Antioxidant and cytotoxicity activity of Cordyceps militaris extracts against human colorectal cancer cell line

Plant materials

Cordyceps militaris fungus was produced by Ganofarm R&D SDN BHD research laboratory (Puchong, Selangor, Malaysia). The isolate of C. militaris (strain CMRU-1) used in the present study was collected from the Department of Plant Protection, Can Tho University, Vietnam.

Extraction of the sample

Fresh fruiting bodies or mycelia of C. militaris were extracted using the maceration technique. 100 g of the sample was macerated in 1,000 ml of water (stirred at 200 rpm) at 90°C for 1 hour (Azrie et al., 2014; Morales et al., 2019). The crude CME was then filtered. With some modification, the CME was mixed with 10% of maltodextrin and blended until homogeneous (Chankana et al., 2013; Chong and Wong, 2015). The mixture was spray-dried with the inlet and exit air temperatures were 170and 80°C (Chankana et al., 2013). The mixture was sprayed through a 1.5 bar atomizer pressure nozzle during the spray-drying process (Chankana et al., 2013). The spray-dried CME was collected and weighed and the percentage yield was determined. The extracts were stored in the desiccator before further analysis. The yield of the CME was calculated using the following equation:

$\mathrm{Yield}\mathrm{of}\mathrm{crude}\mathrm{extract}\mathrm{(\%}\frac{\mathrm{w}}{\mathrm{w}})=\frac{\mathrm{mass}\mathrm{of}\mathrm{crude}\mathrm{extract}\mathrm{(}\mathrm{g}\mathrm{)}}{\mathrm{mass}\mathrm{of}\mathrm{sample}\mathrm{(}\mathrm{g}\mathrm{)}}\times 100(1)$

Total phenolic and total flavonoid content (TFC)

The total phenolic content (TPC) of CME was analyzed using the Folin–Ciocalteu’s method. 1 ml of CME (12.5 mg/ml) was mixed with 50% Folin–Ciocalteu reagent (50 μl) and 2% sodium carbonate (2 ml). The solution was thoroughly mixed before being incubated for 30 minutes at room temperature. Using a UV-Vis spectrophotometer (UV1800, Kyoto, Japan), the solution’s absorbance was measured at 720 nm. Gallic acid was used as a standard and a calibration curve was constructed (6.55–32.79 mg/l). The TPC was measured as a milligram of gallic acid equivalent (GAE) in a gram of dry weight extract. Each experiment was carried out in triplicate, unless otherwise mentioned. The flavonoid–aluminum complex formation was used to assess the TFC of CME. 1 ml of CME (12.5 mg/ml) was combined with 1 ml of methanol and 2% aluminum chloride. After 15 minutes of incubation, the complex was formed and spectrophotometrically analyzed at 430 nm. A standard calibration curve of rutin (6.67–33.33 mg/l) was constructed. TFC was described as a milligram of rutin equivalent (RE) in a gram of dry weight extract.

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay

1 ml of sample was thoroughly mixed with 2 ml of DPPH solution (0.1 mM) at concentrations ranging from 100 to 500 μg/ml. After 30 minutes of incubation, the absorption was measured at 520 nm. In this study, rutin was used since it is commonly used as a positive control in all previous antioxidant assays. The potential to scavenge the DPPH was measured using equation (2), where the regulation and the sample absorbance are A control and A sample, respectively, as follows:

$\mathrm{DPPH}\mathrm{scavenging}\mathrm{activity}\mathrm{(\%)}=\frac{\mathit{Acontrol}-\mathit{Asample}}{\mathit{Acontrol}}\times 100(2)$

Cell culture

Human colorectal cancer cell lines HT-29 were grown in RPMI-1640 (Gibco, Waltham, MA) containing 10% fetal bovine serum (FBS) and 1% of penicillin–streptomycin mixed solution. 0.05% of trypsin-Ethylenediaminetetraacetic acid (EDTA) (GIBCO, Waltham, MA) was used to harvest the confluent cells, which was neutralized with RPMI-1640 supplemented with 10% of FBS (1:1). The cells were routinely cultured in 25 cm² plastic corning flasks (T-25) and kept at 37°C in a humidified atmosphere with 5% of carbon dioxide supply (CO₂) maintained at 37°C as monolayer cultures.

