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Antioxidant and antiulcerogenic activities of Opuntia ficus indica f. inermis root extract in rats.

ABSTRACT

Opuntia ficus indica f. inermis methanolic root extract (ORE) was investigated for phenolic and flavonoids contents, in vitro evaluated for DPPH radical scavenging activity, reducing power and in vivo tested for its gastro-protective ability against 80% ethanol induced ulcer in rats. Phytochemical test of ORE were positive for phenolic and flavonoid contents. DPPH radical scavenging activity and reducing power of ORE showed an [EC.sub.50] of 118.65 [+ or -] 2.51 [micro]g/ml and 300 [micro]g/ml respectively. In vivo the pre-treatment of rats with ranitidine (50 mg/kg) and 200, 400, and 800 mg/kg doses of ORE significantly (p < 0.05) reduced the 80% ethanol induced-ulcer lesion, with a rate of 82.68%, 49.21%, 83.13%, and 92.59% respectively, and prevented the depletion of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH), and inhibited the increase of myeloperoxidase (MPO) and malondialdehyde (MDA) in rat stomach tissues when compared with ethanol group. Also pre-treateatment with ORE marked a dose-dependent attenuation of histopathology changes induced by ethanol. Phenolic and flavonoids wealth, radical scavenging activity, and reducing power, have been implicated for antiulcer property of ORE.

ARTICLE INFO

Keywords:

Gastro-protective Opuntia ficus indica f. inermis Phenolic content Reducing power Ethanol

[C] 2010 Elsevier GmbH. All rights reserved.

Introduction

The ulcer is a lesion of the gastric mucous membrane. Gastric and duodenal ulcers are the two types of ulcer distinguished which affecting a considerable number of people in the world (Ineu et al. 2008). The infection with Helicobacter pylori and the use of non-steroid anti-inflammatory drugs (AINS) are the two essential causes of the development of gastric ulcer (O'Malley 2003). Outwards of these well identified causes certain factors can increase the ulcer development risk, among this factors is ethanol consumption. Excessive ethanol ingestion results in gastritis characterized by mucosal oedema, sub-epithelial haemorrhages, cellular exfoliation, and inflammatory cell infiltration (Guslandi 1987). It has been suggested that reactive oxygen species (ROS), primarily super-oxide anions, hydroxyl radicals, and lipid peroxides, are the harmful species known to cause the gastric ulcer development (Smith et al. 1996). To scavenge ROS, gastric cell have several enzymatic and non-enzymatic antioxidants including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), myeloperoxidase (MPO), and endogenous glutathione (GSH), but excessive generation of ROS enhance lipid peroxidation and depletes these antioxidants enzymes (Cadirci et al. 2007). Although many available synthetic drugs are used to treat gastric ulcers, most of them produce several adverse reactions, when used at long term (Bandyopadhyay et al. 2002). Plant extracts, containing a wide variety of antioxidants such us phenolic and flavonoid compounds, are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of gastric ulcers (Hiruma-Lima et al. 2001). In this line, our laboratory has been interested in the study of the protective potential afforded by natural compounds towards gastric ulcer, and a special interest was given to the cactus plants.

Opuntia sp. belongs to the Cactaceae family and the Centrospermae order. This plant grows wildly in arid and semi-arid regions in Tunisia. The fruit of Opuntia ficus indica f. inermis is a fleshy berry with a number of hard seeds, which are consumed as fresh fruit or used for preparing a traditional jam in our country. The nutraceutical benefits of fruit are believed to their antiulcerogenic activity and antioxidant properties related to ascorbic acid, phenolics, and a mixture of betaxanthin and betacyanin pigments (Tesoriere et al. 2004). Cladodes of this plant named "Nopalitos" are consumed mainly as staple food, but according to Mexican popular medicine, some diseases like diabetes mellitus, blood glucose levels, hyper-lipidemy, obesity and gastrointestinal disorders can be alleviated by eating this vegetable (Corrales-Garcia et al. 2004). Other studies demonstrate that cladodes of this plant are used in folk medicine for their cicatrising (Park and Chun 2001) and anti-ulcerogenic activities (Galati et al. 2001). Hence the various parts of cactus plant fruit and cladodes showed an antioxidant and antiulcero-genic effects. Literature reports few data about cactus root which enhances us to evaluate for the first time its possible antioxidant and anti-ulcerogenic activities.

