Polyphenols and extracts from Zingiber roseum (Roxb.) Roscoe leaf mitigate pain, inflammation and pyrexia by inhibiting cyclooxygenase-2: an in vivo and in silico studies

Abstract

Zingiber roseum (Roxb.) Roscoe, a perennial herb from the Zingiberaceae family, has a long history of traditional use in the treatment of several ailments including pain, inflammation, fever, cough, arthritis, skin diseases, and liver infections. This study sought to confirm the efficacy of Zingiber roseum (Roxb.) Roscoe leaves methanol extract (ZrlME) as reported in traditional usage by evaluating its analgesic, anti-inflammatory, and antipyretic capabilities. In addition, in silico molecular docking of the metabolites identified in ZrlME was studied to verify the experimental outcomes. ZrlME demonstrated strong dose-dependent analgesic efficacy against all analgesic tests. ZrlME (400 mg/kg) showed higher anti-inflammatory activity than the standard in the carrageenan-induced paw edema test model. A significant reduction of rectal temperature (3.97°F↓) was also recorded at the same dose of ZrLME after 24 h of treatment. Seven polyphenolic metabolites were identified and quantified by HPLC-DAD analysis, including 3, 4- dihydroxy benzoic acid, (-) epicatechin, rutin hydrate, p-coumaric acid, trans-ferulic acid, rosmarinic acid, and myricetin. Strong binding affinities (ranges from -5.8 to -8.5 Kcal/mol) between the aforesaid polyphenols and cyclooxygenase-2 were discovered. Moreover, molecular dynamics simulations (MDS) demonstrated that these polyphenols exhibit significant COX-2 inhibitory activity due to their high stability in the COX-2 active site. In computational prediction, the polyphenols were also found to be nontoxic, and a variety of biological activities, such as antioxidant, analgesic, anti-inflammatory, antipyretic, and hepatoprotective, were observed. The results of this study revealed that ZrlME possesses notable analgesic, anti-inflammatory, and antipyretic properties.

Keywords: Zingiber roseum; analgesic activity; anti-inflammatory activity; antipyretic activity; polyphenols.

FIGURE 1

The polyphenols and their respective peaks identified in the HPLC-DAD analysis.

FIGURE 2

Results of Z. roseum leaves methanol extract (ZrlME) and standard (aspirin) in formalin-induced test. Values are stated as mean ± SEM, (n = 5); *p < 0.05, **p < 0.01, and ***p < 0.001 are considered significant compared to control.

FIGURE 3

Effect of methanol extract of Z. roseum leaves (ZrlME) and standard (ketorolac) on the hot plate test. Values are stated as mean ± SEM, (n = 5); Following an ANOVA and Bonferroni test, all data were evaluated. *p < 0.05, **p < 0.01, and ***p < 0.001 are considered significant compared to control.

FIGURE 4

Effect of ZrlME and standard (ketorolac) on the hot plate test. This panel presented the mean percent maximum effect (%MPE) of standard and various dosages of plant extract.

FIGURE 5

2D interactions of identified polyphenols, (A) rutin hydrate, (D) trans-ferulic acid, (G) p-coumaric acid, (B) 3,4 dihydroxy benzoic acid, (E) rosmarinic acid, (H) celecoxib, (C) epicatechin, (F) myricetin, with protein COX-2 (PDB ID:3LN1) (AutoDock Vina anticipated this position).

FIGURE 6

3D configuration of identified polyphenols, (A) rutin hydrate, (D) trans-ferulic acid, (G) p-coumaric acid, (B) 3,4 dihydroxy benzoic acid, (E) rosmarinic acid, (H) celecoxib, (C) epicatechin, (F) myricetin, with protein COX-2 (PDB ID:3LN1) (AutoDock Vina anticipated this position).

FIGURE 7

Analysis of a 10 ns molecular dynamics simulation of Cox-2 with ligands. (A) Root mean square deviation (RMSD), (B) Root mean square fluctuations on a residue-wise basis, and (C) Radius of gyration.

FIGURE 8

The number of hydrogen bonds produced during 10 ns dynamics simulation between COX-2 and the following ligands: (A) celecoxib, (B) rutin hydrate, (C) 3,4 dihydroxy benzoic acid, and (D) myricetin.

FIGURE 9

Possible mode of action for polyphenols identified in ZrlME in reducing inflammation. 5-LOX, 5-lipoxygenase; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 5-HETE, 5-hydroxyeicosatetraenoic acid; LTB4, leukotriene B4; LTC4, leukotriene C4; COX 1, cyclooxygenase 1; COX 2, cyclooxygenase 2; PGE2, prostaglandin E2; MAPK, mitogen-activated protein kinase; IKK, IКB kinase; IКB, inhibitor of kappa B; NF-КB, nuclear factor kappa B; CREB, cAMP response element-binding protein; AP-1, activator protein-1; TNF-α, tumour necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase.