonetheless, a higher significant (p<0.01) relative cell death was recorded for apoptosis than cytotoxicity at 2 mg mL^-1 test concentration (Fig. 1a). The aqueous extract of Curculigo pilosa shows more of significant (p<0.01) cytotoxic deaths than apoptosis in which both (Fig. 1b) exhibited higher relative cell deaths in comparison with the organic extract (Fig. 1a). This gives a good indication that further work might be necessary to further purify the aqueous extract in order to see if its cytotoxic effect could have beneficial uses.

Fig. 1(a-d):	(a) Effect of organic extract of Curculigo pilosa, (b) Aqueous extract of Curculigo pilosa, (c) Organic extract of Ganoderma lucidum and (d) Aqueous extract of Ganoderma lucidum on HeLa cells relative cell death using apoptotic and cytotoxicity assays, **p<0.01, *p<0.05

Fig. 2:	DNA extracted in agarose gel from aqueous and organic solvents of G. lucidum and C. pilosa treated cells

On the other hand, various research works have been done on G. lucidum and it has also been reported that G. lucidum has been used in traditional Chinese medicine as an anti-tumour medication or as an immunomodulator (Sliva et al., 2002). The biologically active compounds isolated and purified from G. lucidum were identified as polysaccharides and the main fraction was designated as branched (1>3)-β-D-Glucans (Sone et al., 1985; Sliva et al., 2002). The polysaccharides were able to induce the expression of IL-1, IL-6, IL-12, IFN-γ, TNF-α, GM-CSF, G-CSF, M-CSF in monocytes-macrophages and T-lymphocytes which might, in part, mediate some of their anti-tumor activity (Muller et al., 2006). Figure 1c shows low cytotoxic and apoptotic effects of the organic extract of G. lucidum whereas the aqueous extract of G. lucidum possess both cytotoxic and apoptotic effects with the apoptotic effect rather higher than the cytotoxic effect as shown in Fig. 1d and 2. These findings corroborates previous works by showing the effect of pro-apoptotic effect of the aqueous methanolic extract of G. lucidum on human cervical adenocarcinoma cell line (HeLa) as depicted by the hallmark of apoptosis test-DNA fragmentation (Fig. 2). More so many reports have showed G. lucidum extracts to possess anti-proliferative effects on many cancers, such as acute myelogenous leukemia (Chui et al., 2006), lung cancer (Kashimoto et al., 2006), breast cancer (Yue et al., 2006), colorectal cancer (Xie et al., 2006), bladder cancer (Lu et al., 2004) and prostate cancer (Jiang et al., 2004).

CONCLUSION

From our findings, the aqueous extract of G. lucidum showed more promise as a pro-apoptotic natural agent in comparison with C. pilosa extracts which only had a cytotoxic effect. This encourages further developmental research into the various phytochemicals present in the aqueous portion of Ganoderma lucidum as a proposed future anti-cancer agents.