In this context, the present study aimed firstly to investigate the phenolic and flavonoid content of Opuntia ficus indica f. inremis root extract (ORE) and to evaluate their antioxidant activities using two in vitro tests; DPPH radical scavenging activity and ferric reducing power assay. Secondly to investigate in vivo the effect of (ORE) on ethanol-induced ulcer in male Wistar rats by measuring the lesion index, the stomach mucus weigh, the malondialdehyde (MDA), the reduced glutathione (GSH), some enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), myelo-peroxidase (MPO) and histological studies.

Materials and methods

Chemicals

Ascorbic acid, 2-thiobarbituric acid (TBA), 2,2 diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT) and aluminium chloride ([A1C1.sub.3]) were purchased from Sigma (Sigma, Aldrich). Gallic acid, rutin and Folin-Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). All other chemicals were purchased locally and were of analytical grade.

Plant material and preparation extract

Roots of Opuntia ficus indica f. inermis was collected from municipal areas of Gafsa, state of Tunisia. Opuntia root was cuted into slices, oven-dried at 40 [degrees]C and grounded with moulinex blinder. Grounded root (50g) was extracted with 300 ml of 50% methanol solution for 24 h at room temperature (37 [degrees]C), with magnetically stirring. The extract solution was centrifuged at 4500 x g for 10 min and the supernatant were dried in the oven at 40 [degrees] C. The 50% methanol Opuntia ficus indica f. inermis root extract (ORE) yielded a brown residue weighing 13 g (26%) were stored at -21 [degrees]C until use.

Determination of total phenolics contents

The total phenolic content was determined by adding 0.5 ml of ORE aqueous solution to 2.5 ml, 10% Folin-Ciocalteu reagent (v/v) and 2.0 ml of 7.5% [Na.sub.2][CO.sub.3]. The reaction mixture was incubated at 45 [degrees]C for 40 min, and the absorbance was measured at 765 nm in Analytik jena 40, spectrophotometer (Singleton et al. 1999). Gallic acid was used as a standard phenol, giving the calibration equation: y = 0.9961x + 0.009 ([R.sup.2] = 0.9986), where x is the gallic acid concentration in [micro]g/ml and y is the absorbance reading at 765 nm. All tests were carried out in triplicate and the results were expressed as gallic acid equivalents (mg GAE/g dry weight of the ORE).

Determination of total flavonoid content

The flavonoid content was determined based on the formation of flavonoid-aluminium complex (Djeridane et al. 2006). One milliliter of sample was mixed with 1 ml of 2% aluminium chloride solution. After incubation for 15 min at room temperature, the absorbance of the reaction mixture was measured at 430 nm. A standard curve was first plotted using rutin (0.5-40 [micro]g/ml) as a standard, giving the calibration equation: y = 0.0088x + 0.0054 ([R.sup.2] = 0.9986), where x is the concentration of rutin in [micro]g/ml, and y is the absorbance reading at 430 nm. The amount of flavonoids was expressed as rutin equivalents (mg RE/g dry weight) and all tests were carried out in triplicate.

