Animals

Adult Wistar strain male albino rats, average body weight 175-180 g were procured from the registered animal breeder (M/s Saha Enterprise, Kolkata, Regn No. 1828/PO/Bt/S/15/CPCSEA) and used for this study. Principles of laboratory animal care guidelines were strictly followed¹². The animals were maintained under 12:12 hours dark: light cycle, controlled temperature (22±3°C), with balanced diet and water ad libitum. Institutional Animal Ethical Committee (544/c/PO/02/CPCSEA) approved the experimental protocol. All the experiments were performed between 08:00-16:00 h.

Test drug preparation

The leaves of Sonneratia apetala Buch.-Ham were collected from the mangrove forest area of Indian Sunderban, West Bengal and identified by Botanical Survey of India, West Bengal. A voucher specimen (DST/537/5G-4/09-05) was deposited in the departmental herbarium. The shade dried and powdered leaves of S. apetala were extracted with hydro-methanol (20-80%) in soxhlet apparatus and concentrated by distilling the solvents and dried under reduced pressure. The extractive value was determined by gravimetric method and expressed as percentage yield. The bioactive phytoconstituents presences in hydro-methanolic extract of S. apetala (SA) were evaluated¹³. In brief, presence of alkaloids in SA was examined by Dragendorff’s test, sterols and triterpenoids by Liebermann-Burchard’s test, saponins by Froth test, tannins by lead acetate test, carbohydrates by Fehling’s test, reducing sugars by Benedict’s test, proteins by Millon’s test, phenolics by ferric chloride test, flavonoids by aluminum chloride test and glycosides by Borntrager’s test.

HPTLC Standardization

Gallic acid (3,4,5-trihydroxybenzoic acid), quercetin (5,7,3^/,4^/ tetrahydroxy flavonol) and coumarin (1,2-benzopyrone), three known biomarkers were used to standardize the extract¹⁴. SA was diluted with methanol (1 mg/ml) and spotted in the form of bands over a pre-coated silica gel plates (Merck, 60F₂₅₄, 20x20 cm) using Camag Linomat 5 applicator. The plates were developed in the solvent system (toluene:ethyl acetate: formic acid = 4.5:3:0.2) for 30 min. The densitometric scanning was performed on Camag TLC Scanner 3 at 280 nm. The amount of gallic acid, quercetin and coumarin present in SA was calculated against respective standards and expressed per mg of extract.

Estimation of phenolic

Total phenolic in SA was determined as described earlier¹⁴. In brief, to 0.2 ml of SA solution (1 mg/ml methanol), 1.0 ml of Folin-ciocalteu reagent was added and incubated in dark for 5 min. Then, 0.8 ml sodium carbonate solution (7.5%) was mixed and kept in dark for 30 min at room temperature. Finally, 3 ml of deionized water was added and read at 765 nm (Ultrospec 2000 UV-visible spectrophotometer, Pharmacia Biotech, USA). Total phenolic content was expressed as gallic acid equivalent (GAE) in µg per mg of extract.

Estimation of flavonoids

Aluminum chloride method was employed for flavonoids estimation¹⁵. In brief, 0.2 ml of SA (1 mg/ml methanol) was added to 0.8 ml methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of deionized water and read after 30 min at 415 nm and expressed as quercetin equivalent (QE) in µg per mg of extract.

Estimation of reducing power

The reducing power of SA was evaluated as described earlier¹⁴. In brief, 0.1 ml of SA was mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6), and 2.5 ml of 1% potassium ferricyanide and incubated at 50°C for 20 min. Then, 2.5 ml of 10% Trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with deionized water (2.5 ml) and ferric chloride solution (0.5 ml, 0.1%) and read at 700 nm. Reducing power was expressed as ascorbic acid equivalent (AAE) in µg per mg of extract.

Estimation of DPPH radical scavenging

The radical scavenging activity of SA was determined using the stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl)¹⁵. In brief, 0.1 ml of SA in methanol at different known concentrations was mixed with 3.9 ml of DPPH solution (0.135 mM) and read after 30 min at 517 nm. Butylated hydroxyl toluene (BHT) was used as positive control. The sample concentration required to scavenge 50% of DPPH (IC₅₀) was determined.

