Vanillin abrogates ethanol induced gastric injury in rats via modulation of gastric secretion, oxidative stress and inflammation

Vanillin is commonly used as an additive in food, medicine and cosmetics, but its effect has not yet been studied in gastric injury. Therefore the effect of vanillin was studied in experimental gastric ulcer. Gastric secretion and acidity were studied in pylorus ligated rats. Ulcer index, levels of gastric mucus, malondialdehyde (MDA), myeloperoxidase activity (MPO), expression of nuclear factor kappa B (NF-κB) p65, and histopathological changes were determined in ethanol induced gastric ulcer. Pre treatment with vanillin significantly reduced gastric secretion (P < 0.001) and acidity (P < 0.0001) and gastric ulcer index scores (P < 0.001). and augmented the gastric mucosal defense. Vanillin significantly restored the depleted gastric wall mucus levels (P < 0.0001) induced by ethanol and also significantly attenuated ethanol induced inflammation and oxidative stress by the suppression of gastric MPO activity (P < 0.001), reducing the expression of NF-κB p65 and the increased MDA levels (P < 0.001). Vanillin was also effective in alleviating the damage to the histological architecture and the activation of mast cells induced by ethanol.Together the results of this study highlight the gastroprotective activity of vanillin in gastric ulcers of rats through multiple actions that include inhibition of gastric secretion and acidity, reduction of inflammation and oxidative stress, suppression of expression of NF-κB, and restoration of the histological architecture. Keywords: Gastric ulcers, Pylorus ligation, Ethanol, Vanillin, Inflammation, Oxidative stres

Efficacy of Lepidium sativum against carbon tetra chloride induced hepatotoxicity and determination of its bioactive compounds by GC–MS

Lepidium sativum seed (LSS) (family: Cruciferae) has been used in traditional medicine for the treatment of jaundice, liver problems, spleen diseases and gastrointestinal disorders. It was also reported to possess antihypertensive, diuretic, anti-asthmatic, antioxidant, and anti-inflammatory activities. Attempt has been made to study hepatoprotective potential of LSS available in Saudi Arabian Market. The aim of the present study was to determine the hepatoprotective effect of ethanolic extracts of LSS against carbon tetrachloride (CCl4) induced acute liver injury in rats. The bioactive compounds responsible for this activity have been analyzed by GC–MS. To evaluate the hepatoprotective activity, six groups (n = 6) of rats were taken. First group was control, second was toxic and other groups received oral test solutions: 100 mg/kg silymarin, or LSS (100, 200, and 400 mg/kg), once daily for 7 consecutive days, followed by hepatotoxicity induction with CCl4. Blood and liver tissues were collected for biochemical, antioxidant and microscopic analyses. The bioactive constituents present in the extract were analyzed by GC–MS. Results showed that pretreatment with LSS and silymarin significantly reduced the level of serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and bilirubin (BIL), which was increased significantly in toxic group treated with only CCl4. Histological analysis of liver tissues in groups pretreated with LSS and silymarin showed mild necrosis and inflammation of the hepatocytes compared to the toxic group. GC–MS analysis of LSS showed the presence of twelve major fatty acids including alpha-linolenic acid as a major constituent. These results indicated that LSS exerts enhance hepatoprotective activity that could be attributed towards its antioxidant activity, coupled together with the presence of anti-inflammatory compounds in LSS extract

<i>Moringa oleifera</i> as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

<div><p>In this study we investigated the anti-cancer effect of <i>Moringa oleifera</i> leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70–90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2–3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of <i>Moringa oleifera</i> could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.</p></div

GC-MS analyses of MO bark.

<p>(A) Typical TIC-GC/MS chromatogram of Moringa bark analyzed on GC system equipped with an apolar 5-MS capillary column attached with Mass Detector. It showed the presence of seventeen compounds. The peaks showing bioactive anticancer compounds in bark such as eugenol and hexadeconoic acid are marked. (B) Chemical structures of the active compounds as mentioned above are shown here.</p

Cell motility assays.

<p>(A) MDA-MB-231 and (C) HCT-8 cell lines. Treatment patterns and annotation of the extracts were the same as descried in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135814#pone.0135814.g003" target="_blank">fig 3</a>. A significant decrease in cell motility was observed upon extracts treatment. Columns of the bar graphs (B&D) showing 80–95% decrease in cell motility. * Statistically significant (<i>P</i>≤0.05).</p

Colony formation assay.

<p>Anchorage dependent colony formation assay shows a significant reduction in colony formation in (A) MDA-MB-231 and (C) HCT-8 cell lines treated with different concentration of plant extracts. Quantitative analyses are given in the form of bar graphs for MDA-MB-231(B) and HCT-8 (D) respectively. A dramatic decrease in colony formation is evident from the figure. Statistically significant values were marked with asterisk. (<i>P</i>≤0.05).</p

Apoptosis assay.

<p>Assessment of apoptosis in (A) MDA-MB-231 and (C) HCT-8 cell lines treated with the extracts of MO leaves (L), bark (B) and seed (S) for 24 h. The cells cultured either in DMEM or in RPMI media were used as control. The apoptotic rates were detected by annexin V-PI dual staining. Q1 quadrant (annexin V−, PI+) represented dead cells; Q2 quadrant (annexin V+, PI+) represented late apoptotic cells; Q4 quadrant (annexin V+, PI–) represented early apoptotic cells; Q3 quadrant (Annexin V−, PI−) represented live cells. The percentage of total apoptotic cells (Q2+Q4) was calculated and shown in the bar graphs MDA-MB-231(B) and HCT-8 (D) cell lines respectively. The extent of apoptosis was significantly high both in leaves and bark treated cell lines. However, few apoptotic cells were also observed only in HCT-8 when treated with the seed extract. * Statistically significant (<i>P</i>≤0.05).</p

Cell cycle assay.

<p>Analysis of cell cycle arrest in plant extract treated (A) MDA-MB-231 and (C) HCT-8 cell lines. Cells were treated with 500μg/ml extracts of leaves (L) Bark (B) and seeds(S). Statistical analyses of the findings were shown in the form of bar graphs for MDA-MB-231(B) and HCT-8 (D) cell lines respectively. A significant G2/M enrichment was observed in leaves (L) and bark (B) treated cell lines. * Statistically significant (<i>P</i>≤0.05).</p

Cell survival assay.

<p>(A) MDA-MB-231 (B) HCT-8 cell lines. Cells were treated with different concentrations of plant extracts; (L) leaves; (B) bark and (S) seed. Equal numbers of cells in 20 μl were taken in 380 μl of cell counting solution. Cell viability was analyzed on Muse cell analyzer. Column (B & D) representing the quantitative analysis of the viable cells. A significant number of dead cells were observed upon extract treatment as compared with control. * Statistically significant (<i>P</i>≤0.05).</p