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Effects of water extract of Usnea longissima on antioxidant enzyme activity and mucosal damage caused by indomethacin in rats.

Abstract

In this study, the antiulcerogenic effect of a water extract obtained from the lichen species Usnea longissima was investigated using indomethacin-induced ulcer models in rats. Experimental groups consisted of six rats. Antiulcerogenic activities of 50, 100 and 200 mg/kg body wt. doses of the water extract were determined by comparing the negative (treated only with indomethacin) and positive (ranitidine) control groups. Although all doses of the water extract of U. longissima showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 100 mg/kg body wt. doses (79.8%). The water extract of U. longissima showed moderate antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. In addition, the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST)] were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of antioxidant enzymes on antiulcerogenic activity. SOD and GST enzymes activities in indomethacin-administrated tissues were reduced significantly by indomethacin in comparison to control groups. These enzymes were activated, however, by the water extracts of U. longissima. In contrast to SOD and GST activities, CAT activity was increased by indomethacin and reduced by all doses of U. longissima and ranitidine. The present results indicate that the water extract of U. longissima has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Lichens; Usnea longissima; Antiulcer; Antioxidant; Antioxidant enzymes

Introduction

Lichens are complex plants, living in symbiotic relationships with fungi and algae, and the pertinent partners are defined as mycobiont and phycobiont, respectively. The fungus forms a thallus or lichenized stroma that may contain characteristic secondary metabolites in all lichens (Ahmadjian, 1993). Secondary metabolites isolated from lichens have been reported to show a wide variety of biological activities, including antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects (Huneck, 1999).

Usnea longissima Ach., has a hanging thallus and lives on trees (epiphytic). U. longissima is one of the species most sensitive to air pollution. In some European countries, its health is accepted as an indicator of air pollution. The presence of U. longissima indicates clear air, while its absence indicates polluted air. It has been used to strengthen hair and in the production of hygienic products for women (Brodo et al., 1999). In the folk medicine of different countries of the world, U. longissima has also been used widely as an expectorant, for wound dressing and to stanch nose bleeding, as well as in the treatment of ulcers. It has also been used in the treatment of injuries to the legs and loins, bone fractures, and skin eruptions (Lal and Upreti, 1995; Blackwell, 1990).

Non-steroidal anti-inflammatory drugs (NSAIDs) are used widely in the treatment of pain, fever and inflammation. However, these drugs have some side-effects, especially in the gastrointestinal tract. Reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulcers (Das et al., 1997). The role of ROS in the development of pathogenesis in acute experimental gastric lesions induced by stress, ethanol and NSAIDs is well-known (Das et al., 1997; Isenberg et al., 1995). Tissue and gastric damage may also be attributed to the presence of superoxide anions ([O*.sub.2.sup.-]). Thus, much attention has been focused recently on oxygen-derived free radicals. Recent developments in biomedical science have also shown that ROS contents such as superoxide, hydroxyl radicals (OH*) and singlet oxygen have some degenerative effects (Ames et al., 1993).

ROS damage membrane proteins by causing lipid peroxidation in membranes by attaching to unsaturated fatty acids (Ames et al., 1993). The damage to membrane proteins decreases the membrane permeability, activities of enzymes and receptors, and activation of cells. When free radicals attack DNA, cancer-causing mutations may occur. Therefore, antioxidant defence systems including antioxidant enzymes, foods and drugs are important in the prevention of many diseases (Yen and Hsieh, 1998; Pietta et al., 1998). Oxygen-handling cells have antioxidant enzymes that are able to protect them against the toxic effects of oxygen-derived free radicals. Antioxidants may also play an important role in the prevention of gastric damage. In recent years, the antioxidant properties of numerous crude extracts, primary and secondary metabolites of many plants have been widely reported (Pietta et al., 1998; Hidalgo et al., 1994).

