Figure 1.
Free radical scavenging activities of mangiferin in cell free system.
Figure 1A stands for the DPPH radical and superoxide radical scavenging activities, Vit C and quercetin were used as positive control respectively. Figure 1B stands for the hydroxyl and nitric oxide radical scavenging activities, mannitol and curcumin were used as standard respectively. Values are represented as the mean of six different experiments in each set. Data represent the mean ± SEM of 6 separate experiments. “*” sign indicates the optimum dose of mangiferin at which it shows its significant radical scavenging activity.
Figure 2.
Dose and time dependent effects.
A: Dose and time dependent effect of Pb(II) on ALT level. Closed circle: ALT level in normal mice, Closed Square: Pb(II) induced ALT level in mice for 2 days, Blank circle: Pb(II) induced ALT level in mice for 4 days, Closed triangle: Pb(II) induced ALT level in mice for 6 days, Closed asterisk: Pb(II) induced ALT level in mice for 8 days at a dose of 1 mg, 3 mg, 5 mg, 7 mg and 9 mg/kg body weight. Panel B: Dose and time dependent effect of mangiferin on ALP level against Pb(II) induced toxicity in the hepatic tissue of the experimental mice. Cont: ALP level in normal mice, Pb(II): ALP level in Pb(II) induced mice, Pb(II)+Mang(25), Pb(II)+Mang(50), Pb(II)+Mang(75), Pb(II)+Mang(100), Pb(II)+Mang(125) and Pb(II)+Mang(150): ALP level in mangiferin (Mang) treated mice for 6 days at a dose of 25, 50, 75, 100, 125 and 150 mg/kg body weight respectively post to Pb(II) administration; Pb(II)+Mang(2D), Pb(II)+Mang(3D), Pb(II)+Mang(4D), Pb(II)+Mang(5D), Pb(II)+Mang(6D), Pb(II)+Mang(7D) and Pb(II)+Mang(8D): ALP level in mangiferin post-treated mice for 2, 3, 4, 5, 6, 7 and 8 days respectively at a dose of 100 mg/kg body weight [at a dose of 5 mg/kg body weight of Pb(II)]. Each column represents mean ± SEM, n = 6. “a” indicates the significant difference between the normal control and toxin treated groups and “b” indicates the significant difference between the Pb(II) treated and mangiferin treated groups. (Pa<0.05, Pb<0.05).
Table 1.
Effect of Pb(II) and mangiferin on the activities of the lipid peroxidation and protein carbonylation in liver tissue.
Table 2.
Status of the thiol based antioxidant in the liver tissue of the Pb(II) and mangiferin treated mice.
Table 3.
Effect of Pb(II) and mangiferin on the activities of the antioxidant enzymes in liver tissue.
Figure 3.
Effect of mangiferin on Pb(II) induced intracellular ROS production.
Cont: normal control, Mang: treated with mangiferin, Pb(II): administered with Pb(II), Pb(II)+Mang: Mangiferin treated post to Pb(II) administration. Data are mean ± SEM, for 6 sets per group and were analyzed by one-way ANOVA, with Student-Newman-Keuls post hoc tests. “a” indicates the significant difference between the normal control and Pb(II) induced groups and “b” indicates the significant difference between the Pb(II) induced and mangiferin treated groups. (Pa<0.05, Pb<0.05).
Figure 4.
Dose and time dependent effects on cell viability.
A. Dose dependent effect of Pb(II) on cell viability. MTT assay was carried out for this purpose. Cell viability in Pb(II)-exposed hepatocytes for 2 h at a dose of 0, 10, 20, 30, 40 and 50 µg/mL. Each point represents mean ± SEM, n = 6 (number of plates). B. Time dependent effect of Pb(II) on cell viability. MTT assay was carried out for this purpose. Cell viability in Pb(II)-exposed hepatocytes for the incubation time of 0 hour, 1 hour, 1.5 hour, 2 hour, 2.5 hour and 3 hour at a dose of 30 µg/mL. Each point represents mean ± SEM, n = 6 (number of plates). C. Dose dependent effect of mangiferin against Pb(II) -induced toxic effect on cell viability. MTT assay was performed for this purpose also. Cont: cell viability in normal hepatocytes, Pb(II): cell viability in Pb(II)-induced hepatocytes for 2 h at a dose 30 µg/mL, Pb(II)&Mang-30, Pb(II)&Mang-40, Pb(II)&Mang-50, Pb(II)&Mang-60, Pb(II)&Mang-70 and Pb(II)&Mang-80: cell viability in simultaneous exposure of Pb(II) and mangiferin in hepatocytes.for 2 h at a dose of 30, 40, 50, 60, 70 and 80 µg/mL. “a” indicates the significant difference between the normal control and toxin-treated cells, “b” indicates the significant difference between Pb(II) -induced and mangiferin-treated cells. Each column represents mean ± SEM, n = 6; (pa<0.05, pb<0.05).
Figure 5.
DNA fragmentation on agarose/ethydium bromide gel. DNA isolated from experimental mouse liver was loaded onto 1% (w/v) agarose gels.
