Rhizophora mucronata Lam. (Mangrove) Bark Extract Reduces Ethanol-Induced Liver Cell Death and Oxidative Stress in Swiss Albino Mice: In Vivo and In Silico Studies

Abstract

The bark extract of Rhizophora mucronata (BERM) was recently reported for its prominent in vitro protective effects against liver cell line toxicity caused by various toxicants, including ethanol. Here, we aimed to verify the in vivo hepatoprotective effects of BERM against ethanol intoxication with the prediction of potential targets employing in silico studies. An oral administration of different concentrations (100, 200 and 400 mg/kg body weight) of BERM before high-dose ethanol via intraperitoneal injection was performed in mice. On day 7, liver sections were dissected for histopathological examination. The ethanol intoxication caused liver injury and large areas of necrosis. The pre-BERM administration decreased the ethanol-induced liver damage marker tumor necrosis factor-alpha (TNF-α) expression, reduced hepatotoxicity revealed by nuclear deoxyribonucleic acid (DNA) fragmentation and decreased oxidative stress indicated by malondialdehyde and glutathione contents. Our in silico studies have identified BERM-derived metabolites exhibiting the highest predicted antioxidant and free radical scavenger activities. Molecular docking studies showed that most of the metabolites were predicted to be enzyme inhibitors such as carbonic anhydrase inhibitors, which were reported to stimulate the antioxidant defense system. The metabolites predominantly presented acceptable pharmacokinetics and safety profiles, suggesting them as promising new antioxidant agents. Altogether, the BERM extract exerts antioxidative activities and shows promising hepatoprotective effects against ethanol intoxication. Identification of related bioactive compounds will be of interest for future use at physiological concentrations in ethanol-intoxicated individuals.

Keywords: Rhizophora mucronata; ethanol intoxication; hepatoprotection; liver injury; metabolites; necrosis; oxidative stress.

Figure 1

BERM pre-administration prevented ethanol intoxication-induced liver necrosis and injury in mice. Histopathological analysis of representative microsections of liver tissues extracted from the untreated (Group 1—Control) and treated mice (Group 2–6). From Group 2, high-dose ethanol-induced liver damage was observed in the tissues, as indicated by the arrows, pointing at damaged hepatic cells with microvacuolation (black arrowhead), granular cytoplasm (red arrow), and necrosis (orange arrow). Ethanol-induced liver injury was also observed, as indicated by the arrows, pointing at central vein damage (black arrow) and immune cell infiltration (green arrow). From Group 3, the oral pre-administration with Silymarin prevented ethanol-induced liver necrosis and injury as indicated by normal tissue architecture (normal central pointed by black arrow) and less damaged hepatic cells, granular cytoplasm, and cell infiltration. From Groups 4–6, the oral pre-administration with BERM (100–400 mg/kg b.w.) gradually prevented ethanol intoxication-induced liver damage as indicated by the absence of histopathologic alterations (no hepatic microvacuolations, pointed at by a black arrowhead) and areas showing recovery of the nucleus (pointed at by a yellow arrow) and normal hepatic artery (pointed at by the blue arrow), as compared to the Group 1 presenting no damage of the liver tissue, confirmed by normal hepatic cells (black arrowhead) and central vein (black arrow).

Figure 2

BERM pre-administration decreased ethanol-induced TNF-α mRNA expression levels and upregulated ethanol-suppressed NRF2 mRNA expression levels. Representative gel electrophoresis showing mRNA expression levels of (A) TNF-α and the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and (B) NRF2 and the internal control β-actin, determined by RT-PCR analysis in the control mouse group, ethanol-treated group, BERM, or Silymarin administration prior to ethanol injection. The bar graphs show the expression levels of TNF-α mRNA related to GAPDH and of NRF2 related to β-actin. The data are presented as the mean ± standard deviation (SD) of three independent experiments. ** p < 0.01 and *** p < 0.001 vs. Control.

Figure 3

BERM pre-administration decreased ethanol-induced programmed cell death. (A) Representative photomicrographs showing apoptotic DNA fragments containing digoxigenin-labeled nucleotides were revealed using the TUNEL assay. The arrows point at examples of brownish apoptotic cells. (B) The bar shows the percentage TUNEL-positive apoptotic cells. The results are presented as the mean ± SEM based on three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 signify a statistically significant difference compared with the control.

Figure 4

Effect of BERM pre-administration on hepatic tissue GSH and MDA levels following to ethanol-induced liver damage. The bar graphs show hepatic tissue levels of GSH and MDA measured using colorimetric methods involving specific substrates such as 5-5’-dithio-bis(2-nitrobenzoic acid) (DNTB) and 2-thiobarbituric acid (TBA) solutions, respectively. Refer to the methods section for more information. The letters a, b and ab, clearly indicate that they are statistically significant with the control (untreated) mouse group, with the ethanol group, and with the control and ethanol groups, respectively. The results are presented as the mean ± SEM based on five independent experiments.

Figure 5

Chemical analysis using Liquid Chromatography with Mass Spectrometry (LC-QTOF). The secondary metabolites in BERM were tentatively identified as (1) n-Hexadecanoic acid, (2) 4H-1-benzopyran-4-one, 7-hydroxy-3-methoxy-2-pheyl, (3) Elaidic acid, isopropyl ester, (4) 2-Cyclohexen-1-one, (5) Lupeol, (6) Oleic acid, (7) Flavone, (8) O-methyl-d-glucose, and (9) Ethyl iso-allocholate. Means m/z implies measured m/z.

Figure 6

The molecular docking of Silymarin and BERM-derived Metabolites into CA II Enzyme. (A) Overlay of Silymarin (Violet), Metabolite 5 (faded orange) and Metabolite 7 (green) in the binding pocket of CA II enzyme; (B) surface representation of CA II enzyme with the docked Metabolites occupying the binding pocket; (C) the molecular interactions of Silymarin (Violet), Metabolite 5 (faded orange), and Metabolite 7 (green) with the amino acids in the binding site.

Figure 7

The bioavailability radar for the identified bioactive Metabolites in BERM. LIPO: Lipophilicity, Size: Molecular weight, POLAR: solubility, INSOLU: insolubility, INSATU: insaturation, and FLEX: flexibility. The properties within the colored zone are preferred for orally active drugs.

Figure 7

The bioavailability radar for the identified bioactive Metabolites in BERM. LIPO: Lipophilicity, Size: Molecular weight, POLAR: solubility, INSOLU: insolubility, INSATU: insaturation, and FLEX: flexibility. The properties within the colored zone are preferred for orally active drugs.