Nevertheless, the antiulcerogenic and antioxidant properties of lichens, which are used widely in traditional medicine, are poorly known. Properties of the crude extracts of some lichen species have been reported recently (Hidalgo et al., 1994; Gulcin et al., 2002; Suleyman et al., 2003). No reports have appeared, however, on the antiulcer and antioxidant activities of U. longissima Ach., used in the treatment of ulcers in folk medicine. In view of this fact, in the present study, we aimed to determine the in vivo antiulcerogenic activity on indomethacin-induced gastric ulcer models in rats and the in vitro antioxidant activity of water extract of U. longissima (WEUL), and to expose the possible relationship between antiulcer and antioxidant activities. In addition, the activities of three antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST)] in rat stomach tissues were evaluated to determine the effects of these enzymes on the ulceration process.

Material and methods

Plant material

U. longissima Ach. (KKEF-374) was collected from the Giresun region of Turkey in 2000. The lichen sample was identified by Dr. Ali Aslan (Aslan, 2000) and a voucher specimen has been deposited in the herbarium of the Kazim Karabekir Education Faculty, Ataturk University, Erzurum (Turkey).

Extraction of plant material

Hundred grams of lichen sample were extracted with distilled water (60-80 [degrees]C, 200 ml X 4) for 2 days in a water bath with a shaking attachment. The extract was lyophilized under 5 [micro]m Hg pressure and stored at -18 [degrees]C. The experiments were carried out using an appropriate amount of lyophilized material.

Animals

A total of 36 male, albino Wistar rats (weighing 190-200 g each), were used for the experiments. The animals, which were provided by the Experimental Animal Laboratory of Ataturk University, Medicinal Faculty, Department of Pharmacology, were grouped before the experiments and kept under standard conditions.

Indomethacin-induced gastric damage

In this series of experiments, the effects of WEUL and ranitidine on indomethacin-induced gastric damage were determined (Guidobono et al., 1997). The protective effect of WEUL was compared with ranitidine, [H.sub.2] receptor blocker. WEUL (50, 100 and 200 mg/kg body wt.) and ranitidine (150 mg/kg body wt.) were administered orally to the assigned groups of rats. The 25 mg/kg body wt. dose of indomethacin was administered to all animals orally, 5 min after WEUL administration. After 6 h, animals were sacrificed using high dose anaesthesia (thiopental sodium, 50 mg/kg body wt.). The stomachs were removed and the gastric damage in it was evaluated macroscopically. The protective effects of WEUL were determined by comparison with the results obtained for the ranitidine and control groups.

The macroscopic analyses of stomach tissues

The rat stomachs, subjected to the macroscopic evaluation to designate gastric lesions, were opened along the greater curvature and washed with serum physiological water. The ulcer rates were then determined macroscopically. The area size was determined using millimetric paper and a magnifier.

Biochemical investigation of stomach tissues

Following the macroscopic analyses, rat stomachs were kept at -20 [degrees]C for the biochemical investigations for 3 days and then the SOD, CAT and GST enzyme activities in stomach tissues were determined. To prepare the tissue homogenates, stomach tissues were ground with liquid nitrogen in a mortar. In all, 0.5 g was weighed for each group and treated with 4.5 ml buffer. This mixture was homogenized on ice using an ultraturraks homogenizer for 15 min. Homogenates were filtered and centrifuged using a refrigerator centrifuge at 4 [degrees]C. Then, these supernatants were used in the determination of the enzymatic activities and total protein amounts. All assays were carried out at room temperature.

Enzymatic activity assays

SOD activity: It was measured according to Sun et al. (1988). SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacts with nitro blue tetrazolium to form formazan dye. SOD activity was then measured at 560 nm by the degree of inhibition of this reaction, and is expressed as units per milligram of protein (EU/mg protein).

CAT activity: Decomposition of [H.sub.2][O.sub.2] in presence of CAT was followed at 240 nm (Aebi, 1984). One unit (U) of CAT was defined as the amount of enzyme required to decompose 1 nmol of [H.sub.2][O.sub.2] per min, at 25 [degrees]C and pH 7.8. Results are expressed as millimole per minute per milligram protein (mmol/min/mg protein).

