Role of phenolics from Spondias pinnata bark in amelioration of iron overload induced hepatic damage in Swiss albino mice

Abstract

Background: Crude Spondias pinnata bark extract was previously assessed for its antioxidant, anticancer and iron chelating potentials. The isolated compounds gallic acid (GA) and methyl gallate (MG) were evaluated for their curative potential against iron overload-induced liver fibrosis and hepatocellular damage.

Methods: In vitro iron chelation property and in vivo ameliorating potential from iron overload induced liver toxicity of GA and MG was assessed by different biochemical assays and histopathological studies.

Results: MG and GA demonstrated excellent reducing power activities but iron chelation potential of MG is better than GA. Oral MG treatment in mice displayed excellent efficacy (better than GA) to significantly restore the levels of liver antioxidants, serum markers and cellular reactive oxygen species in a dose-dependent fashion. Apart from these, MG exceptionally prevented lipid peroxidation and protein oxidation whereas GA demonstrated better activity to reduce collagen content, thereby strengthening its position as an efficient drug against hepatic damage/fibrosis, which was further supported by histopathological studies. Alongside, MG efficiently eliminated the cause of liver damage, i.e., excess iron, by chelating free iron and reducing the ferritin-bound iron.

Conclusions: The present study confirmed the curative effect of GA and MG against iron overload hepatic damage via their potent antioxidant and iron-chelating potential.

Keywords: Antioxidant enzymes; Hemosiderosis; Lipid peroxidation; Liver fibrosis; Oxidative stress.

Fig. 1

Iron chelation and reducing power activity of the isolated compounds. a iron chelation activity of the compounds, b Standard EDTA c Reducing power assay. ‘As acid’ represents Ascorbic Acid. The results represent the mean ± S.D. (n = 6). *p < 0.05, **p ≤ 0.01 and ***p < 0.001 vs. control

Fig. 2

Chemical structure of the identified compounds. a Gallic acid and b Methyl gallate from S. pinnata bark

Fig. 3

Iron removal potential of GA and MG. a Hepatic iron content, b Serum ferritin content. Mouse groups (B; C; R2; S2; R4; S4; R8; S8; D) were treated as described in the ‘Experimental design and tissue preparation’ section. Values are expressed as the mean ± SD of six mice. **p ≤ 0.01, ***p ≤ 0.001 compared with the blank and ^### p ≤ 0.001 compared with the control

Fig. 4

Iron release from ferritin. a Iron release from ferritin b Correlation between iron released from ferritin with reducing power. Iron released in response to increasing amounts (100–500 μg) of both GA and MG was plotted against reducing power displayed at the same doses

Fig. 5

Effect of GA and MG treatment on liver antioxidant enzyme levels. a SOD, b Catalase, c GST, d GSH. Mouse groups (B; C; R2; S2; R4; S4; R8; S8; D) were treated as described in the ‘Experimental design and tissue preparation’ section. Values are expressed as the mean ± SD (n = 6). *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with the blank and ^# p ≤ 0.05, ^## p ≤ 0.01, ^### p ≤ 0.001 compared with the control

Fig. 6

Restoration of ROS levels in liver, spleen and serum after GA and MG treatment. a Relative ROS levels in liver homogenates, b Relative ROS levels in spleen homogenates c. Relative ROS levels in serum. Mouse groups (B; C;R2; S2; R4; S4; R8; S8; d were treated as described in the ‘Experimental design and tissue preparation’ section. Values are expressed as the mean ± SD (n = 6). *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with the blank and ^## p ≤ 0.01, ^### p ≤ 0.001 compared with the control

Fig. 7

Effect of GA and MG treatment on liver damage parameters. a Lipid peroxidation levels, b Protein carbonyl content, c Collagen content. Mouse groups (B; C; R2; S2; R4; S4; R8; S8; d were treated as described in ‘Experimental design and tissue preparation’ section. Values are expressed as the mean ± SD (n = 6). *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with the blank and ^### p ≤ 0.001 compared with the control

Fig. 8

Microscopic observation of mouse liver sections that had been stained with hematoxylin and eosin at × 400. Liver sections from control mice with normal cytoarchitecture. b Iron-overloaded (iron dextran, 100 mg/kg b.w.) liver section demonstrates degeneration of cellular boundaries, fatty ballooning deterioration, inflammation (I), and necrosis (N). c Desirox-treated liver sections demonstrate reduced necrotic area. d Liver section from the R2 mouse group improved histology with portal inflammation (PI). e Liver section from the R4 mouse group (f) Liver section from the R8 mouse group (g). Liver section from the S2 mouse group (h). Liver section from the S4 mouse group (i). Liver section from the S8 mouse group demonstrates improved histology with minimal necrotic area

Fig. 9

Microscopic observation of liver sections stained with Perls’ Prussian blue at × 400. a Liver section from control mice demonstrates normal hemosiderin deposition patches (very low). b Liver sections from iron-overloaded mice display excess blue patches. c Desirox-treated liver section d Liver section from the R2 mouse group with lesser blue patches. e Liver section from the R4 mouse group (f). Liver section from the R8 mouse group (g). Liver section from the S2 mouse group (h). Liver section from the S4 mouse group (i). Liver section from the S8 mouse group. MG demonstrates improved histology, and a gradual reduction of blue patches indicates effective iron removal from the liver

Fig. 10

Microscopic observation of liver sections stained with Masson’s Trichrome × 100. a Liver sections from control mice displayed normal cellular integrity with no fibrosis. b Liver sections from iron-overloaded mice displayed elongated fibrous septa and collagen accumulation (blue). c Desirox-treated liver section d. Liver section from the R2 mouse group with lesser blue patches. e Liver section from the R4 mouse group (f). Liver section from the R8 mouse group (g). Liver section from the S2 mouse group (h). Liver section from the S4 mouse group (i). Liver section from the S8 mouse group. MG demonstrates a nearly negligible collagen accumulation and healthy liver. Higher doses of both GA and MG demonstrate reduced collagen deposition, fibrous septum and necrotic cells in periportal veins, indicating a trend of restoration of normal cellular integrity