Evaluation of ORE antioxidant activity

Free radical-scavenging activity on DPPH

The free radical-scavenging activity of ORE was evaluated using the stable radical DPPH, according to the method of Grzegorczyk et al. (2007). Aqueous solution of ORE (1 ml) at various concentrations (50-300 [micro]g/ml) was added to 1 ml of a 0.1 mM methanolic solution of DPPH and allowed to stand for 30 min at 27 [degrees]C. The absorbance of the sample was measured at 517 nm. DPPH radical-scavenging activity (RSA), expressed as percentage was calculated using the following formula:

RAS (%) = [A.sub.DPPH] - ([A.sub.sample] - [A.sub.control])/[A.sub.DPPH] x 100

where [A.sub.DPPH] is the absorbance of DPPH solution without sample extract, [A.sub.sample] is the absorbance of sample extract mixed with DPPH solution and [A.sub.control] is the absorbance of the sample extract tested without DPPH. DPPH radical-scavenging activity (RSA) of ORE was compared with ascorbic acid (AA) and ranitidine (Ran) used as standards.

Ferric reducing power (FRAP) of ORE

The ferric reducing power (FRAP) of ORE was evaluated using the method described by Chu et al. (2000). 2.5 ml of Potassium phosphate buffer (0.1 M, pH 6.6) and 2.5 ml of 1% (w/v) potassium ferricyanide were mixed with 1.0 ml of ORE solution at varying concentrations (5-500 [micro]g/ml). The reaction mixture was incubated at 50 [degrees]C for 20 min, after which 2.5 ml of 10% (w/v) trichloroacetic acid was added. Water (2.5 ml) and 0.5 ml of 0.1% (w/v) [FeCl.sub.3] was then added to 2.5 ml of the reaction mixture, and incubated at 28 [degrees]C for 30 min to facilitate colour development. The absorbance was measured at 700 nm, plotted against ORE concentration ([micro]g/ml) and compared with ascorbic acid (AA) and ranitidine (Ran) used as standards.

Effect of ORE on ethanol-induced ulcers in rats

Animals

Adult male albino rats of Wistar strain (n = 112) weighing 180-200g purchased from SIPHAT (Tunis, Tunisia) were used in this study. Before any experience, all animals were kept for 2 weeks under the same laboratory conditions of temperature (22 [+ or -] 2 [degrees]C), relative humidity (70 [+ or -] 4%), and a 12 h light/dark cycle, and received a nutritionally standard diet (SICO, Sfax, Tunisia) and tap water. Animals were cared for, under the Tunisian code of practice for the Care and Use of Animals for Scientific Purposes.

Acute toxicity of ORE

A total of 32 rats were randomly divided into four groups (n = 8). First group served as normal control. Groups two three and four received ORE dissolved in distilled water respectively at the dose level of (1000, 2000, 3000 mg/kg p.o.). All animals were observed for toxic symptoms and mortality for 72 h.

Antiulcer test

A total of 80 rats were divided into 10 groups of eight animals. Group I was kept as control without any treatment and all other groups were fasted for 24 h and administered 80% ethanol. Group II (Ethanol) received 0.5 ml of 80% ethanol solution alone. Groups III, IV, V and VI were treated with ORE (200 mg/kg, 400 mg/kg, and 800 mg/kg) and ranitidine (50 mg/kg), respectively, 1 h prior to the administration of 0.5 ml of 80% ethanol by gastric instillation. The animals were sacrificed under ether anaesthesia 1 h after ethanol administration. The stomach was removed and opened along the greater curvature, the mucus covering each stomach was gently scraped using a glass slide and weighted. The mucosal lesion area ([mm.sup.2]) was measured by planimetry using a transparent grid. The ulcer index (UI) for each rat was taken as the mean lesion area ([mm.sup.2]). The percentage of inhibition (1%) was calculated as described by (Nguelefack et al. 2008) using the following formula:

1% = [[UI.sub.EtOH] - [UI.sub.Treated]/[UI.sub.EtOH]] x 100

where [UI.sub.EtOH] is the ulcer index of the rats treated with ethanol and [UI.sub.Treated] is the ulcer index of the rats treated with ORE or ranitidine and ethanol.

