Gastroprotective effect of plumbagin and ethanolic extract of plumbaginales in experimentally-induced ulcer

Gastroprotective effect of plumbagin and ethanolic extracts of Plumbaginales Journal of HerbMed Pharmacology, Volume 5, Number 3, July 2016 http://www.herbmedpharmacol.com 93 tus, cigarette smoking, family history of peptic ulcer and infection with H. pylori were all associated with increased risk of peptic ulcer. Separate analyses were performed by sex and occupational group to avoid confounding by cigarette smoking and age. Multivariate analyses showed that for all females and males, only family history was significantly predictive of peptic ulcer or duodenal ulcer. This study was designed to evaluate the anti-ulcer effects of ethanolic extracts of Plumbaginales namely P. auriculata, P. indica and P. zeylanica and Plumbagin in Aspirin and Ethanol induced gastric ulcer models. Materials and Methods Plant authentication Samples of P. auriculata, P. indica & P. zeylanica were collected from different regions of Kerala. They were then authenticated (Voucher No. 1105, 1102, 1101) by School of Ecological Sciences, M.G. University, Kottayam, Kerala. Plumbagin extract Roots of P. indica were oven dried at 40 ̊C and powdered which was extracted with chloroform for 5 hours in a Soxhlet apparatus. The solution was evaporated to dryness. The extract obtained was dissolved in methanol and again evaporated to dryness to yield yellow crystals (4). Preparation of Plumbagin free alcoholic extract Roots of P. auriculata, P. indica and P. zeylanica (1 kg) were dried, coarsely powdered and soaked in limewater till red color of the lime water disappears. They were then dried again, finely powdered and extracted using Soxhlet’s apparatus for 6 hours using ethanol as solvent. The extract obtained was concentrated and dried at room temperature. The extraction efficiency was defined as follows: Percentage extraction (w/w) = Mass of extracts Mass of dried root Estimation of the amount of Plumbagin in the extracts One ml alcoholic KOH (10%) was added to 1ml of standard stock solution (1 mg/mL) of Plumbagin and the volume was then adjusted to the 5 ml with absolute alcohol. The absorbance of the colored solution was observed by UV/Visible spectrophotometer in the range of 400 to 800 nm against the reagent blank. The blank was prepared similarly in which volume of standard Plumbagin was replaced by an equal volume of absolute alcohol. The maximum absorbance was obtained at 520 nm. In a series of 5 mL volumetric flask, 0.2, 0.3, 0.4, 0.8 and 1.2 mL of standard stock solution of Plumbagin were mixed with 1 mL of 10% alcoholic KOH and volume was made upto the mark with absolute alcohol. The absorbance of colored solution was measured at 520 nm against a reagent blank. The absorbance was plotted against concentration of Plumbagin and the concentration of unknown solution was computed from the calibration graph or from the regression equation. 10 mg/mL of the four extracts were prepared as the standard sample solution with measuring absorbance at 520 nm (5). In vitro Methods DPPH assay A stock solution of DPPH (1.3 mg/mL in methanol) was prepared such that 75 μl of it in 3 ml methanol gave an initial absorbance of 0.9. After 30 minutes, decrease in the absorbance in the presence of sample extract and standard at different concentrations was noted. A blank reading was taken using methanol instead of sample extract. Absorbance at 517 nm was determined after 30 minutes using UV-visible Spectrometer. The IC50 (Inhibitory concentration to scavenge 50% free radicals) was also determined. Lower absorbance levels of the reaction mixture indicate higher free radical scavenging activity. The IC50 value denotes the concentration of sample required to scavenge 50% of the DPPH free radicals. The IC50 was calculated from equation of line obtained by plotting a graph of concentration vs. % inhibition. The ability to scavenge the DPPH radical was calculated using the following equation (6): Percentage Inhibition = C-T/A × 100 Where C = Absorbance of DPPH alone, and T = Absorbance of DPPH along with different concentrations of extracts. Lipid peroxidase assay Lipid peroxidation product (i.e. malondialdehyde or MDA) was measured according to the method of Ohkawa et al (7). 