Phytochemistry and Evaluation of the Anti-Inflammatory Activity of the Hydroethanolic Extract of Virola elongata (Benth.) Warb. Stem Bark

Abstract

Background: Previous studies of the hydroethanolic extract of Virola elongata inner stem bark (HEVe) have demonstrated its antioxidant, gastroprotective, and antiulcer properties, but have not evaluated its anti-inflammatory potential.

Methods: HEVe was obtained by maceration and phytochemically analyzed. Its systemic anti-inflammatory activity was assessed by its effect on lipopolysaccharide (LPS)-induced peritonitis in mice. HEVe gel (HEgVe) was employed to evaluate topical anti-inflammatory activity by measuring the ear edema resulting from croton-oil-induced dermatitis in mice. A cell viability assay was conducted to determine the non-cytotoxic concentrations of the HEVe. RAW 264.7 cells were stimulated by LPS to determinate cytokine and nitric oxide production.

Results: A phytochemical analysis of the HEVe revealed the presence of phenolic acids, neolignans, flavonoids, and monomeric catechins. The oral treatment of acute peritonitis with HEVe reduced the total leukocytes, neutrophils, TNF-α, and IL-1β and elevated IL-10 levels. The application of the HEgVe reduced local edema. The HEVe on the RAW 264.7 cells exhibited no cytotoxicity, and the cells with HEVe displayed reduced TNF-α, IL-1β, and NO levels and increased IL-13 levels.

Conclusions: HEVe demonstrated systemic and topical multitarget anti-inflammatory activity, likely due to the combined effects of secondary metabolites. HEVe emerges as a promising herbal remedy for inflammation with minimal cytotoxicity, emphasizing its potential therapeutic significance.

Keywords: Virola elongata; cytotoxicity; extract; inflammation; leukocytes; phytochemistry.

Figure 1

UHPLC-PDA-ESI-IT-MSⁿ analysis of the hydroethanolic extract of Virola elongata inner stem bark (HEVe) was conducted, with detection using base peak ion (BPI). With a signal correspondence value of 1.86 × 10⁵. The highlighted peaks represent some substances highlighted in Table 1.

Figure 2

Effect of oral administration of vehicle (Vehi 0.1 mL/10 g distilled water), hydroethanolic extract from the inner stem bark of Virola elongata (HEVe 5, 25, and 100 mg/kg), and dexamethasone (Dexa 0.5 mg/kg) on total cells, mononuclear (MN) cells, and polymorphonuclear (PMN) cells present in the intraperitoneal fluid of mice with LPS-induced peritonitis (250 ng/0.2 mL/well). Each column represents the mean ± SE of eight animals/group. One-way ANOVA, followed by the Student–Newman–Keuls test, ^### p < 0.001 vs. naive, ** p < 0.01, and *** p < 0.001 vs. vehi.

Figure 3

Effect of oral administration of the vehicle (Vehi 0.1 mL/10 g of distilled water), hydroethanolic extract from the inner stem bark of Virola elongata (HEVe 5, 25, and 100 mg/kg), and dexamethasone (Dexa 0.5 mg/kg) on the concentrations of (A) tumor necrosis factor-alpha (TNF-α) and (B) interleukin-1 beta (IL-1β) in the peritoneal lavage of mice with peritonitis induced by intraperitoneal injection of LPS (250 ng/well/0.2 mL). The naive group received distilled water (0.1 mL/10 g p.o.) and 0.9% saline (0.2 mL/well i.p.). Each column represents the mean ± SE of eight animals/group. One-way ANOVA, followed by the Student–Newman–Keuls test, * p < 0.05 vs. Vehi, ** p < 0.01 vs. Vehi, *** p < 0.001 vs. Vehi, ^### p < 0.001 vs. naive.

Figure 4

Effect of oral administration of the vehicle (Vehi 0.1 mL/10 g of distilled water), hydroethanolic extract from the inner stem bark of Virola elongata (HEVe 5, 25, and 100 mg/kg), and dexamethasone (Dexa 0.5 mg/kg), on the concentration of interleukin 10 (IL-10) in the peritoneal lavage of mice with peritonitis induced by intraperitoneal injection of LPS (250 ng/well/0.2 mL). The naive group received distilled water (0.1 mL/10 g p.o.) and 0.9% saline (0.2 mL/well i.p.). Each column represents the mean ± SE of eight animals/group. One-way ANOVA, followed by the Student–Newman–Keuls test, *** p < 0.001 vs. vehi.

Figure 5

Difference in weight of the ears of female mice after topical treatment with the vehicle (Vehi 20 µL of 2.5% croton oil in acetone:water (70:30)), hydroethanolic extract gel from the inner stem bark of Virola elongata (HEgVe 1, 3, and 9%), and dexamethasone (Dexa 0.05 mg/ear/20 µL). Each column represents the mean ± SE of eight animals/group. One-way ANOVA, followed by the Student–Newman–Keuls test, *** p < 0.001 vs. vehi.

Figure 6

Cytotoxicity assessment of the hydroethanolic extract from the bark of the stem of Virola elongata (HEVe 6.25–400 µg/mL) and doxorubicin (Doxo 0.0058–58 µg/mL in RAW 264.7 cells stimulated with lipopolysaccharide (LPS 1 µg/mL) for 24 (A), 48 (B), and 72 h (C). Inhibitory concentration at 50% (IC₅₀ ± S.E.). Nonlinear regression analysis (curve fitting).

Figure 7

Effect of in vitro treatment with hydroethanolic extract from the bark of the stem of Virola elongata (HEVe 1, 5, and 20 µg/mL) and Nω-Nitro-L-arginine-methyl-ester (L-NAME 10 mM) on nitric oxide (NO) concentrations in RAW 264.7 cells stimulated with lipopolysaccharide (LPS 1 μg/mL) for 24 h. Cells in the basal group were treated with DMEM culture medium with 10% fetal bovine serum (FBS). One-way ANOVA, followed by the Student–Newman–Keuls test, ^### p < 0.001 vs. Basal, *** p < 0.001 vs. LPS.

Figure 8

Effect of in vitro treatment with hydroethanolic extract from the inner stem bark of Virola elongata (HEVe 1, 5, and 20 µg/mL) and dexamethasone (Dexa 10 mM) on the concentration of the cytokines (A) TNF-α and (B) IL-1β in LPS (1 µg/mL)-stimulated RAW 264.7 cells. Cells in the basal group were treated only with DMEM culture medium with 10% fetal bovine serum (FBS). One-way ANOVA, followed by the Student–Newman–Keuls test, * p < 0.05 vs. LPS, ** p < 0.01 vs. LPS, *** p < 0.001 vs. LPS, ^### p < 0.001 vs. Basal.

Figure 9

Effect of in vitro treatment with hydroethanolic extract from the inner stem bark of Virola elongata (HEVe 1, 5, and 20 µg/mL) and dexamethasone (Dexa 10 mM) on the concentration of the cytokine IL-13 in LPS (1 µg/mL)-stimulated RAW 264.7 cells. Cells in the basal group were treated only with DMEM culture medium with 10% fetal bovine serum (FBS). One-way ANOVA, followed by the Student–Newman–Keuls test, *** p < 0.001 vs. LPS, ^### p < 0.001 vs. Basal.