Ascorbic acid was also tested against ethanol-induced ulcer in rats, groups VII, VIII, IX and X (n = 8 rats), received respectively four doses (50, 200, 400 and 800 mg/kg) of ascorbic acid (AA) and treated with 80% ethanol solution in the same manner of ORE and ranitidine groups. In this test all ascorbic acid (AA) doses used have no effect in ulcer generated with ethanol in rats. In order to compare with ranitidine effect, the dose 50 mg/kg of ascorbic acid (AA) was chosen for the biochemical and histological studies.

Stomach tissues preparation

After the macroscopic analyses, the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), myeloperoxidase (MPO) enzyme activities and the levels of total glutathione (GSH), and malondialdehyde (MDA) in rat stomach tissues were determined. To prepare the tissue homogenates, stomach tissues were grinded with liquid nitrogen in a mortar. The grinded tissues (0.5 g each) were then treated with 4.5 ml of appropriate buffer. The mixtures were homogenized on ice using an ultra-turraks homogeniser for 15 min, then filtered and centrifuged at 4 [degrees]C. The supernatants were used for the determination of the enzymatic activities. All assays were carried out at room temperature in triplicate.

Biochemical estimations

SOD activity was estimated according to the method described by Misra and Fridovich (1972). CAT activity was measured using Aebi's (1984) method. GPx activity was determined according to the method described by Sazuka et al. (1989). MPO was estimated using the method of Bradley (Bradley et al. 1982). Reduced GSH in the gastric mucosa was measured by Ellman's reaction using 5,5'-dithiobis 2-nitrobenzoic acid (Moron et al. 1979). MDA was estimated using the thiobarbituric acid test (Ohkawa et al. 1979). The protein content of the enzymes was determined by Lowry's method (Lowry et al. 1951).

Histological studies

After digital pictures of each stomach were obtained, tissues were immersed for 48 h at 4 [degrees]C in the fixative solution (4% formaldehyde, in phosphate buffer, pH 7.6). Paraffin sections, 5 [micro]m thick, were made and stained with hematoxylin-eosin solutions (H&E). Tissue preparations were observed and micro-photographed under a light [BH.sub.2] Olympus microscope.

Statistical analysis

Statistical analysis was performed using one-way ANOVA followed by Tukey's test and significance of difference between treatments was accepted at p < 0.05. Data are expressed as mean [+ or -] standard deviation of the mean.

Results and discussion

Currently, gastric ulcer therapy faces a major drawback due to the unpredictable side effects of the long-term uses of commercially available drugs. Hence, the search on to find a drug possessing antioxidant and antiulcer properties is still. In this fact the 50% methanolic root extract of Opuntia ficus indica f. inermis (ORE) were phytochemical studied for their total phenolic and flavonoids contents, in vitro evaluated for their antioxidant activity and in vivo investigated for their antiulcerogenic activity.

The Folin-Ciocalteu's assay is a fast and simple method used to rapidly determine phenolic contents in samples (Singleton et al. 1999). The methanolic root extract of Opuntia ficus indica f. inermis (ORE) exhibited high total phenolic content (57.56 [+ or -] 0.51 mg GAE/g of ORE) which appears higher then that the phenolic content of swallow root (Decalepis hamiltonii) extract (34 mg GAE/g) suggested to have an antiulcerogenic effect (Naik et al. 2007). The total flavonoids content in ORE was determined based on the formation of flavonoid-aluminium complex (Djeridane et al. 2006). The amount of total flavonoids assessed in ORE was 23.5 [+ or -] 0.67 mg RE/g of ORE this content appears higher then that the flavonoids content detected in Opuntia ficus indica (L.) Mill fruit juice (652.5 [+ or -] 38 ([micro]g/ml), known for their antioxidant and antiulcerogenic activities (Galati et al. 2003). The higher amount of total phenolic and flavonoids contents detected in ORE may attribute for this extract a potential antioxidant activity.

