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Protective effect of Anzer honey against ethanol-induced increased vascular permeability in the rat stomach
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 57 (2005) 173–178
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Protective effect of Anzer honey against ethanol-induced increased vascular permeability in the rat stomach Aslı Dog˘an, Du¨rdane Kolankaya Department of Biology, Faculty of Science, Hacettepe University, 06532 Beytepe Campus, Ankara, Turkey Received 9 December 2004; accepted 5 April 2005
Abstract The purpose of this study was to determine the protective effect of Anzer honey on ethanol-induced increased vascular permeability in rats. Evan’s Blue (EB) dye, administered intracardiacly and extravasation of EB into the stomach, served as an indicator of vascular permeability following exposure to alcohol. Ethanol was given orally to the ethanol group for 90 days, and N-etylmaleimide (NEM) was given subcutaneously to the NEM group, and we observed increased extravasation of EB in the stomach in both groups. For this reason, we used NEM as a positive control for ethanol. Anzer honey, which contains 25.44 mg/g ascorbic acid, was given to the honey+ethanol group orally 30 min before beginning the 90-day ethanol administration. The mean amount of EB that leaked into the stomach of rats in the ethanol group and the NEM group was higher than that of the control group. Furthermore, if compared to the control, EB values in the stomachs were significantly reduced when receiving honey before administration of ethanol in rats. Histopathologically, the incidence and severity of gastric mucosal congestion were significantly reduced in the honey+ethanol group when compared to the ethanol group. These result indicate that Anzer honey is able to protect the stomach of the rat against ethanol-induced increased vascular permeability, which may be correlated with the ascorbic acid content. r 2005 Elsevier GmbH. All rights reserved. Keywords: Anzer honey; Ethanol; Ascorbic acid; Vascular permeability; Rat stomach; Evans blue
Introduction Honey is produced by honey bees from sugars present in the nectar of various plants. Honey is made up mostly of carbohydrates (70–80%), while it also contains preservative substances such as a-tocopherol, ascorbic acid and other phenolics and enzymes such as glucose
Corresponding author. Fax: +90 312 299 2028.
E-mail address: [email protected] (A. Dog˘an). 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.04.004
oxidase, catalase and peroxidase. Many of these substances have antioxidant properties (Gharzouli et al., 2001; Mckibben and Engeseth, 2002). Furthermore, honey has an in vitro antioxidant capacity similar to those of many fruits and vegetables on a fresh weight basis, as measured by the oxygen radical absorbance capacity (ORAC) assay (Gheldof et al., 2002). Honey is considered an antioxidant because of the presence of ascorbic acid, flavonoid and a-tocopherol. Rize-I˙kizdere-Anzer honey contains 6267 mg/g ascorbate and can be used as an alternative source of dietary
ARTICLE IN PRESS 174
A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
antioxidant (Kolankaya et al., 1999). This amount of ascorbic acid contains the recommended daily allowance of vitamin C for a 70 kg man. This is the reason we used Anzer honey in this study. It has been reported that natural honey is able to protect the rat stomach against acute ethanol-induced lesions (Ali et al, 1990; Ali and Al-Swayeh, 1997). Similarly, as an antioxidant drug, sucralfate, has also been shown to afford protection against ethanolinduced gastric lesions. Following the oral administration of ethanol, there was a marked increase in vascular permeability that preceded the development of visible mucosal damage (congestion or hemorrhage). It has been postulated that such an increase is a precursor of ethanol-induced macroscopic mucosal damage (Woods et al., 1988). Although the mechanisms by which luminal ethanol causes gastric mucosal injury are unclear, several factors have been implicated: products of arachidonate metabolism, oxygen-derived radicals, and mast cell secretory products (Kvietys et al., 1990). The present study was undertaken to examine the protective effect of Anzer honey against ethanol-induced increased vascular permeability and gastric lesion in rats. We used Evan’s Blue (EB) to demonstrate increased vascular permeability in the rat stomach. The azo dye EB is widely used to demonstrate pathologically increased microvascular permeability (Saria and Lundberg, 1983). When injected into the circulation, the dye binds within seconds to serum proteins (mainly albumin), forming a dissociable complex. An increase in local capillary permeability to macromolecules, due to inflammation or other types of damage, will therefore be detected by the extravasation and deposition in the interstitial tissue of protein–EB complex (Lange et al., 1994).
