Metabolites of endophytic fungi isolated from leaves of Bauhinia variegata exhibit antioxidant activity and agonist activity on peroxisome proliferator-activated receptors α, β/δ and γ

Abstract

Diabetes mellitus is a metabolic disorder that affects millions of people worldwide and is linked to oxidative stress and inflammation. Thiazolidinediones (TZD) improve insulin sensitization and glucose homeostasis mediated by the activation of peroxisome proliferator-activated receptors γ (PPARγ) in patients with type 2 diabetes. However, their use is associated with severe adverse effects such as loss of bone mass, retention of body fluids, liver and heart problems, and increased risk of bladder cancer. Partial PPARγ agonists can promote the beneficial effects of thiazolidinediones with fewer adverse effects. Endophytic fungi colonize plant tissues and have a particularly active metabolism caused by the interaction with them, which leads to the production of natural products with significant biological effects that may be like that of the colonized plant. Here, we identify seven endophytic fungi isolated from Bauhinia variegata leaves that have antioxidant activities. Also, one of the extracts presented pan-agonist activity on PPAR, and another showed activity in PPARα and PPARβ/δ. A better understanding of this relationship could help to comprehend the mechanism of action of antioxidants in treating diabetes and its complications. Moreover, compounds with these capabilities to reduce oxidative stress and activate the receptor that promotes glucose homeostasis are promising candidates in treatment of diabetes.

Keywords: Bauhinia variegata; antioxidant; diabetes; endophytic fungi; peroxisome proliferator-activated receptors.

Figure 1

Phylogenetic tree constructed for PCR amplicons of the ITS1-5.8S-ITS2 rDNA of the endophytic fungi by genetic similarity comparison with data acquired with data from GenBank (NCBI). Phylogenetic tree constructed on the basis of the similarity of the PCR amplicons of the internal transcribed spacer 1 and 2 rDNA regions and sequences found in GenBank (NCBI), analyzed by the grouping neighbor-joining method, with 1,000 bootstrapping repetitions. The number of ramifications presents the bootstrap value (Felsenstein, 1985). The analysis involved 31 FASTA sequences. Rhizopus oryzae was used to root the phylogenetic tree. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021).

Figure 2

Polyphenols and flavonoids content and antioxidant effects. (A) Polyphenols and flavonoids contents in the extracts produced by the endophytic fungi isolated from Bauhinia variegata leaves. (B) Hydrogen-donating ability of the fungi extracts (BvFI, BvFII, BvFIII, BvFV, BvFVII, BvFVIII, and BvFIX) was evaluated by the DPPH· method. The concentration range of the extracts used in the reaction medium was 0.036 to 150.6 µg.mL^-1. Standard compounds, ascorbic acid, BHT, and quercetin concentrations in the reaction medium also were used in the same concentrations. (C) Evaluation of the ability to inhibit lipid peroxidation by extracts of fungi. The emulsion of linoleic acid with polysorbate 20 in the presence and absence of extracts (150 µg.mL^-1) and standards with the antioxidant activity known [tocopherol (125 µg.mL^-1) and BHT (125 µg.mL^-1)]. (D) Endophytic fungi extract ability to scavenge superoxide anion radicals. Extracts’ ability in scavenge superoxide anion radicals was evaluated using the xanthine/xanthine oxidase system as the source of the superoxide. ${\text{O}}_2^{-}·$ produced reduces NBT to formazan detected by spectrophotometry at 560 nm. Antioxidants scavenge superoxide anion radicals, decreasing the reduction of NBT and, consequently, formazan production. Results were expressed as the percentage of inhibition of the lipid peroxidation process, as mean ± standard deviation of three different assays. The statistical test applied was the analysis of variance (one-way ANOVA) followed by the Newman-Keuls multiple comparison test, ***, significantly different, p<0.0001 vs. all compounds.

Figure 3

Extracts BvFII and BvFVII do not promote adipogenesis in 3T3-L1. Cells were induced to differentiate with insulin and exposed to vehicle (DMSO), Rosi 10^-5 mol.L^-1, Extract BvFII 20 or 25 µg.mL^-1 and Extract BvFVII 3.4 or 7.8 µg.mL^-1. (A) After 14 days, the cells were fixed, stained with oil red O, and photo documented. (B, C) Human mesangial cells were co-transfected with the expression vectors for PPARα or PPARγ, and the plasmid containing the luciferase reporter gene and treated with vehicle (DMSO:MeOH, 1:3), bezafibrate 3.10^-4 mol.L^-1for PPARα, β and rosiglitazone 10^-5 M for PPARγ (positive controls) or increasing concentrations of extracts of fungi BvFII (B) and BvFVII (C). (D) Real-time quantitative PCR to evaluate the expression of Fabp4 (aP2) after 8 days of differentiation. (E) Western blot to evaluate the expression of FABP4 (aP2) protein after 14 days of differentiation. Data are presented as the mean (SD) of three independent experiments conducted in triplicate and expressed as activation relative to transcript levels in vehicle samples (DMSO). *p ≤ 0.05; **p ≤ 0.001.

Figure 4

LBD proteolysis pattern of PPARγ by trypsin after treatment with different ligands. (A) Trypsin activity on PPARγ LBD was evaluated by using the recombinant protein his-LBD-hPPARγ, which was incubated with vehicle (DMSO), 10 µmol.L^-1 rosiglitazone, fungus BvFII extract, or fungus BvFVII extract and with trypsin (0, 100, 250 and 350 μg.mL^-1) for 60 minutes. LBD proteolytic patterns of PPARγ were analyzed by polyacrylamide gel electrophoresis under denaturing conditions, followed by staining the gel with Coomassie blue. (B) The proteolytic activity of trypsin on gelatin was evaluated after incubation of the enzyme with vehicle (DMSO) or with extracts of fungus BvFII (400 µg.mL^-1) or fungus BvFVII (400 µg.mL^-1). Polyacrylamide gel with 0.25% gelatin was left in contact with filter paper discs soaked in 20 µL of trypsin solution (200 µg.mL^-1) for 1 h. Proteolytic patterns were analyzed by staining the gel with Coomassie blue. Dark areas show degraded gelatin. The quantification of the inhibition rate of vehicle and extracts BvFII and BvFVII is shown on the right. The data presented represent the mean ± standard deviation of the mean. The statistical test applied was the analysis of variance (one-way ANOVA) followed by the Newman-Keuls multiple comparison test, with no statistically significant difference.