The radical-scavenging activity of ORE was tested using a methanolic solution of the "stable" free radical, 2,2 diphenyl-1-picrylhydrazyl (DPPH) and compared with ascorbic acid (AA) and ranitidine (Ran) used as standards. From the analysis of Fig. 1 we can conclude that the radical scavenging activity (RSA) of ascorbic acid (AA), ORE and ranitidine (Ran) on DPPH radicals increased as dose dependant manner and respectively reach the maxima of inhibition 90.32 [+ or -] 3.81%, 78.01 [+ or -] 4.11% and 55.27 [+ or -] 3.71 for the same concentration 200 [micro]g/ml. The [EC.sub.50] values calculated from the graph (Fig. 1) shows that the radical scavenging activity (RSA) of ORE ([EC.sub.50] = 118.65 [+ or -] 2.51 [micro]g/ml), appeared significantly (p < 0.05) lower than that of the ascorbic acid ([EC.sub.50] = 3.28 [+ or -] 0.49 [micro]g/ml), higher than that of the ranitidine (Ran) ([EC.sub.50] = 151.79 [+ or -] 3.11). Fig. 2 shows that reducing power of ORE increased with concentration and reaches a maximum at 500 [micro]g/ml which is significantly lower (p < 0.05) than that of ascorbic acid (200 [micro]g/ml). However the reducing power of ORE appears significantly higher (p < 0.05) than that of ranitidine for the same concentration 500 [micro]g/ml. The [EC.sub.50] of ORE and ascorbic acid (AA) providing 0.5 of absorbance calculated from the graph (Fig. 2) was respectively 300 [micro]g/ml and 80 [micro]g/ml. On the other hand the reducing activity of ranitidine (Ran) appears weak and cannot reach the absorbance 0.5 for the same range of concentration (5-500 [micro]g/ml) used. These results suggest that ORE exhibited a reducing activity for ferric iron ([Fe.sup.3+]) as dose dependant manner more than ranitidine (Ran) but it is weak when compared with ascorbic acid (AA). In order to determine the relative importance of ORE phenolic and flavonoids contents in radical scavenging activity (RSA) correlation analysis were made (Fig. 3a and b). A high correlation existed between the total phenolic content (r = 0.982), flavonoids contents (r = 0.977) of ORE and DPPH radical scavenging activity (RSA). Also the ferric reducing power (FRAP) of ORE was tested using [Fe.sup.3+]-ferricyanide complex according to the method described by Chung et al. (2002). A positive linear correlation between ferric reducing power (FRP) phenolic (r = 0.983) and flavonoids (r = 0.981) contents in ORE was observed (Fig. 3c and d). This result demonstrate that ORE exhibited a high DPPH radical-scavenging activity and a ferric reducing power (FRAP), and it seams that this activities is due to phenolic and flavonoids contents, detected, or added to the effect of other unknown compounds.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The acute toxicity studies demonstrate that oral administration of ORE at 1000-3000 g/kg body weight did not cause any toxic effect and no mortality was observed in treated rats.

The gastroprotective effects of ORE on ethanol-induced gastric damage were macro-scopically studied in rats (Table 1 and Fig. 4). The gastric lesions in the stomachs of rats, treated with 80% ethanol increased significantly when compared to the normal group. The pre-treatment of rats with ranitidine (50mg/kg) and 200, 400, 800 mg/kg doses of ORE reduced the ulcer lesion with a rate of 82.68%, 49.21%, 83.13%, and 92.59% respectively when compared with ethanol group (p < 0.05). In addition the reduction of the ulcer lesion, in the groups described above, was accompanied by a significant (p < 0.05) decrease of ulcer index as dose dependant manner when compared with ethanol group. Whereas pretreatment with ascorbic acid (50mg/kg) have no effect on ethanol-induced ulcer lesion and expressed an ulcer index near ethanol group. Stomach from rats pre-treated with moderate and high dose of ORE (400mg/kg, 800mg/kg), showed an absence of ulcer crater and a small cicatrized lesions (Fig. 4C and D circle). The reduction of the ulcer index (UI) was accompanied by a significant (p < 0.05) increase of mucus production which reach a maximum amount of 205.99 [+ or -] 1.74 mg in stomach of rats treated with 800mg/kg of ORE, where as in ascorbic acid (AA) and only ethanol treated rats we monitored 89.77 [+ or -] 1.33 and 92.37 [+ or -] 0.87 mg of mucus respectively, our results are in agreement with those described by Galati et al. (2001). The reducing of ulcer lesion and the increase of mucus production are both related to increase of ORE concentration administered, which in turn provides rats stomach with potent antioxidants compounds.