Glutathione S-transferase (GST) activity: Total GST activity was determined as described by Habig and Jakoby (1981). Briefly, the enzyme activity was assayed spectrophotometrically at 340 nm in a 4 ml cuvette containing 0.1 M PBS (pH 6.5), 30 mM glutathione, 30 mM 1-chloro-2,6-dinitrobenzene and tissue homogenate. Enzyme activity was expressed as nanomole per minute per milligram protein (nmol/min/mg protein).

Determination of protein concentration

In all tissues studied, protein concentration was determined according to the method of Bradford (1976) using bovine serum albumin as a standard.

Total antioxidant activity assays

Antioxidant activity of the lyophilized lichen extract was determined using the thiocyanate method (Mitsuda et al., 1996). Briefly, the sample (1 mg) in 1 ml distilled water was mixed with 5 ml linoleic acid emulsion (0.02 M, pH 7.0) and 5 ml phosphate buffer (0.2 M, pH 7.0). Linoleic acid emulsion was prepared by mixing 0.5608 g of linoleic acid with 0.5608 g of Tween 20 as emulsifier, and 100 ml phosphate buffer, and the mixture was then homogenized. The reaction mixture was incubated at 37 [degrees]C. Aliquots of 0.1 ml were taken at different intervals during incubation. The degree of oxidation was measured according to the thiocyanate method by sequentially adding 4.7 ml ethanol (75%), 0.1 ml ammonium thiocyanate (30%), 0.1 ml sample solution, and 0.1 ml ferrous chloride (0.02 M, in 3.5% HCl). The mixture stood for 3 min and the peroxide value was then determined by reading the absorbance at 500 nm using a UV-visible spectrophotometer (Thermo-Spectronic-HE[lambda]IOS [beta]). A control was performed with linoleic acid but without the extract. Trolox and ascorbic acid solutions, prepared under the same conditions described above, were used as positive control. Inhibition % was calculated using the following equation:

([1 - absorbance of sample at 500 nm]/[absorbance of control at 500 nm]) 100.

Determination of total phenolic content

The amount of total phenolic compounds in the lyophilized lichen extract was determined with the Folin-Coicalteu's reagent according to a published method (Slinkard and Singleton, 1977) using gallic acid as standard. Samples (500 [micro]l) (three replicates) were introduced to test cuvettes, and then 2.5 ml Folin-Ciocalteu's reagent (diluted 1:10, v/v) and 2 ml [Na.sub.2]C[O.sub.3] (7.5%) were added. The absorbance of all samples was measured at 765 nm using a UV-visible spectrophotometer after incubation at 30[degrees]C for 90 min. Results were expressed as milligrams of gallic acid equivalents (GAE) per milliliter lyophylisates.

Reducing power (RP) assay

RP was determined according to the method previously reported (Yen and Chen, 1997). Lyophilized lichen extract (5 mg) was solved in 5 ml distilled water and then 0.5 ml this solutions was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [[K.sub.3]Fe(CN)[.sub.6]] (1%). This mixture was incubated at 50 [degrees]C for 30 min. Afterwards, 2.5 ml trichloroacetic acid (10%) was added into the mixture, which was centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of the supernatant fractions were mixed with 2.5 ml distilled water and 0.5 ml Fe[Cl.sub.3] (0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased RP.

Statistical analyses

The results are shown as mean[+ or -]S.E.M. All the results were analyzed using one-way analysis of variance. Differences between groups were considered significant at p<0.05.