1 mL of sample was mixed with 0.2 mL 4% (w/v) sodium dodecyl sulfate, 1.5 mL 20% acetic acid in 0.27 M hydrochloric acid (pH 3.5) and 15 mL of 0.8% thiobarbituric acid (TBA, pH 7.4). The mixture was heated for 1 hour at 85°C in a hot water bath. The intensity of the pink color developed was read against a reagent blank at 532 nm (7). Acid-neutralizing capacity The acid-neutralizing capacity of the solute was carried out at a temperature of 37 ± 3°C. A pΗ meter was standardized using 0.05 M potassium biphthalate and 0.05 M potassium tetra oxalate standardized buffers. A magnetic stirrer was used to produce stirring rate of 300 ± 30 rpm. 0.5 g of the extract was transferred to a 250-mL beaker, and then 70 mL of distilled water added. Mixing with the magnetic stirrer continued for 1 minute. Then 30 mL 1 N HCl was added to the test solution with continuous stirring for 15 minutes. Excess HCl was titrated with 0.5 N NaOH to attain stable pΗ of 3.5. The number of mEq of acid consumed was calculated by the folowing formula (7): Total mEq(ANC) = (30 × N of HCL) – (V of NaOH × N of NaOH) N = Normality; V = Volume Evaluation of ethanol-induced anti-ulcer activity Samples of small intestine from goat were collected from a slaughterhouse. They were washed with the tyrode’s soluIttiyavirah SP et al Journal of HerbMed Pharmacology, Volume 5, Number 3, July 2016 http://www.herbmedpharmacol.com 94 tion to remove unwanted food materials and mesenteries. To keep the tissues active and live, they remained in the tyrode’s solution supplied with air. The intestine samples were examined for any lesions or ulcers. Ethanol (80%) was used as an ulcerogenic agent. The goat small intestine was cut into six 1.5-2.0 cm long segments. The segments were opened upwards with mucous, washed and stretched on a dish containing the tyrode’s solution. The dishes were supplied with air under constant temperature (8). The segments were divided into 2 groups, 3 segments each, and treated as following: Group 1control, treated with 1 mL of normal saline, Group 2treated with 1 mL of chloroform extract (Plumbagin), Group 3treated with 1 mL of P. indica extract, Group 4treated with 1 mL of P. zeylanica extract, Group 5treated with 1 mL of P. auriculata extract. After 30 minutes of treatment of the groups, 1 mL of 80% of ethanol was added to each dish. Each intestinal segment was then examined for the presence of lesions and ulcers using a magnifying lens. Statistical analysis The statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison tests with the normally-distributed data. Non-parametric tests were also used for the ordinal data. All the samples were compared themselves and statistical significance was noted. In vivo Methods Animals The rats were housed in polyacrylic cages and maintained at 27 ± 2°C. They were fed with standard pellet diet (Hindustan Lever Ltd. Bangalore, India) and water ad libitum. All animal procedures were approved by the Animal Ethical Committee (No: 008/MPH/UCP/CVR/12) in endogenous defense mechanism Aspirin-induced model Animals were randomly divided into three groups, each with six animals. Group I served as positive control, Group II acted as standard and received Ranitidine (20 mg/kg). The group III was treated with ethanolic extract by oral route in a dose of 300 mg/kg for a period of 5 days in aspirin-induced ulcer model. On day 5, aspirin at a dose of 200 mg/kg was administered to the animals of all groups, 1 hour after the administration of last dose of the extract/ ranitidine. After 4 hours, the animals were sacrificed and the stomach was then excised and cut along the greater