[FIGURE 4 OMITTED]
Table 1
Effects of ORE and ranitidine on ethanol induced ulcers in rats.

     Treatment and dose     N     UI ([mm.sup.2])

Control (distilled water)   8   0.00 [+ or -] 0.00
EtOH (80%) (0, 5 ml/rat)    8  26.74 [+ or -] 0.33 *
ORE (200 mg/kg) + 80% EtOH  8  13.58 [+ or -] 0.94 [dagger]
ORE (400 mg/kg) + 80% EtOH  8   4.51 [+ or -] 0.79 [dagger]
ORE (800 mg/kg) + 80% EtOH  8   1.98 [+ or -] 0.53 [dagger]
Ran (50 mg/kg) + 80% EtOH   8   4.63 [+ or -] 0.97 [dagger]
AA (50 mg/kg) + 80% EtOH    8  27.39 [+ or -] 0.81 *

    Treatment and dose      (%) I    Mucus weight (mg)

Control (distilled water)   -      128.22 [+ or -] 0.73
EtOH (80%) (0, 5 ml/rat)    -       92.37 [+ or -] 0.87 *
ORE (200 mg/kg) + 80% EtOH  49.21  118.41 [+ or -] 1.24 [dagger]
ORE (400 mg/kg) + 80% EtOH  83.13  167,28 [+ or -] 0.97 [dagger]
ORE (800 mg/kg) + 80% EtOH  92.59  205.99 [+ or -] 1.74 [dagger]
Ran (50 mg/kg) + 80% EtOH   82.68  148.24 [+ or -] 1.02 [dagger]
AA (50 mg/kg) + 80% EtOH    -       89.77 [+ or -] 1.33 *

N = rats number/group = 8, UI = ulcer index, %I = inhibition
percentage, EtOH = ethanol, ORE = Opuntia ficus indica f. inermis root
extract, Ran = ranitidine, AA = ascorbic acid. Values are expressed as
means [+ or -] S.D. of eight animals compared with ANOVA followed by
Tukey's test.
* p < 0.05 were compared with control group.
[dagger] p< 0.05 were compared with ethano) group.


The release of oxygen-derived free radicals (ROS) has drawn attention as a possible pathogenic factor of gastric mucosal injury associated with ethanol consumption (Smith et al. 1996). Superoxide produced by peroxidase in the stomach tissues might damage cell membranes and cause ulcer by increasing MDA level (Cadirci et al. 2007). Preventive antioxidants such as SOD, CAT, GPx, and GSH are the first line of defence against ROS. Therefore, in the present study the activities of SOD, CAT, GPx, MPO antioxidant enzymes and the levels of GSH and MDA in rat stomach tissue were studied to explore the effects of ORE on oxidative damage. As shown in Table 2, the administration of ethanol significantly (p < 0.01) decreased the levels of SOD, CAT, GPx, and GSH antioxidants and increased the MPO activity and MDA in the stomach tissues of rats in comparison to healthy rats. Based on the present results it can be concluded that ethanol caused the gastric lesion by reducing the levels of SOD, CAT, GPx and GSH which were associated with increase of oxidative damage, our result is in agreement with those of Cadirci et al. (2007). In contrast to ethanol, ranitidine and all doses of ORE showed significant increased levels, as dose dependant manner, of gastric SOD, CAT, GPX and GSH and decreased effect on lipid peroxidation MDA. In addition, it has been shown that exposure of gastric mucosa to ethanol induced a significant increase of gastric neutrophil infiltration, depicted by the increase of MPO activity (La Casa et al. 2000). According to our results, ORE inhibited the increase in MPO activity and thus protected gastric mucosa from the deleterious effect of ethanol. Although the great antioxidant activity proved by ascorbic acid in vitro, their effect against the ethanol-induced ulcer in vivo appears weak, which is explained by the diminution of the levels of SOD, CAT, GPX and GSH and the increase of the MDA and MPO contents as the same manner of ethanol-treated group. Due to their antioxidant capacity proved above, their capacity to preserve the integrity of mucosal tissue near normal and their ability to prevent the depletion of antioxidant enzymes it is clear that ORE have a great anti-ulcerogenic activity.
Table 2
Effect of ORE, ranitidine (RAN) and ascorbic acid (AA) on stomach
antioxidants enzymes and the contents of MDA and GSH in ethanol-treated
rats.