Estimation of superoxide anion radical scavenging

The incubation mixture contained 3 ml of Tris-HCl buffer (0.1 M, pH 7.4), 0.75 ml of nitroblue tetrazolium (300 µM) solution, 0.75 ml of NADH (936 µM) solution and 0.3 ml of SA at different concentrations (10-100 µl in methanol). The reaction started after adding 0.75 ml of phenazine methosulphate (120 µM) to the mixture. The reaction mixture was exposed to light at room temperature for 5 min and thereafter, read at 560 nm¹⁴. L-Ascorbic acid was used as positive control. The percent inhibition of super oxide anion generation of SA was calculated and was expressed as IC₅₀.

Estimation of nitric oxide radical scavenging

Test extract (10-100 µl in methanol) was mixed to 0.5 ml of 1 M phosphate buffer saline, 2 ml of 10 mM sodium nitroprusside and kept in room temperature for 150 min. Potassium nitrite solution was used as standard. After incubation, 0.5 ml of the reaction mixture containing nitrite was transferred and mixed with 1 ml of 0.33% sulphanilic acid reagent and allowed to stand for 5 min for completing diazotization. Then, 1 ml of 1% naphthylethylene diamine dihydrochloride was added and read after 30 min at 540 nm¹⁶.

Estimation of hydroxyl radical scavenging

The reaction mixture contained 0.1 ml deoxyribose (2.8 mM), 0.1 ml ferric chloride (0.1 mM), 0.1 ml EDTA (0.1 mM), 0.1 ml hydrogen peroxide (1 mM), 0.1 ml ascorbate (0.1 mM), 0.1 ml phosphate buffer (20 mM, pH 7.4) and 1 ml of SA (10-100 µl in methanol). The mixture was incubated at 37°C for 1 h. At the end of the incubation period, 1.0 ml thiobarbituric acid (1%) was added and heated at 95°C for 20 min. After cooling, the thiobarbituric acid reacting substances (TBARS) formation was measured at 532 nm¹⁴. The percent TBARS production for positive control (H₂O₂) was fixed at 100% and the relative percent TBARS was calculated for the extract treated groups.

Estimation of ABTS radical scavenging

In brief, stock solution included ABTS (2,2’-azino-bis-3-ethyl benzthiazoline-6-sulphonic acid) solution (7 mM) and ammonium persulphate (2.45 mM) and working ABTS⁺ solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12-16 h at room temperature in the dark. The final working ABTS⁺ solution was obtained after appropriate dilution with methanol (OD 0.7 at 734 nm). SA (10-100 µl in methanol) was added to 1 ml of ABTS⁺ solution and after 7 min read at 734 nm¹⁵. The percent inhibition was calculated and expressed as IC_50.

Antiulcer activity in rats

Antiulcer activity of SA was evaluated on alcohol-induced gastric ulcers in rats as described by Morimoto and coworkers (1991)¹⁷ with slight modification^18-19. Omeprazole (20 mg/kg, p.o) was used as standard prototype agent. All rats (body weight 150-175 g) were divided into five groups (N=6) as follows: Group I (normal control), Group II (treated control), Group III (SA 125 mg/kg), Group IV (SA 250 mg/kg) and Group V (Omeprazole 20 mg/kg). All animals were pre-treated according to above protocol for 10 days by oral route. On day 10, 50% alcohol at the dose of 5 ml/kg was administered orally to all rats (except normal control). Normal control rats were received distilled water (5 ml/kg). The last dose of all test drugs was given 1 h prior to alcohol administration. All animals were sacrificed under anesthesia (sodium pentothal 40 mg/kg, i.p) two hours after alcohol ingestion. The stomach was incised along the greater curvature and examined for gastric lesions. The gastric ulcer was scored and indexed as follows²⁰.

Ulcer Index = (number of lesions I) + (number of lesion II) X 2 + (number of lesion III) X 3

I=presence of edema, hyperemia and single submucosal punctiform haemorrhages

II=presence of submucosal punctiform hemorrhagic lesions with small erosions

III=presence of deep ulcer with erosions and invasive lesions

Moreover, gastric mucosal tissues were dissected out and a portion was homogenized in cold phosphate buffer (0.1 M, pH 7.2), centrifuged and used for estimation of glutathione, catalase, lipid peroxides and protein^19-22.

Statistical analysis

Data have been summarized by routine descriptive statistics. All statistical analyses were performed using statistical software package (SPSS, version 17.0, IBM, USA). Differences between and within groups have been assessed for statistical significance by standard parametric and non-parametric tests, as appropriate. ‘P’ value < 0.05 was taken as level of statistical significance in all tests.