Results

Effect of the water extract of U. longissima on indomethacin-induced gastric damages

In the present study, the antiulcerogenic activity of 50, 100 and 200 mg/kg body wt. doses of water extract of U. longissima (WEUL) on indomethacin-induced ulcers in rats was studied, and compared to the antiulcerogenic effect of ranitidine (150 mg/kg body wt. dose) as positive control. The results of macroscopic analyses are presented in Table 1. As shown in Table 1, ulcer areas decreased with 50, 100 and 200 mg/kg body wt. doses of WEUL as compared to the indomethacin-induced tissue model. The effective dose in protection from gastric damages was a 100 mg/kg body wt. dose of WEUL. Ranitidine and the 50, 100 and 200 mg/kg body wt. doses of WEUL reduced the ulcer areas at rates of 86.4%, 60.8%, 78.9% and 74%, respectively, in comparison to control groups.

Comparison of antioxidant enzymes activities in rat stomach tissues

In order to explore the effects of antioxidant enzymes (SOD, CAT and GST) on the ulceration process in all rat stomach tissues, the activities of these enzymes were evaluated according to appropriate methods reported previously. The results obtained for 50, 100 and 200 mg/kg body wt. doses of WEUL, ranitidine, indomethacin and healthy rat groups are shown in Table 2. These results show that, although indomethacin reduced the activity of SOD enzyme, its inhibition effect (p<0.05) was increased significantly by ranitidine and 50, 100 and 200 mg/kg body wt. doses of WEUL, as compared to healthy rat groups. Among the doses of WEUL, the highest increasing effect on the SOD activity was shown with 100 mg/kg body wt. doses. Likewise, GST activity, decreased by indomethacin, was increased by all doses of WEUL. The increasing effect of the 50 mg/kg body wt. dose of WEUL on GST activity was, however, not statistically significant (p>0.05). Interestingly, ranitidine inhibited the activity of GST strongly. In contrast, similar results were not obtained for the activity of CAT enzyme. CAT enzyme was activated significantly by indomethacin (p<0.05). The increased activity of CAT was reduced by ranitidine and all doses of WEUL.

Antioxidant potential of the water extract of U. longissima

Reactive-oxygen species (ROS) had negative effects on the gastric ulcer. Because of this, the antioxidant sources may also have antiulcerogenic activity. Therefore, in this study, the antioxidant activity and RP of WEUL were also determined and compared to the positive controls trolox and ascorbic acid. The results are shown in Table 3. When compared to control groups, WEUL and trolox reduced peroxide production by 47.08% and 99.52%, respectively (p<0.05). On the other hand, the inhibition effect on peroxide formation with linoleic acid emulsion (22.59%) of ascorbic acid is not statistically important (p>0.05). In general, the phenolic compound(s) are responsible for the antioxidant activity of crude extracts obtained from plant samples. Therefore, total phenolic content was also determined and expressed as milligrams of GAE per milliliter lyophylisates (Table 3). The total phenolic content of WEUL was found to be 18.3[+ or -]0.010 mg GAE/g lyophylisate.

Discussion and conclusion

Although it has been reported that U. longissima is used in treating ulcer in folk medicine, no experimental evidence has been reported. Thus, in the present study, the antiulcerogenic effects of three doses (50, 100 and 200 mg/kg body wt.) of WEUL and ranitidine (150 mg/kg body wt. dose) on indomethacin-induced gastric damage in rats were studied. The 50, 100 and 200 mg/kg body wt. doses of WEUL showed antiulcerogenic activity against gastric damage (Table 1). Among the doses of WEUL administered, the most effective was the 100 mg/kg body wt. dose. Although the antiulcerogenic activity of this dose is lower than that of ranitidine, this difference is not statistically significant (p>0.05).