Cytotoxicity assay

To dilute the human cell lines to a concentration of 5 × 10³ cells ml⁻¹, serum-free RPMI-1640 (GIBCO, Waltham, MA) was used. A total of 0.1 ml of cell suspension was pipetted into each of the 96-well microtiter plate’s allocated wells. In this study, the blank control group consisted of three wells containing a culture medium. In a 5% CO₂ incubator at 37°C, the plate was incubated for 24 hours. The culture medium was pipetted out after incubation, and 0.1 ml of serum-free culture medium containing CME with different concentrations from 0.625 to 10.000 μg/ml was distributed in triplicate into specified wells. The positive control (cisplatin) was used in this study with a concentration of 10 μg/ml (Sigma, Cream Ridge, NJ). For 48 hours, the plate was incubated at 37°C in a 5% CO₂ incubator. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent was then applied to each well in a volume of 20 μl. This plate was then incubated in a CO₂ incubator at 37°C for another 4 hours before the dye was clear. The supernatant was then was pipetted out, and each well was filled with 0.1 ml of Dimethyl Sulfoxide (DMSO). At 540 nm, the microplate Enzyme-linked immunosorbent Assay (ELISA) reader (Corona Microplate Reader SH1000, Hitachi) was used to measure the absorbance. Equation (3) was used to calculate the concentration inhibition (CI, %) as follows:

CI (%) = 1 – {(As – Ab)/(Ac – Ab)}100 (3)

where the absorbance of blank (Ab), negative control (Ac), and sample (As) were denoted in the equation. Determination of the half-maximal inhibitory concentration (IC₅₀) of each extract was made by interpolating the linear regression of percentage of mortality versus the concentration of extract. The experiment was carried out in triplicate.

Acridine orange (AO) and propidium iodide (PI) double staining

The cell lines at the concentration of 10⁶ cells/ml were treated with CME (at the concentration of the IC₅₀. On the other hand, cells with no application of CME were used as the negative control. Both of the treated and untreated cells were incubated in a 25 cm² tissue culture flask for 48 hours at 37°C. A 20 μl of AO/PI mixture in PBS at 1:1 (v/v) ratio was used to stain the cells and was visualized using a Leica fluorescence microscope DM 2500 (Leica Microsystem, Wetzlar, Germany) at 100× magnification. Alpha Imager (AlphaInnotech, San Leandro, CA) was used to capture the images of the cells.

Antioxidant activity on the DPPH, TPC, and TFC

The percentage yield of spray-dried CME obtained was 10.2 w/w%. Based on the C analysis of variance analysis in Table 1, all the antioxidant activities which were DPPH, TPC, and TFC of CME were significantly (p < 0.05) higher than fresh CM. The higher percentage of inhibition of the antioxidant activity presence and lowest half-maximal IC₅₀ in CME is due to the high content of TPC (160 ± 0.74 mg GAE/100 g) and TFC (6.6 ± 1.13 mg RE/100 g) present in the extract. While CM samples only showed a moderate inhibition on the DPPH activity which was 54.3% (IC₅₀ = 2.95 mg/ml) compared to CME 83.8% (IC₅₀ = 0.60 mg/ml). The CME demonstrated strong antioxidant efficacy by reducing the initial violet color of the DPPH solution to subtle violet and consequently brighter yellow. The DPPH (a stable free radical) is converted to 1,1-diphenyl-2-picrylhydrazyl due to the reaction with antioxidants (Iqbal et al., 2017). The level of discoloration suggests the antioxidant’s radical-scavenging potential. In this step, the DPPH reacts with compounds that convert it to a solid diamagnetic molecule by contributing hydrogen (H) atoms. The antioxidant activity of CME was comparable to Agaricus blazei (97.1% at 2.5 mg/ml), Ramaria botrytis polysaccharides (82.67% at 1.4 mg/ml), and Coprinus comatus (84.5% at 5 mg/ml) (Huang et al., 1999; Li et al., 2017; Tsai et al., 2007).

