A new triterpene and protective effect of <i>Periploca somaliensis</i> Browicz fruits against CCl₄-induced injury on human hepatoma cell line (Huh7)

<div><p>The potential hepatoprotective effect of the methanolic extract of <i>Periploca somaliensis</i> Browicz fruits, its different fractions (<i>n</i>-hexane, chloroform and <i>n</i>-butanol) and the major isolated compound ursolic acid was evaluated using the human hepatoma cell line (Huh7) based on the changes in the activity of aspartate aminotransferase, alanine transaminase, glutathione and superoxide dismutase. Each sample was tested at three different concentrations (1000, 100 and 10 μg/mL). All tested samples exhibited a potent concentration-independent cytoprotective effect relative to silymarin as a reference standard. Chromatographic fractionation of the chloroform-soluble fraction of the methanol extract of <i>P. somaliensis</i> Browicz fruits afforded two known triterpenes, namely ursolic acid, and 11α,12α-epoxy-3β-hydroxy-olean-13β,28-olide, and a newly discovered one, namely 3β-hydroxy-urs-11-en-13β,28-olide. The structures of the isolated compounds were elucidated by the analysis of 1D and 2D NMR spectral data.</p></div

Effect of BCA on hepatic metabolic capacity.

<p>(<b>A</b>) CYP2E1 activity levels; (<b>B</b>) CYP1A1 expression levels; (<b>C</b>) SULT1A1 relative quantitation (RQ) versus group mean plot. <i>a</i>: significantly different from control gp. <i>b</i>: significantly different from CCl₄ gp.</p

Histopathological findings followed by grading of liver damage.

<p>(x40) (<b>A</b>) control: normal histological structure of portal area and surrounding hepatocytes; (<b>B</b>) CCl₄: loss of architecture with severe ballooning degeneration (arrow1), necrosis (arrow2) and fatty changes (arrow3) accompanied with fibrosis; (<b>C</b>) BCA+CCl₄: much less damage than in CCl₄ gp (<b>D</b>) BCA alone: similar to control gp.</p

Effect of BCA on hepatic synthetic capacity.

<p>n = 8.</p>*<p>p<0.05 compared to CCl₄ group.</p>**<p>p<0.01 compared to CCl₄ group.</p>***<p>p<0.001 compared to CCl₄ group.</p

Representative images of liver sections of different experiments.

<p>(<b>A–E</b>): Immunohistochemical detection of COX-2, iNOS, NF-κB, MMP-9 and α-SMA (x100). Control gp: minimal expression; CCl₄ gp: extensive expression; BCA+CCl₄ gp: less than CCl₄ gp; BCA gp: minimal expression. Image analysis was performed by examining 6 fields/slide. (<b>F</b>): Masson trichrome stain (x40). Control gp: shows absence of collagen fibers (stained blue) between hepatic lobules; CCl₄ gp: extensive fibers deposition with pseudolobules formation (bridging fibrosis); BCA+CCl₄ gp: less fibers than CCl₄ gp; BCA gp: absence of fibers. The bar chart represents levels of hydroxyproline expressed as µg/gm of wet tissue measured by Reddy’s method. <i>a</i>: significantly different from control gp. <i>b</i>: significantly different from CCl₄ gp.</p

Effect of BCA on oxidative stress markers.

<p>n = 8.</p>*<p>p<0.05 compared to CCl₄ group.</p>**<p>p<0.01 compared to CCl₄ group.</p>***<p>p<0.001 compared to CCl₄ group.</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

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