        Treatments                SOD                    CAT

Control (distilled water)  3.51 [+ or -] 0.22     7.03 [+ or -] 0.15

EtOH (80%) (0.5 ml/rat)    1.37 [+ or -] 0.21 **  2.73 [+ or -] 0.05 **

ORE (200 mg/kg) + EtOH     2.29 [+ or -] 0.13 *   3.98 [+ or -] 0.28 *

ORE (400 mg/kg) + EtOH     3.48 [+ or -] 0.10 **  6.39 [+ or -] 0.13 **

ORE (800 mg/kg) +EtOH      3.61 [+ or -] 0.19 **  6.92 [+ or -] 0.21 **

Ran(50 mg/kg) + EtOH       3.40 [+ or -] 0.16 *   6.23 [+ or -] 0.19 **

AA(50 mg/kg) + EtOH        1.58 [+ or -] 0.23     3.04 [+ or -] 0.11

     Treatments               GPx                      MPO

Control (distilled    39.57 [+ or -] 0.86      6.32 [+ or -] 0.39
water)

EtOH (80%)            10.42 [+ or -] 0.43 **  27.44 [+ or -] 0.53 **
(0.5 ml/rat)

ORE (200 mg/kg) +     27.82 [+ or -] 0.80 **  12.64 [+ or -] 0.8 **
EtOH

ORE (400 mg/kg) +     36.32 [+ or -] 0.72 **   6.39 [+ or -] 0.72 **
EtOH

ORE (800 mg/kg) +     38.59 [+ or -] 0.65 **   6.33 [+ or -] 0.3 **
EtOH

Ran(50 mg/kg) + EtOH  29.14 [+ or -] 0.49 **   7.01 [+ or -] 0.31 **

AA(50 mg/kg) + EtOH   10.09 [+ or -] 0.66     26.89 [+ or -] 0.71

       Treatments                 GSH                    MDA

Control (distilled water)  2.41 [+ or -] 0.12     2.18 [+ or -] 0.09

EtOH (80%) (0.5 ml/rat)     0.8 [+ or -] 0.07 **  7.38 [+ or -] 0.22 **

ORE (200 mg/kg) + EtOH     1.51 [+ or -] 0.05 **     5 [+ or -] 0.59 *

ORE (400 mg/kg) + EtOH     2.31 [+ or -] 0.02 **  2.35 [+ or -] 0.08 **

ORE (800 mg/kg) +EtOH      2.35 [+ or -] 0.03 **  2.27 [+ or -] 0.36 **

Ran(50 mg/kg) + EtOH       1.85 [+ or -] 0.05 **  3.78 [+ or -] 0.48 **

AA(50 mg/kg) + EtOH        0.69 [+ or -] 0.09     8.22 [+ or -] 0.16

Values are expressed as mean [+ or -] S.D. for eight animals in each
group. Ethanol solution (80%) was administered in order of 0.5 ml/rat.
Ethanol group was compared with control group. ORE, ranitidine (Ran),
and ascorbic acid (AA) treated groups were compared with ethanol group.
SOD, units/mg protein (one unit of the SOD activity is the amount of
enzyme required to give 50% inhibition of epinephrine auto oxidation);
CAT, [micro] mol of [H.sub.2][O.sub.2] consumed/min/mg protein; GPx,
nmol GSH oxidized/min/mg protein; MPO [micro]mol/min/mg protein; GSH,
[micro]g of GSH/mg protein; MDA, nmol of MDA/mg protein.

