3.1 Bioactive compounds in C. militaris extract
The bioactive compounds detected in C. militaris extract, including cordycepin, adenosine, carotenoids, phenolics and flavonoids, are shown in Table 2. After analysis by HPLC, the highest amount of adenosine and cordycepin was found in the aqueous fraction of C. militaris FS and FBS, with values of 2.156 ± 0.012 and 3.315 ± 0.007 mg/g extract, respectively. Moreover, the ethanolic and methanolic extracts of C. militaris FS showed values of adenosine and cordycepin of 0.873 ± 0.024 and 2.030 ± 0.002 mg/g extract, respectively (Fig. S1 and S2). A high content of total carotenoids was found in the aqueous and methanolic extracts of C. militaris FBS with values of 8.593 ± 0.236 and 8.044 ± 0.116 mg BCT/g extract, respectively. The ethanolic extract of C. militaris FBS had the highest total phenolic content of 24.032 ± 3.470 mg/g extract, followed by C. militaris FS aqueous extract of 22.318 ± 3.912 mg GAE/g extract. A high level of flavonoids was present in the ethanolic and methanolic extracts of C. militaris FS, of 1.332 ± 0.089 and 0.918 ± 0.080 mg QUE/g extract, respectively.
Table 2
The content of bioactive compounds in C. militaris extract.
C. militaris | Adenosine (mg/g extract) | Cordycepin (mg/g extract) | Carotenoids (mg BCT/g extract) | Phenolics (mg GAE/g extract) | Flavonoids (mg QUE/ g extract) |
Water extracts FB FBS | 2.156 ± 0.012* 0.339 ± 0.067 | 1.209 ± 0.004 3.315 ± 0.007* | 4.842 ± 0.155 8.593 ± 0.236* | 22.318 ± 3.912 21.150 ± 4.338 | Not detected 0.155 ± 0.016 |
Ethanol extracts FB FBS | 0.873 ± 0.024 0.113 ± 0.041 | 1.763 ± 0.003 0.874 ± 0.008 | 2.103 ± 0.360 3.953 ± 0.534 | 15.962 ± 1.527 24.032 ± 3.470* | 1.332 ± 0.089* 0.431 ± 0.012 |
Methanol extracts FB FBS | 0.516 ± 0.061 0.064 ± 0.003 | 2.030 ± 0.002 1.679 ± 0.054 | 0.092 ± 0.056 8.044 ± 0.116 | 14.671 ± 0.815 17.290 ± 1.196 | 0.918 ± 0.080 0.047 ± 0.004 |
The results are presented as mean ± SD values of triplicate independent experiments. *The statistical data show the highest value in each compound (P < 0.05). Abbreviations are indicated as follows: fruiting body (FB), and fruiting body with substrate (FBS). |
3.2 Antioxidant activity of C. militaris extract
The radical scavenging activities of C. militaris extract were investigated against DPPH free radicals using DPPH radical scavenging assay. The results show that the aqueous extract of FBS had the highest antioxidant activity of 5.105 ± 0.457 mg GAE/g extract (Table 3). Furthermore, the antioxidant activity in the FBS methanol and FB ethanol extracts could be scavenged for DPPH radicals at values of 3.318 ± 0.057 and 3.123 ± 0.190 mg GAE/g extract, respectively (Table 3).
Table 3
Antioxidant activity of C. militaris extract.