Cytotoxic effect of CME on HT-29 cells

The MTT cytotoxicity results showed that CME has the highest inhibitory activities followed by synthetic anticancer drug cisplatin in a dose-dependent manner (Fig. 1). On the contrary, cisplatin expressed low inhibitory activities against HT-29 cells. The IC₅₀ values for CME and cisplatin were to be 1.53 and 3.11 mg/ml, respectively. The obvious difference in the mean percentage inhibition of the five concentrations (p < 0.0001) of CME and cisplatin using Tukey’s Honest significant difference (HSD) post-hoc analysis stipulated that the 10 mg/ml of extract had significantly decreased the value of the absorbance as the extract concentration increased. However, the inhibition percentage increases with an increase in CME. This result showed that the compound has a high cytotoxic effect on HT-29 cells even at the lowest concentration of the extract, which is 0.625 and 1.25 mg/ml with 64.66% and 52.14% cell inhibition as compared to cisplatin with 52.52% and 49.14% cell inhibition, considering that the CME is a crude extract.

Table 1. The antioxidant activity of CME and fresh CM. [Click here to view]

The chemopreventive activity of cordycepin, a major compound responsible for anticancer properties in CME against human colon, liver, bladder, and renal cancer, lung, and breast cancer cell lines, has been reported (Choi et al., 2011; Lee et al., 2009; Shao et al., 2016; Tao et al., 2016; Yamamoto et al., 2015; Yoon et al., 2018). Nevertheless, the study has been limited to its effect on mitochondrial dehydrogenase activity. It was confirmed that the crude CME exhibits powerful concentration-dependent growth inhibitory activity against HT-29 cells. Furthermore, the results showed that CME was more cytotoxic against colorectal cancer (IC₅₀ of 1.53 mg/ml) than cisplatin (IC₅₀ = 3.11 mg/ml). Furthermore, changes in the HT-29 cell shape to round in shape (from polygonal shape), reduction of cell adherence (due to cell death), increment of cell debris, and decrease in cell density were observed, indicating the cytotoxicity effect of CME (Fig. 2).

Disruption of cell membrane integrity results in the increment of red fluorescence and the reduction of green fluorescence image in the AO/PI assay. The fluorescent microscopy study was executed to investigate the mode of HT-29 cell death by CME. The HT-29 morphological observation demonstrates features of chromatin condensation and nuclear margination which were the main characteristics of apoptosis together with the loss of cell membrane integrity after 48 hours of incubation. Chromatin condensation and nuclear margination due to apoptotic trigger were observed in both early (indicated by the chromatin condensation and nuclear fragmentation) and late apoptosis (formation of apoptotic bodies and membrane loss) features as shown in Figure 3.

It was observed that the HT-29 cells treated with CME exhibited early apoptotic behavior with the formation of condensed chromatin and marginated nuclear indicated with a bright-green color stain after 48 hours of treatment together with membrane blebbing. In addition, late stages of apoptosis also appeared after the treatment as green-orange fluorescence stain was observed. Treatment with CME also denatures the deoxyribonucleic acid of the cell as observed in red in the morphological image analysis due to the binding with AO.

Figure 1. Viability (%) of HT-29 cells 48 hours after treatment with different concentrations of CME. [Click here to view]

Figure 2. Morphological changes of HT-29 cancer cell. Cells are imaged by inverted phase-contrast microscope. [Click here to view]

Figure 3. Morphological changes of HT-29 cancer cell detected with AO/PI staining method and imaged by fluorescence microscope. [Click here to view]