Phenolic phytochemistry, in vitro, in silico, in vivo, and mechanistic anti-inflammatory and antioxidant evaluations of Habenaria digitata

Abstract

Excessive and imbalance of free radicals within the body lead to inflammation. The objective of the current research work was to explore the anti-inflammatory and antioxidant potential of the isolated compounds from Habenaria digitata. In this study, the isolated phenolic compounds were investigated for in vitro and in vivo anti-inflammatory potential along with the antioxidant enzyme. The anti-inflammatory and antioxidant potential of the phenolic compounds was assayed via various enzymes like COX-1/2, 5-LOX and ABTS, DPPH, and H₂O₂ free radical enzyme inhibitory assay. These compounds were also explored for their in vivo antioxidant activity like examining SOD, CAT, GSH-Px, and MDA levels in the brain, heart, and liver. The anti-inflammatory potential was evaluated using the carrageenan-induced pleurisy model in mice. On the basis of initial screening of isolated compounds, the most potent compound was further evaluated for the anti-inflammatory mechanism. Furthermore, the molecular docking study was also performed for the potent compound. The phenolic compounds were isolated and identified by GC-MS/NMR analysis by comparing its spectra to the library spectra. The isolated phenolic compounds from H. digitata were 5-methylpyrimidine-24,4-diol (1), 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (2), 2-isopropyl-5-methylphenol (3), 3-methoxy-4-vinylphenol (4), and 2,6-dimethoxy-4-vinylphenol (5). In in vitro antioxidant assay, the most potent compound was compound 1 having IC₅₀ values of 0.98, 0.90, and 5 μg/mL against ABTS, DPPH, and H₂O₂, respectively. Similarly, against COX1/2 and 5-LOX ,compound 1 was again the potent compound with IC₅₀ values of 42.76, 10.70, and 7.40 μg/mL. Based on the in vitro results, compound 1 was further evaluated for in vivo antioxidant and anti-inflammatory potential. Findings of the study suggest that H. digitata contains active compounds with potential anti-inflammatory and antioxidant effects. These compounds could be screened as drug candidates for pharmaceutical research, targeting conditions associated with oxidative stress and inflammatory conditions in medicinal chemistry and support their ethnomedicinal use for inflammation and oxidative stress.

Keywords: Habenaria digitata; anti-inflammatory; antioxidant; mechanism; molecular docking; phenolic.

FIGURE 1

Structure of isolated compounds (compounds 1–5).

FIGURE 2

SOD activity of the brain, heart, and liver of mice in different treatment groups. Different lowercase letters mean significant difference in SOD activity between the same organ among treatment groups, and two-way ANOVA was conducted, followed by the Bonferroni test. Values that showed significant differences compared to the positive control are indicated as follows: n = 3, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001; ns indicates non-significant results.

FIGURE 3

GSH-Px activity in the brain, heart, and liver of mice among treatment groups. Different lowercase letters mean significant difference in GSH-Px activity between the same organ of different treatment groups (p < 0.05), and two-way ANOVA was conducted, followed by the Bonferroni test. Values that showed significant differences compared to the positive control are indicated as follows: n = 3, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001; ns indicates non-significant results.

FIGURE 4

MDA activity in the brain, heart, and liver of mice among treatment groups. Different lowercase letters mean a significant difference in MDA activity between the same organ of different treatment groups, and two-way ANOVA was conducted, followed by the Bonferroni test. Values that showed significant differences compared to the positive control are indicated as follows: n = 3, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001; ns indicates non-significant results.

FIGURE 5

CAT activity of the brain, heart, and liver of mice in different treatment groups. Different lowercase letters mean a significant difference in CAT activity between the same organ among treatment groups, and two-way ANOVA was conducted, followed by the Bonferroni test. Values that showed significant differences compared to the positive control are indicated as follows: n = 3, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001; ns indicates non-significant results.

FIGURE 6

(A) Percentage inhibition produced by histamine and compound 1 (20 mg/kg) in the histamine-induced paw edema model. (B) Percentage inhibition produced by bradykinin and compound 1 (20 mg/kg) in bradykinin-induced paw edema. (C) Percentage inhibition produced by tested compound 1 (20 mg/kg) in prostaglandin E2-induced paw edema. (D) Percentage inhibition produced by tested compound 1 (20 mg/kg) in leukotriene-induced paw edema. Each percentage point signifies mean ± SEM for a group of eight mice. Statistical analysis involved ANOVA, followed by Dunnett’s post hoc test. Asterisks denote significance: *p < 0.05, **p < 0.01, and ***p < 0.001; “ns” indicates non-significant values.

FIGURE 7

3D and 2D visualization of synthesized compounds with cyclooxygenase COX-I, (A) compound 1, (B) compound 2, and (C) compound 5.

FIGURE 8

3D and 2D visualization of isolated compounds with cyclooxygenase COX-II, (A) compound 1, (B)compound 2, and (C) compound 5.

FIGURE 9

3D (A) and 2D (B) interactions of the synthesized compound 1 with 5-lipoxygenase 5-LOX.

FIGURE 10

Presenting the 3D and 2D visualization of synthesized compounds with 5-lipoxygenase 5-LOX, (A) compound 2, and (B) compound 5.