Evaluation of Antioxidant and Antiulcer Potential of Cucumis sativum L. Seed Extract in Rats

MATERIALS AND METHODS

Plant Material
The present study was conducted at Rayat Institute of Pharmacy Department, Near Railmajra, Ropar-144533, India. Seeds were purchased from Local Grain Market Sector 26, Chandigarh (UT). Healthy looking seeds were chosen for examination (without mechanical damages and bacterial infection). The seeds were authenticated and the voucher specimen No. 0355 has been deposited in the Botanical and Environmental Science Department, Guru Nanak Dev University, Amritsar (PB). The seeds were cleaned, washed, dried at room temperature for two days and powdered. The sample was kept in air tightened light-protected containers. Further, extraction process and pharmacological evaluation was carried out during March, 2008 to Jan, 2009 period.

Drugs and Chemicals
DPPH (1,1-diphenyl-2-picrylhydrazyl) was obtained from Sigma Chemical Co. Ranitidine was obtained as free sample from Jackson Laboratories, Amritsar. Pentobarbitone (Neon pharmaceuticals), methanol, ethyl acetate, hexane and sodium hydroxide were of analytical grade and purchased from SD fine chemicals, Merk, Qualigen and Loba chemicals.

Preparation of Extract
Powdered seeds were defatted with hexane. Then extraction was carried out with different solvents of increasing polarity such as chloroform, ethyl acetate and methanol by maceration process for 16 h at room temperature. The solvent was evaporated to dryness under reduced pressure and obtained crude extracts were used for further investigation.

Preliminary Phytochemical Screening
The preliminary phytochemical screening of the extracts was carried out to know the different constituents present as per the standard procedures. The extracts were tested for alkaloids, sterols, triterpenes, saponins, flavonoids, tannins, carbohydrates, protein and amino acids (Harborne, 1973).

Experimental Animals
Wistar albino rats (160-180 g) of either sex were purchased from Agricultural University, Ludhiana (PB). Animals were maintained under standard environmental condition like temperature (24.0±1.0°C), relative humidity: 55-65% and 12 h light/dark cycle and had free access to feed and water ad libitum. The animals were acclimatized to laboratory condition for 1 week prior to experimentation. All animal experiments were carried out in accordance with the guidelines of CPCSEA and study was approved by the IAEC (Institutional Animal Ethical Committee) with registration No. 874/ac/05/CPCSEA).

Quantitative Scavenging Activity of DPPH Radical
Cucumis sativum L. seed extracts (0.5 mL) of different concentration (500-700 μg mL^-1) were added to 1.5 mL of freshly prepared methanolic solution of DPPH (0.05 mM). The change in absorbance at 517 nm was measured 30 min later by a spectrophotometer (Shimadzu UV-1700 Pharma Spec). A blank was used to remove the influence of the colour of the samples. A methanolic solution of DPPH was used as negative control. Ascorbic acid was used as a reference drug. Percentage inhibition was calculated by using the following equation:

where, A₀ and As are the values for the absorbance of the negative control and the absorbance of the sample respectively. Tests were carried out in triplicate.

Antiulcer Activity
Experimental Design for Pyloric Ligation Induced Gastric Ulcer
Animals were divided into 6 groups, each comprising of 6 rats.

Methanolic seed extract (150 and 300 mg kg^-1) was administered for a period of 8 days. On 8th day normal saline, ranitidine and MECL were administered 1 h prior to pyloric ligation. Animals were anaesthetized using pentobarbitone (35 mg kg^-1, i.p.) and the abdomen was opened and pylorus was ligated without causing any damage to its blood vessels. The stomach was replaced carefully and the abdominal wall was closed with interrupted sutures. (Shay et al., 1945; Muralidharan and Srikanth, 2009). After 4 h of ligation, the animals were sacrificed by cervical dislocation. The abdomen was opened and a ligature was placed around the cardiac sphincter. The stomach was removed (Khayum et al., 2009). Gastric volume, free and total acid content of gastric juices were determined. Mean ulcer score for each animal was expressed as ulcerative index and the percentage ulcer protection was also calculated (Bose et al., 2003).

Experimental Design for Water Immersion Stress Induced Gastric Ulcer
Animals were divided into 5 groups, each comprising of 6 rats.

Gastric ulcer was induced by water immersion stress as described by Paul et al. (2004). Briefly, they were immersed vertically up to the level of xiphoid in plastic containers containing water maintained at 23°C for 4 h. Animals were fasted for 24 h prior to the experiment. All the drugs were administered by oral route. After 4 h animals were removed, they were then sacrificed and stomach was opened along the greatest curvature, washed with normal saline (0.9% w/v NaCl). Then ulcerative index and percentage ulcer protection were calculated.

Estimation of Gastric Volume and Free and Total Activity Changes in PL Model
Gastric Volume
Four hours after ligation, stomachs were dissected out and contents were collected into measuring cylinder to measure the volume of gastric content.

Free and Total Acidity
The gastric contents were centrifuged and subjected to titration for estimation of free and total acidity. One milliliter of the supernatant liquid was pipette out and diluted to 10 mL with distilled water. The solution was titrated against 0.01 N NaOH using Topfer’s reagent as indicator, to the endpoint when the solution turned to orange colour. The volume of NaOH needed was taken as corresponding to the free acidity (Rajkapoor et al., 2002). Titration was further continued by adding 1% solution of phenolphthalein till the solution gained pink colour. The volume of NaOH required was noted and was taken as corresponding to the total acidity. The sum of the two titrations was total acidity (Rajkapoor et al., 2002). Acidity was expressed as:

Estimation of Gastric Ulcerative Index Changes in PL and WIS Model
Ulcerative index was measured by method of Takagi et al. (1969). Briefly, the stomach was opened along the greater curvature. The stomach was washed with running tap water. Then it was placed on a flat wooden plate to count the ulcerative area.