C. militaris extracts | Antioxidant activity (mg GAE/g extract) |
Water extracts FB FBS | 2.153 ± 0.312 5.105 ± 0.457* |
Ethanol extracts FB FBS | 3.123 ± 0.190 2.162 ± 0.777 |
Methanol extracts FB FBS | 3.070 ± 0.225 3.318 ± 0.057 |
The results are presented as mean ± SD values of triplicate independent experiments. *The statistical data show the highest value in each extract (P < 0.05). Abbreviations are indicated as follows: fruiting body (FB), and fruiting body with substrate (FBS). |
3.3 Antibacterial activity of C. militaris extract and bioactive compounds
The antibacterial activity of C. militaris extract was evaluated by broth dilution method, and the bioactive compounds were also determined, including cordycepin and carotenoids. The results show that the ethanolic and methanolic extracts of FB and FBS, as well as the bioactive compounds cordycepin and carotenoids, had the ability to inhibit all enteric pathogenic bacteria, including E. coli, E. coli O157:H7, S. dysenteriae, S. Typhi, V. cholerae and B. cereus. In contrast, the aqueous extracts of FB and FBS did not inhibit all tested bacteria. The ethanolic and methanolic extracts of FB and FBS exhibited the lowest MIC and MBC doses of inhibition against all tested bacteria at values of 31.25–250 mg/ml and 7.81–250 mg/ml, respectively (Table 4). In addition, the methanolic extract of FS exhibited the lowest MIC and MBC values of 7.81 mg/ml against B. cereus. Cordycepin and carotenoids were able to inhibit all bacteria with the lowest MIC and MBC of 0.5–1.0 and 0.625–5.0 mg/ml, respectively. Cordycepin showed the lowest MIC and MBC of 0.5 mg/ml against S. dysenteriae and V. cholerae. Carotenoids showed the greatest inhibition, demonstrated in B. cereus, with the lowest MIC and MBC of 0.625 mg/ml (Table 4).
Table 4
MIC and MBC values of C. militaris extract and bioactive compounds against enteric pathogenic bacteria.
Extracts | MIC/MBC values (mg/ml) |
E. coli | E. coli O157:H7 | S. dysenteriae | S. Typhi | V. cholerae | B. cereus |
Ethanol extracts FB FBS | 31.25/250 31.25/125 | 62.50/250 62.50/250 | 62.50/125 62.50/125 | 62.50/250 62.50/250 | 62.50/250 62.50/250 | 62.50/62.50 62.50/125 |
Methanol extracts FB FBS | 31.25/250 31.25/250 | 31.25/250 31.25/250 | 31.25/125 31.25/125 | 31.25/250 62.50/125 | 31.25/250 31.25/250 | 7.81/7.81 31.25/31.25 |
Cordycepin | 0.5/1.0 | 1.0/1.0 | 0.5/0.5 | 0.5/1.0 | 0.5/0.5 | 0.5/1.0 |
Carotenoids | 0.625/5.0 | 1.25/5.0 | 1.25/1.25 | 1.25/5.0 | 1.25/5.0 | 0.625/0.625 |
Gentamycin (1 mg/ml) | 1.95/3.91 | 1.95/3.91 | 0.12/0.98 | 3.91/3.91 | 1.95/3.91 | 1.95/3.91 |
The results are presented as mean ± SD values of triplicate independent experiments. Abbreviations are indicated as follows: fruiting body (FB), and fruiting body with substrate (FBS). |
3.4 Effect of C. militaris extract and bioactive compounds on adhesion and invasion of bacteria in the epithelial cell
Non-toxic concentrations of the ethanolic and methanolic extracts of C. militaris (10 µg/ml) were selected to be tested against the adhesion and invasion of bacteria in the Caco-2 cell. Additionally, non-toxic concentrations of cordycepin and carotenoids at 25 µg/ml and 40 µg/ml were used in this study. The methanolic extract of FB showed the greatest adhesion inhibition of 86.05% against E. coli adherent on Caco-2 cells (Fig. 1). Subsequently, the ethanolic extracts of FBS could inhibit the adhesion of E. coli and S. dysenteriae on Caco-2 cells by values of 85.68% and 85.13%, respectively. The methanolic extract of FB inhibited the adherence of S. dysenteriae to Caco-2 cells by 85.30%. Moreover, the ethanolic extracts of FB and FBS had the ability to inhibit the adhesion of all tested bacteria to Caco-2 cells by 48.62 − 85.68%. The methanolic extracts of FB and FBS had the ability to inhibit the adhesion of all tested bacteria on Caco-2 cells by 57.56–86.05%. Moreover, FB and FBS extracts had the ability to block the invasion of bacteria in Caco-2 cells by 13.94–99.96% (Fig. 1). For their bioactive compounds, cordycepin and carotenoids were able to interfere with the attachment of bacteria on Caco-2 cells by 52.31–87.17% for cordycepin, and 58.47–84.51% for carotenoids.
