, St.Louis, MO, USA) [30] and imaged with a laser scanning confocal system (Zeiss LSM 510 META, Germany) and the stacked images through multiple slices were captured. Four slides were prepared for each rat from each group and only the representative images are presented.The digitized images were then

analyzed using image analysis system (ImageJ, NIH Software, Bethesda, MI) and the total collagen area fraction of each image was measured and expressed as the % collagen volume. The fundic part of the gastric mucosa was suspended in phosphate-buffered saline containing protease inhibitors, minced, and incubated for 10 min at 4 °C. It was centrifuged at 10,000 rpm for 10 minutes. The pellet was extracted in the lysis buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100 plus protease inhibitors) selleck products NVP-BKM120 manufacturer and centrifuged at 10,000 rpm for 10 minutes. Tissue extracts were preserved at -20 °C and used in future studies. For determination of pro-MMP-9 activity, mucosal extracts were electrophoresed in SDS-polyacrylamide gel containing 1 mg/ml gelatin under non-reducing conditions. To determine exactly the band of pro-MMP 9, rat uterine sample was loaded as a marker which has rich store of pro-MMP 9. The gels were washed in 2.5% Triton-X-100 and incubated in digestion buffer (40 mM Tris-HCl,

pH 7.4, 0.2 M NaCl, 10 mM CaCl2) overnight at 20 °C and stained with 0.1% Coomassie Blue followed by destaining. The zones of gelatinolytic activity came as negative staining. Enzymatic activity was determined by measuring the area produced by each band at 92 kDa region with the help of Image J software. This procedure was adopted from [43] with slight modifications. The methods of [38] were used to determine the total phenols and flavonoid content of the extract. Total phenols were expressed as mg gallic acid equivalents (GAE/g extract) where gallic acid was used as standard. MycoClean Mycoplasma Removal Kit Flavonoid content was expressed as mg catechin equivalents (CE/g extract) where catechin was used as standard. Alkaloid contents

were estimated [41] and expressed as mg/g bismuth nitrate. Total tannins were determined according to the method of [33] and expressed as tannic acid equivalent (TAE/g extract). GC-MS analysis [39] was carried out using Agilent Technologies 6890 N Network GC system & interfaced to Agilent Technologies 5973 Inert Mass Selective Detector (MSD) employing the following conditions: column DB-1 ms fused silica capillary column (30X0.25 I.D.X 0.10 Film, composed of 100% dimethylpolysiloxane) (chosen for improved signal to noise ratio for better sensitivity and mass spectral integrity), operating in electron impact mode; helium (5.0) was used as carrier gas at a constant flow of 1 ml/min. The injector, MS Source & MS quadrapole temperature were fixed at 250 °C, 230 °C & 150 °C, respectively, and turbo speed of the pump was 100%.