One of the main factors of indomethacin-induced gastric damage is the inhibition of prostaglandin synthesis (Tegeder et al., 2000; Whittle, 1981). ROS also has an important role in the mucosal damages caused by ethanol, indomethacin and other agents (Elliot and Wallace, 1998). In recent studies, it has been shown that pro-oxidants expeditiously block the antioxidant systems of mucosal cells, which cause ROS formation. As a consequence of this process, oxidative damages occur (Takeuchi et al., 1991; Yoshikawa et al., 1997). Organisms do, however, have enzymatic and non-enzymatic defense mechanisms against the toxicity and tissue damage of ROS (Smith and Kvietys, 1988). SOD, GST and CAT are some of the antioxidant enzymes and part of the enzymatic defense mechanisms. It has been reported that SOD activity in rat stomach tissues is decreased by NSAIDs (Djahanguiri, 1969; El-Missiry et al., 2001; Alarcon et al., 2002; Basiviredy et al., 2003). Our results are in agreement with these findings. In our study, SOD activity was inhibited (35.8%) by indomethacin, and all doses of WEUL and ranitidine increased SOD activity near to control group levels (p<0.05) (Table 2). These results indicate that SOD plays an important role in eliminating gastric damage by partially preventing oxidative damage.

Likewise, the results for GST activity assays are similar to those for SOD. Although GST activity was inhibited by indomethacin, all doses of WEUL increase the activity of this enzyme (Table 2). However, the inhibition effect of indomethacin on GST activity was low in comparison with SOD activity. Thus, all doses of WEUL may be seen as chemopreventive agents which activate the GST enzyme. Our results agree with previous studies. It has been shown that many agents isolated from numerous plant species control lipid peroxidation in tissues by reducing the levels of lipid peroxides (Van Lieshout et al., 1996), and many of these compounds also have catalyzing effects on glutathione (GSH) (Aruna and Sivaramakrishnan, 1990). GSH and GSH-bound enzymes in tissues, especially, glutathione peroxidase (G[P.sub.x]) and GST, have been proposed as potential chemopreventive agents for their antioxidant and detoxification properties (Aruna and Sivaramakrishnan, 1990; Hayes and Pulford, 1995).

Indomethacin may also produce superoxide via reacting with [H.sub.2][O.sub.2] by peroxidases present in tissue. This superoxide might damage the membranes and cause the ulcer by increasing lipid peroxidation (Miura et al., 2002). Reports support that lipid peroxidation has an important role in the formation of gastric damage apart from the inhibition of COX enzymes. Thus, in the treatment, the concentration of [H.sub.2][O.sub.2] in tissue must be reduced quickly to inhibit the lipid peroxidation to be caused by indomethacin. [H.sub.2][O.sub.2] is a substrat of CAT enzyme. The increase in [H.sub.2][O.sub.2] concentration will activate the CAT enzyme in damaged gastric tissues. Our results are in agreement with these findings. Table 2 shows that the activity of CAT increased in indomethacin-administrated tissues. CAT activity was decreased by all doses of WEUL (p<0.05) and ranitidine, which can be attributed to a decrease in [H.sub.2][O.sub.2] concentration. It can be concluded that the effects of indomethacin on [H.sub.2][O.sub.2] concentration were eliminated by all doses of WEUL and ranitidine in the prevention of gastric damage.

Some researchers have shown recently that some antioxidant compounds increased the activities of some antioxidant enzymes (El-Missiry et al., 2001; Kahraman et al., 2003). As shown in Table 2, all doses of WEUL activated the SOD and GST enzymes. In living tissues, the inhibition and scarcity of antioxidants cause ROS accumulation. Therefore, antioxidants might help the elimination of the negative effects on the gastric damage of ROS. Antioxidants have various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging (Ames et al., 1993). The present results indicated that WEUL has also a moderate antioxidant activity (see Table 3) and its antioxidant activity might play an important role in prevention of gastric damage in rats. Antioxidant activity may be due to its phenolic and/or non-phenolic contents. Recently, antioxidant properties of many phenolic compounds have been reported (El-Missiry et al., 2001; Kahraman et al., 2003).

In conclusion, WEUL has a gastroprotective effect on gastric lesions induced by indomethacin. Further, the present results also indicate that the antiulcerogenic effect could be related to its antioxidant potantial. The component(s) responsible for the antiulcerogenic and antioxidant activities were not, however, determined in the present study. Our future studies are therefore focused on the fractionation and isolation of the crude extracts of the lichen samples containing the active components responsible for the antiulcerogenic and antioxidant activities.