3.5 Effect of C. militaris extract and bioactive compounds on cell viability of Caco-2 cells
The optimal concentrations of C. militaris extract and bioactive compounds on viable Caco-2 cells were assessed by MTT assay, as shown in Fig. 2. Caco-2 cells were treated with various concentrations of aqueous (40 − 5,000 µg/ml), ethanol (2.5-1,250 µg/ml) and methanol (2.5-1,250 µg/ml) C. militaris extracts and the bioactive compounds cordycepin (6.25–100 µg/ml) and carotenoids (2.5–40 µg/ml) for 24 h. The maximum dose of extract and bioactive compounds did not show cytotoxicity on Caco-2 cells compared to the control group, which expressed over 80% cell viability after treatment. Doses of aqueous, ethanolic and methanolic extracts of 80–160 µg/ml of FB and 2.5-5.0 µg/ml of FBS did not have any toxic effect on the cells. In addition, doses of 12.5–25 µg/ml cordycepin and 20–40 µg/ml carotenoids were not toxic on the cells. Non-toxic doses of C. militaris extract and bioactive compounds were further investigated for anti-inflammatory activity.
3.6 Effects of C. militaris extract and bioactive compounds on nitric oxide (NO) production from LPS stimulus in Caco-2 cells
LPS-stimulated Caco-2 cells were treated with C. militaris extract and bioactive compounds for 24 h, and the NO production was determined using Griess reagent. The results show that the aqueous extracts of FS and FBS at concentrations of 80–160 µg/ml had significant ability to attenuate NO production from the LPS-stimulated group. The aqueous extracts of FS significantly decreased NO production by 33.58–73.21%. Moreover, FBS significantly decreased NO production by 2.24–50.96% (Fig. 3). The methanolic extract of FBS (5.0 µg/ml) could significantly decrease the NO release from LPS-stimulated Caco-2 cells by 71.09%. Cordycepin (12.5-5.0 µg/ml) and carotenoids (20–40 µg/ml) expressed significant inhibitory activity on NO from LPS-stimulated Caco-2 cells, with the percentage of NO production from 2.84–28.97% (Fig. 3).
3.7 Effect of C. militaris extract and bioactive compounds on inflammatory gene expression in LPS-stimulated Caco-2 cells
The aqueous extract of C. militaris and bioactive compounds cordycepin and carotenoids significantly decreased NO production in LPS-stimulated Caco-2 cells. Thus, the expression of inflammatory genes, including iNos, Cox-2, NF-κB, TNF-α, AP-1, TLR-4, IL-1ß and IL-6, were also investigated by qRT-PCR. The results demonstrate that FB and FBS extracts (80–160 µg/mL) had significant ability to downregulate the inflammatory gene expressions by 1.8–6.2 and 1.2–6.2 folds, respectively. In contrast, FBS extract did not decrease the expression of the Cox-2 gene compared to the LPS-stimulated control (Fig. 4). Cordycepin and carotenoids found in the aqueous extract of FB and FBS were able to support the downregulation of inflammatory genes. Cordycepin (12.5–25 µg/ml) showed significant inhibitory activity to downregulate the expression of Cox-2, NF-κB, TNF-α, AP-1, TLR-4, IL-1ß and IL-6 genes by 0.7–6.2 folds. Carotenoids (20–40 µg/ml) could significantly decrease the expression of inflammatory genes, including TNF-α, AP-1, TLR-4, IL-1ß and IL-6, by 1.3–6.2 folds. Moreover, carotenoids at a concentration of 40 µg ml− 1 could significantly downregulate the expression of iNos, Cox-2, NF-κB genes by 0.4–3.1 folds (Fig. 4).