The ulcer index was determined by using the formula:

where, X = Total mucosal area/Total ulcerated area

Percentage ulcer protection was calculated using the formula:

Where:

Statistical Analysis
All the biochemical results were expressed as mean±standard error of means (SEM). Data were analysed by Tukey’s multiple range tests using Sigma Stat Version-3.5 software. A probability value of p<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

Preliminary phytochemicals screening of different extracts of Cucumis sativum L. seeds showed the presence of sterols, triterpenes, carbohydrates, tannins, protein and amino acid but methanolic extract showed the presence of proteins, amino acids, triterpenoids, carbohydrates, phytosterols and tannins (Table 1). Further, free radical scavenging activity of all the extracts were evaluated by DPPH method. All the extract showed concentration dependent antioxidant activity but maximum activity was reported in the methanolic extract of Cucumis sativum L. seeds (MECS). DPPH scavenging effect of MECS extract was 76.2% at a concentration of 700 μg mL^-1 which was comparable to the scavenging effect of ascorbic acid (Fig. 1). Hence, MECS was further used to evaluate its antiuncerogenic potential in pyloric ligated and water immersion stress induced peptic ulcer models.

Table 1:	Phytochemical screening of Cucumis sativum L. seed extracts
+: Presence of compounds, - : Absence of compounds

Fig. 1:	Percentage DPPH radical scavenging activity

Table 2:	Effect of MECS on gastric secretion, free acidity and total acidity in pylorus ligation induced gastric ulcer in rats
Values are Mean±SEM, n = 6 animals in each group; ap<0.05 as compared with sham control group. bp<0.05 compared with disease control groups, cp<0.05 compared with ranitidine treated group

Table 3:	Effect of MECS on ulcerative index and percentage inhibition in PL and WIS induced gastric ulcer in rats
Values are Mean±SEM, n = 6 animals in each group; ap<0.05 compared with sham control group, bp<0.05 compared with PL and WIS groups respective coloums, cp<0.05 compared with ranitidine treated group

Various mechanisms are thought to be involved in the ulcer production in different experimental models (Parmar and Desai, 1993; Bose et al., 2003). Hence, it is not possible to propose a single mechanism for antiulcer effect of a particular drug. Digestive effect of the accumulated gastric juice is believed to be responsible for producing ulcers in the pyloric ligated rats. In addition to gastric acid secretion, reflex or neurogenic effect has also been suggested to play an important role in the formation of gastric ulcer in this model (Goswani et al., 1997; Bose et al., 2003). Ulcer index parameter was used for the evaluation of anti-ulcer activity since ulcer formation is directly related to factors such as gastric volume, free and total acidity (Khayum et al., 2009). In pyloric ligated rats, there was an increase in the gastric volume, free and total acidity and ulcerative index as compared to the sham control group. MECS showed reduction in gastric secretion, free and total acidity and ulcerative index, but only highest dose i.e., 300 mg kg^-1 showed significant reduction in the above parameters which was comparable to the standard drug ranitidine (Table 2, 3).

Water immersion stress induced ulcer model provides both emotional stress as well as physiological stress (Malairajan et al., 2008). Ulcers are formed as a result of disturbance of gastric secretion, alteration in microcirculation of gastric mucosa and abnormal gastric motility (Kitagawa et al., 1979; Bose et al., 2003). It affect gastrointestinal defence and causes increase accumulation of acid due to influx of H⁺ into the lumen of the stomach by parietal cell plasma membrane bound H⁺ K⁺-ATPase leading to auto digestion of the gastric mucosa and generation of free radicals which further increase the ulcers in the body (Das et al., 2008; Goel and Bhattacharya, 1991; Ivy, 1988). There was an increased ulcer formation in WIS model which was indicated by high ulcerative index as compared to the sham control group. Whereas, MECS showed reduction in ulcerative index, but only highest dose i.e., 300 mg kg^-1 showed significant reduction in the above parameter which was comparable to the standard drug ranitidine (Table 3).

It is well known that free radicals are involved in the progression of ulcers and in PL and WIS models increase in the oxidative and decrease in the antioxidative biomarkers have been reported by Halliwell (1981) and Singh et al. (2008). Moreover, triterpenoids isolated from various species of cucurbita family has been reported to possess anti-inflammatory and anti-cancer activities (Mohammad, 2009). Wound healing property of Cucumis sativus L. fruits has also been studied (William et al., 1991). Isolation of gibberellins was carried out from seeds of Cucumis sativus L. (Delbert et al., 1972). Literature also revealed the antioxidant property of various cucurbitacins isolated from cucurbita seeds. Hence, as our results indicated maximum in vitro free radical scavenging activity along with ameliorative effect on various ulcerative parameters of MECS extract so, this antioxidant potential may be responsible for its anti-ulcerogenic activity.

CONCLUSION

In the present study it may be concluded that the methanolic extract of the Cucumis sativum L. seeds possessed anti-ulcer effect due to its anti-oxidative potential and can be used as a future natural anti-ulcerogenic agent.

ACKNOWLEDGMENT

Thanks to Professor A.C. Rana and all faculty members of Rayat Institute of Pharmacy for their encouragement and support. We are also grateful to Rayat and Bahra Educational and Research Trust for their unconditional help to carryout this project.