* p < 0.05.
** p< 0.001.


Histological studies demonstrate that the gastric mucosa of rats treated with 80% ethanol showed morphologic changes such as erosive lesions (Fig. 4A 1 and 2). The surface epithelial cells were disrupted and became more reddish. The interglandular spaces were dilated and became deeper our results are in agreement with those of Galati et al. (2002). However, rats pre-treated with ORE showed a marked dose-dependent attenuation of histopathology changes induced by ethanol. The gastric layer became more regular in the "lamina propria" of gastric mucosa, fibroblasts were visible near the glandular portion (Fig. 4C 4 and 5). The gastric mucosa of rats treated with ranitidine and distilled water (healthy intact group), appeared intact and showed a continuous epithelial surface and a thin layer of mucus in the neck cells (Fig. 4E and F). However the gastric mucosa of rats pre-treated with ascorbic acid (AA) showed a disrupted epithelial cell and deeper interglandular spaces when compared with control group (Fig. 4G 6). This result indicate that ascorbic acid (AA) have no effect on ulcer generated by ethanol in rats. Our study shows that ascorbic acid (AA) has a great antioxidant activity in vitro but its in vivo effect appears weak. On the contrary ranitidine (Ran) has a weak antioxidant effect in vitro and a large anti-ulcerogenic activity in vivo. But ORE has an intermediate antioxidant activity in vitro and a potent anti-ulcerogenic activity in vivo.

In Conclusion this is the first evidence that methanolic root extract from Opuntiaficus indicat f. inermis (ORE), has an antioxidant and a gastro-protective effect on ethanol induced gastric lesions. Due to the richness of ORE in flavonoids and phenolic compounds their moderate antioxidant activity in vitro, and their potent anti-ulcerogenic activity in vivo we suggest that ORE can have an antioxidant/ranitidine-like pathway(s) in their anti-ulcerogenic activity. Further studies are also in progress to evaluate the unknown bioactive(s) compound(s) of ORE and to found his real mechanism of action before suggesting the use of Opuntia ficus indica f. inermis root extract as a new therapeutic drug for gastric disorders.

Acknowledgements

This research was funded by the Tunisian Ministry higher education, Scientific Research and Technology through the Research Unit of Macromolecular Biochemistry and Genetics, Faculty of Sciences of Gafsa.

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* Corresponding author. Tel.: +216 98328439; fax: +216 72 590566.

E-mail address: khemais.benrhouma@fsb.rnu.tn (K.B. Rhouma).

0944-7113/$--see front matter[C] 2010 Elsevier GmbH. All rights reserved.

Hichem Alimi (a), (b), Najla Hfaiedh (a), Zouhour Bouoni (a), Mbarka Hfaiedh (a), Mohsen Sakly (b), Lazhar Zourgui (a), Khemais Ben Rhouma (b), *

(a) Unite de Biochimie Macromoleculaire et Genetique. Faculte des Sciences de Gafsa, Cite Zurroug, 2112 Gafsa, Tunisia

(b) Laboratoire de Physiologie Integree. Faculte des Sciences de Bizerte, 7021 Jarzouna, Tunisia

doi:10.1016/j.phymed.2010.05.001
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Author:Alimi, Hichem; Hfaiedh, Najla; Bouoni, Zouhour; Hfaiedh, Mbarka; Sakly, Mohsen; Zourgui, Lazhar; Rho
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:6TUNI
Date:Dec 1, 2010
Words:5712
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