Hepatoprotective and antioxidant effect of ellagitannins and galloyl esters isolated from <i>Melaleuca styphelioides</i> on carbon tetrachloride-induced hepatotoxicity in HepG2 cells

<p><b>Context</b> In a previous study, the total extract of <i>Melaleuca styphelioides</i> Sm. (Myrtaceae) showed a significant hepatoprotective effect in a CCl₄-induced toxicity model in mice. However, the active components responsible for the activity of the extract were not identified.</p> <p><b>Objective</b> To determine the <i>in vitro</i> hepatoprotective activity of the isolated pure compounds from <i>M. styphelioides</i> leaves using the CCl₄-challenged HepG2 cell model.</p> <p><b>Materials and methods</b> The hepatoprotective activity of the compounds (at concentrations of 100, 50 and 25 μm), the total extract and silymarin (Sil) (100, 50 and 25 μg/ml) was determined by measuring the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after pretreatment with the tested samples for one hour. Glutathione (GSH) and superoxide dismutase activity (SOD) were estimated to determine the mechanisms of the hepatoprotective activity.</p> <p><b>Results</b> Some compounds showed marked hepatoprotection, including tellimagrandin I, which produced 42, 36 and 31% decrease in ALT and 47, 43 and 37% decrease in AST, at the tested concentrations, respectively, pedunculagin (32, 32 and 30% decrease for ALT and 48, 48 and 45% for AST), tellimagrandin II (38, 32 and 26% decrease for ALT and 45, 40 and 34% for AST) and pentagalloyl glucose (30, 28 and 26% decrease for ALT and 45, 38 and 36% for AST). Tellimagrandin I and II showed the highest increase in GSH (113, 105 and 81% and 110, 103 and 79%, respectively), which was comparable to Sil. Pedunculagin produced the highest increase in SOD (497, 350 and 258%).</p> <p><b>Conclusion</b> This study highlights promising natural hepatoprotective candidates derived from <i>M. styphelioides</i>.</p

Effects of 3-NPA and/or genistein on striatal, cortical and hippocampal AChE activity of ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 6). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (one-way ANOVA followed by Tukey test).</p

Effects of 3-NPA and/or genistein on step through passive avoidance in ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (5, 10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Training was performed on day 5. First and second retention latencies were assessed at days 6 (A) and 8 (B). Data are presented as medians and quartiles (n = 8). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (Kruskal-Wallis nonparameteric test followed by Dunn’s test).</p

Effects of 3-NPA and/or genistein on locomotor activity in ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (5, 10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 8). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (two-way ANOVA followed by Bonferroni test).</p

H & E staining of the cortices of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D and H showed no histological alterations, C showed severe hemorrhage (h), E showed focal gliosis (g) F showed focal gliosis (g) and G showed congested blood vessel (C).

<p>H & E staining of the cortices of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D and H showed no histological alterations, C showed severe hemorrhage (h), E showed focal gliosis (g) F showed focal gliosis (g) and G showed congested blood vessel (C).</p

Effects of 3-NPA and/or genistein on striatal, cortical and hippocampal AChE activity of ovariectomized rats.

<p>3-NPA was administered i.p. (20 mg/kg) for 4 consecutive days. 17β-estradiol (2.5 mg/kg body weight, s.c) and genistein (10 and 20 mg/kg body weight, i.p) were administered for 8 days, beginning 4 days before and continued for 4 days one hour before 3-NPA injections. Data are presented as means ±S.E.M. (n = 6). ^x statistically significant compared to sham group at P< 0.05, * statistically significant compared to control group at P< 0.05, ^# statistically significant compared to 3-NPA-treated group at P< 0.05 (one-way ANOVA followed by Tukey test).</p

H & E staining of the hippocampi of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D, F, G and H showed no histological alterations, C showed severe neurodegeneration (d) and hemorrhage (h) and E showed some degenerated hippocampal cells (d).

<p>H & E staining of the hippocampi of rats belonging to the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (5 mg/kg)- and 3-NPA-treated group (E), genistein (10 mg/kg)- and 3-NPA-treated group (F), genistein (20 mg/kg)- and 3-NPA-treated group (G) and genistein alone-treated group (H): A, B, D, F, G and H showed no histological alterations, C showed severe neurodegeneration (d) and hemorrhage (h) and E showed some degenerated hippocampal cells (d).</p

Immunohistochemical staining of cortical COX-2-positive cells immunized with goat-anti-rabbit antibodies of the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (10 mg/kg)- and 3-NPA-treated group (E), genistein (20 mg/kg)- and 3-NPA-treated group (F) and genistein alone-treated group (G).

<p>Immunohistochemical staining of cortical COX-2-positive cells immunized with goat-anti-rabbit antibodies of the sham group (A), control group (B), 3-NPA-treated group (C), 17β-estradiol- and 3-NPA-treated group (D), genistein (10 mg/kg)- and 3-NPA-treated group (E), genistein (20 mg/kg)- and 3-NPA-treated group (F) and genistein alone-treated group (G).</p