Lane 1: Marker (1 kb DNA ladder); Lane 2: DNA isolated from normal liver; Lane 3: DNA isolated from mangiferin treated liver; Lane 4: DNA isolated from Pb(II) exposed liver; Lane 5: DNA isolated from Mangiferin treated post to Pb(II) administration.
Figure 6.
Impact of Pb(II) and mangiferin (Mang) on mode of cell death in hepatocytes.
Percent distribution of apoptotic and necrotic cells has been presented in all four quadrants. Cell distribution was analyzed by means of Annexin V binding (taken as x axis) and PI uptake (taken as y axis). The FITC and PI fluorescence were measured using a flow cytometer with FL-1 and FL-2 filters respectively. Results were expressed as dot plot representing as one of the six independent experiments.
Figure 7.
Immunoblot analysis of mitogen activated protein kinases (MAPKs) in response to Pb(II) and mangiferin treatment, both in liver and hepatocytes.
Hepatocytes were treated with Pb(II) (30 µg/mL) and mangiferin (50 µg/mL) for 2 h. Figure 7: phosphorylated p38 (phospho- p38) and total p38 MAPK, phosphorylated JNK(phospho- p38) and total JNK MAPK, phosphorylated ERK (½) MAPK [phospho-ERK (½)] and total ERK (½) MAPK. β actin was used as an internal control Cont: normal control, Mang: treated with mangiferin, Pb(II): administered with Pb(II), Pb(II)+Mang: Mangiferin treated post to Pb(II) administration, Pb(II)&Mang: Simultaneous exposure of Pb(II) and mangiferin in hepatocytes. Data are mean ± SEM, for 6 sets per group and were analyzed by one-way ANOVA, with Student-Newman-Keuls post hoc tests. Differences were attributed at p<0.05, and homogeneous subgroups share common superscripted letters.
Figure 8.
Immunoblot analysis of NF-κB, IκBα and IKKα proteins in response to Pb(II) and mangiferin treatment, both in liver and hepatocytes.
Hepatocytes were treated with Pb(II) (30 µg/mL) and mangiferin (50 µg/mL) for 2 h. β actin was used as an internal control Cont: normal control, Mang: treated with mangiferin, Pb(II): administered with Pb(II), Pb(II)+Mang: Mangiferin treated post to Pb(II) administration, Pb(II)&Mang: Simultaneous exposure of Pb(II) and mangiferin in hepatocytes. Data are mean ± SEM, for 6 sets per group and were analyzed by one-way ANOVA, with Student-Newman-Keuls post hoc tests. Differences were attributed at p<0.05, and homogeneous subgroups share common superscripted letters.
Figure 9.
Immunoblot analysis of Bax and Bcl-2 in response to Pb(II) and mangiferin treatment, both in liver and hepatocytes.
Hepatocytes were treated with Pb(II) (30 µg/mL) and mangiferin (50 µg/mL) for 2 h. β actin was used as an internal control Cont: normal control, Mang: treated with mangiferin, Pb(II): administered with Pb(II), Pb(II)+Mang: Mangiferin treated post to Pb(II) administration, Pb(II)&Mang: Simultaneous exposure of Pb(II) and mangiferin in hepatocytes. Data are mean ± SEM, for 6 sets per group and were analyzed by one-way ANOVA, with Student-Newman-Keuls post hoc tests. Differences were attributed at p<0.05, and homogeneous subgroups share common superscripted letters.
Figure 10.
Effect of mangiferin on Pb(II) induced mitochondrial membrane potential in liver tissue.
(Figure 10A). Figure 10B: Immunoblot analysis of Cytochrome C, Apaf-1, Caspase 9 and Caspase 3 in response to Pb(II) and mangiferin treatment, both in liver and hepatocytes. Hepatocytes were treated with Pb(II) (30 µg/mL) and mangiferin (50 µg/mL) for 2 h. β actin was used as an internal control Cont: normal control, Mang: treated with mangiferin, Pb(II): administered with Pb(II), Pb(II)+Mang: Mangiferin treated post to Pb(II) administration, Pb(II)&Mang: Simultaneous exposure of Pb(II) and mangiferin in hepatocytes. Data are mean ± SEM, for 6 sets per group and were analyzed by one-way ANOVA, with Student-Newman-Keuls post hoc tests. Differences were attributed at p<0.05, and homogeneous subgroups share common superscripted letters.
Figure 11.
Histopathological changes in liver tissue (stained with haematoxylin and eosin dye).
Cont: liver section of normal mice liver (×100); Mangiferin: liver section from animals treated with mangiferin only; Pb(II): liver section from the Pb(II) exposed group, arrows indicate the apoptotic changes (×100); Pb(II)+Mangiferin: liver from the animal post treated group (×100) showing almost normal morphology.
Figure 12.
Reaction of mangiferin with free radicals (ROS) and resonating canonical structures of two phenoxyl radicals.
Figure 13.
Schematic representation of Pb(II) induced hepatotoxicity and its protection by mangiferin.