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M. Halici (a), F. Odabasoglu (a,*), H. Suleyman (b), A. Cakir (c), A. Aslan (d), Y. Bayir (a)

(a) Department of Biochemistry, Faculty of Pharmacy (Eczacilik Fakultesi), 25240-Campus, Ataturk University, Erzurum, Turkey

(b) Department of Pharmacology, Faculty of Medicine, Ataturk University, Turkey

(c) Department of Chemistry, Kazim Karabekir Education Faculty, Ataturk University, Turkey

(d) Department of Biology, Kazim Karabekir Education Faculty, Ataturk University, Turkey

Received 23 February 2004; accepted 6 June 2004

*Corresponding author. Tel.: +90 442 2311 590; fax: +90 442 2360962.

E-mail address: fodabasoglu@yahoo.com (F. Odabasoglu).
Table 1. Effects of 50, 100 and 200 mg/kg body wt. doses of lyophylized
water extract of U. longissima (WEUL) on indomethacin-induced gastric
lesions

 Dose (mg/kg Ulcer area
Treatments body wt.) N ([mm.sup.2]) % inhibition P

U. longissima 50 6 17.33 [+ or -] 3.39 60.8 <0.05
 100 6 9.33 [+ or -] 2.16 78.9 <0.05
 200 6 11.50 [+ or -] 1.87 74.0 <0.05
Ranitidine 150 6 6.00 [+ or -] 2.10 86.4 <0.05
Control - 6 44.17 [+ or -] 2.86 -

Control: The average means ([+ or -]S.E.M.) of indomethacin-induced
ulcer areas in rat stomachs.
Ranitidine: The average means ([+ or -]S.E.M.) of raniditine-induced
ulcer areas in rat stomachs.
N: Number of rats.

Table 2. Enzyme activities in the stomachs of rats administered
ranitidin, indomethacin and the water extract of U. longissima (WEUL)

 Dose (mg/kg SOD activity
Treatment body wt.) N EU/mg protein

U. longissima 50 6 34.1 [+ or -] 0.0*
 100 6 44.7 [+ or -] 2.6*
 200 6 39.5 [+ or -] 1.8*
Ranitidine 150 6 51.7 [+ or -] 0.8
Indomethacin 25 6 32.1 [+ or -] 0.7*
Control - 6 50.0 [+ or -] 0.3

 CAT activity GST activity
 mmol/min/ nmol/min/
Treatment mgprotein mgprotein

U. longissima 54.6 [+ or -] 1.2* 28.0 [+ or -] 0.0
 51.4 [+ or -] 0.1* 31.7 [+ or -] 1.1*
 53.9 [+ or -] 0.9* 27.0 [+ or -] 0.5*
Ranitidine 57.2 [+ or -] 0.3* 17.4 [+ or -] 0.8*
Indomethacin 60.9 [+ or -] 1.9* 25.8 [+ or -] 1.3*
Control 34.7 [+ or -] 0.1 29.4 [+ or -] 0.3

The measurements were calculated from three replicates.
*Significant at p<0.05.

Table 3. Antioxidant activity, reducing power and total phenolic content
of WEUL

 Total antioxidant activity
Samples Means of absorbance (48 h, 500 nm) % Inhibition

U. longissima 0.671 [+ or -] 0.027 47.08*
Trolox 0.006 [+ or -] 0.001 99.52*
Ascorbic acid 0.990 [+ or -] 0.025 22.59
Control 1.270 [+ or -] 0.012 -

 Reducing power Total phenolic content
Samples Means of absorbance (700 nm) (mg GAE/g lyophylisate)

U. longissima 0.100 [+ or -] 0.010 18.3 [+ or -] 0.010
Trolox - -
Ascorbic acid - -
Control - -

The measurements were calculated from three replicates.
*Significant at p<0.05.
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Author:Halici, M.; Odabasoglu, F.; Suleyman, H.; Cakir, A.; Aslan, A.; Bayir, Y.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2005
Words:4884
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