Antioxidant activity of mycelia methanolic extracts of endophytic fungi BvFV and BvFIX isolated from leaves of Bauhinia variegata

Abstract

Endophytes are considered an essential source of natural products. Skin is the body's largest organ; its primary function is the protection of other organs, and aging is one of the most relevant problems associated with this organ. UV radiation generates reactive oxygen species (ROS), which lead to skin degeneration and consequent aging. The main endogenous antioxidants that neutralize ROS are enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase, and non-enzymatic antioxidants, such as glutathione and α-tocopherol. Nuclear receptors are involved in molecular mechanisms that control the aging process, especially peroxisome proliferator-activated receptors (PPAR), which regulate the function and expression of genes that modulate the balance between matrix metalloproteinases (MMP) activity and the expression of collagen. Some natural compounds, such as polyphenols, can activate PPAR and reduce the activation of MMP and collagen degradation. In this work, the antioxidant activity of the mycelia methanolic extracts of two endophytic fungi isolated from leaves of Bauhinia variegata, named BvFV and BvFIX, their action as PPAR agonists, and their effect on the activity of antioxidant defense system enzymes were evaluated. The mycelia methanolic extract of BvFV showed a weak agonist effect on PPARβ/δ, a high capability to inhibit lipid peroxidation, increased catalase activity, and increased superoxide dismutase activity by approximately 64%. In contrast, BvFIX increased catalase activity and increased superoxide dismutase activity in a dose-dependent manner, with an increase of 49.62% ± 7.87%, 56.64% ± 12.27%, and 240.46% ± 26.11% at concentrations of 25 µg/mL, 50 µg/mL and 100 µg/mL, respectively, in human dermal fibroblasts submitted to oxidative stress. These results suggest that the metabolites of the mycelia of endophytic fungi studied are promising to act in the chemoprevention of skin aging.

Keywords: Bauhinia variegata; PPAR; antioxidant; endophytic fungus; fibroblasts; skin aging.

Figure 1

Total polyphenols and flavonoids contents and antioxidant activity of the fractions of the crude extracts of the BvFV and BvFIX mycelia. The total polyphenolic contents of samples were expressed as gallic acid equivalents per mg of extract (EAG/mg). The total flavonoid contents of samples were expressed as quercetin equivalent per mg of extract (EQ/mg). Antioxidant activity was a percentage of DPPH· reduction compared to the control. The results presented represent mean ± S.E.M. The statistical test applied was the analysis of variance (two-way ANOVA) followed by Bonferroni posttest. *, p < 0.05 vs. ethyl acetate fraction; #, p < 0.001 vs. methanolic fraction - comparisons among the different fractions of the same fungus. +, p < 0.001 - comparison between the fractions of the different fungi.

Figure 2

Antioxidant activity – determination of IC₅₀. The IC₅₀ value was determined for the extracts using eight different concentrations. A dose vs. response curve was drawn, and the IC₅₀ value was determined in GraphPad Prism 5. (A) IC₅₀, results represent mean ± S.E.M. *, p < 0.05 vs. quercetin, (B) Quercetin dose vs. response curve, (C) BvFV dose vs. response curve, and (D) BvFIX dose vs. response curve. The statistical test applied was the analysis of variance (one-way ANOVA) followed by Turkey’s multiple comparison tests.

Figure 3

Evaluation of cytotoxicity of the fungal extracts. Human dermal fibroblasts were treated for 48 h with different concentrations of fungal extracts or with the solvent used to solubilize the samples (MeOH), (A) BvFV, and (B) BvFIX. HeLa cells were treated for 24 h with different concentrations of fungal extracts or with the solvent used to solubilize the samples (MeOH), (C) BvFV, and (D) BvFIX. The results presented represent mean ± S.E.M. in the percentage of viable cells.

Figure 4

Assessment of lipid peroxidation. The linoleic acid method was used to evaluate the inhibition of lipid peroxidation. The results presented represent mean ± S.E.M. in the percentage of lipid peroxidation inhibition. (A) BvFV and (B) BvFIX. The statistical test applied was the analysis of variance (one-way ANOVA) followed by Dunnett’s test. *, p < 0.05 vs. tocopherol at 100 μg/mL.

Figure 5

Evaluation of agonist activity of extracts against PPAR receptor. HeLa cells were treated for 22 h with different concentrations of extracts, with the solvent used to solubilize the samples (MeOH – negative control) only or with a PPAR agonist (positive control). The results presented represent the mean ± S.E.M of the PPAR activation rate. (A) PPARγ - BvFV, (B) PPARγ - BvFIX, (C) PPARα - BvFV, (D) PPARα - BvFIX, (E) PPARβ/δ - BvFV, and (F) PPARβ/δ - BvFIX. The statistical test applied was the analysis of variance (one-way ANOVA) followed by Dunnett’s test. *, p < 0.05 vs. MeOH (0.2 mol/L).

Figure 6

Assessment of antioxidant system in cells treated with fungal extracts. (A-F) Human dermal fibroblasts were treated for 48 h with different fungal extracts or only with the solvent used to solubilize the extracts (samples without extracts) and then inducted to oxidative stress by treatment with H₂O₂. (A, B) Results presented represent mean ± S.E.M of MDA concentration per mg of protein in cells treated or not with fungus extract: (A) BvFV, (B) BvFIX. (C, D) Results represent the mean ± S.E.M. of SOD enzyme activity: (C) BvFV, (D) BvFIX. (E, F) Results represent the mean ± S.E.M. of catalase enzyme activity: (E) BvFV. (F) BvFIX. (G, H) Human dermal fibroblasts were treated for 48 h with different concentrations of fungal extracts or only with the solvent used to solubilize the extracts (DMEM). Results represent the mean ± S.E.M. of GSH concentration: (G) BvFV, (H) BvFIX. The statistical test applied was the analysis of variance (one-way ANOVA) followed by Dunnett’s test. * and #, p < 0.05. * vs. DMEM or samples without extract, # vs. H₂O₂.