curvature, washed carefully with 5.0 mL of 0.9% NaCl and ulcers were scored (9). Ethanol-induced model Animals were randomly divided into three groups, each with six animals. • Group I: Untreated group received distilled water (p.o) for 9 days followed by ethanol (5 mL/kg, p.o) on 11th day. • Group II: Ranitidine group (20 mg/kg, p.o) for 9 days followed by ethanol (5 mL/kg, p.o) on 11th day. • Group III: Test group (300 mg/kg, p.o) for 9 days followed by ethanol (5 mL/kg, p.o) on 11th day. All ethanol-treated animals were fasted for 36 hours before administration of ethanol. The animals in the standard drug group and aqueous extract (test drug group) were pretreated with respective drugs for 9 days. Later, food and water were withdrawn for 36 hours. The respective drugs were administered 1 hour before ethanol administration. Ethanol (90%) was administered to all animals at a dose of 1 mL/200 g. After 1 hour, the animals were sacrificed, stomach was removed slightly inflated by injecting 15% formalin solution for 10 minutes. The stomachs were then opened along the greater curvature with ulcer scoring and percentage inhibition (10). Ulcer Score The number of ulcers was noted and the severity recorded with the following scores (11): Ulcer index Ulcer index (UI) was calculated using the formula UI = US+ UN+ UP×10-1, where, US = Mean severity of ulcer score; UN = Average number of ulcers per animal; and UP = Percentage of animals with ulcer incidence. Percentage protection Percentage protection of ulcers = CUI-TUI/CUI, where CUI = Ulcer index of control groups, and TUI = Ulcer index of treated groups. The mean scores for each group were then calculated with analyzing the results. Histological studies A portion of the ulcer region in the stomach was dissected out and fixed in 5% buffered neutral formalin solution for histological observations. Following fixation, tissues were embedded in paraffin and sections with a 5 μm thickness were cut from the paraffin block and then stained with hematoxylin and eosin (H & E). The sections were examined with the help of a light microscope and photomicrographs were taken and scored (12). Statistical analysis The statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Dennett multiple comparisons for the data which were normally distributed. For the data of ordinal type, a non-parametric test was used. Statistically significant results were noted. Results Plumbagin extraction A yellow crystal of Plumbagin was obtained. The amount of Plumbagin obtained was 12.6 g with a percentage yield of 1.26%. The percentage yield of Plumbagin in P. zeylanica extract was found to be 1.37%. That in P.auriculata extract, and P.indica was 0.78% and 1.5%, respectively. DPPH assay The obtained percentage inhibition of different concentrations of Plumbaginales is shown in Table 1. The IC50 value of Plumbagin, P. indica, P. zeylanica and P. auriculata was found to be 33.94, 20.93, 29.03 and 20.11 μg/mL, respectively. Gastroprotective effect of plumbagin and ethanolic extracts of Plumbaginales Journal of HerbMed Pharmacology, Volume 5, Number 3, July 2016 http://www.herbmedpharmacol.com 95 Table 1. Average percentage inhibition of P. indica, P. zeylanica, P. auriculata and Plumbagin in DPPH assay and lipid peroxidase assay Assay method Control 10 20 30 40 Plumbagin DPPH assay 18.75± 0.79 34.32±1.26 39.12±0.79 60.94±1.41 Lipid peroxidase 6.64± 2.50 23.31±1.57 34.14±1.96 49.98±2.32 Plumbago indica DPPH assay 31.24±1.09 53.13±0.76 65.71±0.70 71.87±1.21 Lipid peroxidase 16.91±2.14 36.65±2.12 49.98±1.37 62.46±2.86 Plumbago zeylanica DPPH assay 21.25±0.93 33.12±0.67 51.87±1.02 68.12±0.60 Lipid peroxidase 8.26±3.62 33.27±3.49 42.48±2.09 64.40±10.46 Plumbago auriculata DPPH assay 37.19±0.30 51.24±0.87 62.5±0.05 71.24±1.39 Lipid peroxidase 24.10±3.78 47.52±0.69 57.79±3.04 68.30±2.06 Table 2. Number of ulcer spots in ethanol induced in vitro models Sl No. Group Ulcer No. Average Ulcer No (Mean ± SD) % Protection