Materials and methods
Choosing Anzer honey The Anzer honey was obtained from the RizeI˙kizdere-Anzer region of Turkey. We conducted a preliminary study with five different Anzer honey samples that contain various quantities of ascorbic acid according to spectrophotometric measurement. The different samples of Anzer honey were administered 30 min before ethanol (45%) treatment on five groups of rats. Then 1 ml of EB was injected (intracardiacly) 15 min before killing the animals. As a result of this study, we found that the EB accumulation was the highest for the animals, which were fed with the least ascorbic acid-containing honey. Consistent with this value, as the ascorbic acid quantity in those honey samples increased, EB accumulation in the stomach decreased (Table 1). According to this result, we chose the honey containing the highest quantity of ascorbic acid for the 90-day experiment.
Animal treatment The first group of animals served as a control. The ethanol (45%), at a dose of 2 ml/kg body weight/day, was administrated orally each day for 90 days to the second group of rats. The third group received 0.275 g/ kg of honey 30 min before administration of the same value of ethanol (45%). The last group was administered 0.050 g/kg NEM at the very beginning of the 90-day period. In each group, 1 ml of EB (0.5%) was injected intracardiacly 30 min before killing the animals, and the EB that had accumulated in the gastric contents and in the mucosa was quantified by the method described by Lange et al. (1994). At the end of the treatment, the animals were killed by cervical dislocation. After removing the stomachs, we opened each along its greater curvature. The corpus mucosa was scraped off using a glass slide and placed
Animals and experimental design Female Wistar-albino rats (Rattus rattus) 2.3–3 months of age and weighing 200–250 g were obtained from Production Center of Experimental Animals in Hacettepe University, Anakara, Turkey. The rats were fed a commercial pelleted feed and tap water throughout the experiment. After one week of acclimation, the rats were randomly divided into four groups of ten animals each and were housed five per cage. Laboratory conditions were kept at 20 1C and 50% relative humidity. Procedures were performed in the following four groups of rats; first: control, second: ethanol, third: Anzer honey and ethanol, fourth: N-ethylmaleimide (NEM). The study conforms the National Research Council guidelines for animal experimentation (National Research Council, 1996).
Table 1. A preliminary study on effect of pretreatment with Anzer honey on the ethanol-induced increased vascular permeability in the rat stomach Groups
Ascorbic acid values in honey (mg/g)
Evans Blue values in stomach (mg/g)
Control Ethanol Honey 1–ethanol Honey 2–ethanol Honey 3–ethanol Honey 4–ethanol Honey 5–ethanol
— — 6.66 8.63 13.03 13.49 25.44
0.725 3.00 1.388 0.925 0.413 0.362 0.0625
ARTICLE IN PRESS A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
into tubes containing 5 ml of distilled water. The extraction of EB from the gastric contents and mucosa was performed separately by adding 5 ml of formamide to each tube. The tubes were kept in a shaking water bath at a temperature of 50 1C for 24 h. After incubation of the samples, they were centrifuged at 3000 rpm for 15 min, and then supernatant was separated for analysis. The absorbance of supernatant was measured against a blank at 612 nm on a spectrophotometer. The amount of EB in the samples was calculated from a previously created standard curve and expressed as microgram per stomach.
Histopathological studies For histopathological examinations, all stomach samples were fixed in Bouin’s solution. These tissues
Table 2. Effects of exposures to NEM and ethanol (45%) with or without pretreatment with Anzer honey (0.275g/kg) on the vascular permeabiltiy in the rat stomach Groups
Amount of EB (mg/stomach)
% decrease () or increase (+)
Control Ethanol Honey–ethanol NEM
0.5170.27a 1.2970.34b,c,d 0.2670.15a,d 0.7370.11a,c
— +152 49 +43
a
Significantly different from second group P ¼ 0:05. Significantly different from first group P ¼ 0:05. c Significantly different from third group P ¼ 0:05. d Significantly different from fourth group P ¼ 0:05. b
175
were embedded in paraffin wax and sectioned (6 mm). The tissue sections were then stained with Harris Haematoxylin & eosin to evaluate gastric morphology.
Statistical analysis We studied EB results using one way analysis of variance (ANOVA) followed by Levene’s test. All values were expressed as the mean7standard deviation. Comparisons were made between the control and experimental groups. Values of Pp0:05 were regarded as statistically significant. Results of the incidence and severity of congestion in the stomach of control and treatment groups were analysed with the Fisher’s exact test. Results were considered significant at a P-value of less than 0.05.
Results The quantitative values of EB in gastric mucosa of four groups of rats that were given ethanol, Anzer honey+ethanol, NEM and control are presented in Table 2. The mean amount of EB that leaked into the stomach of rats in the ethanol group and the NEM group was higher than that of the control group. On the other hand, if compared to the control, EB values in the stomachs were significantly reduced when receiving honey before administration of ethanol in rats. Histologicaly, the stomach of the animals in the control group has a characteristic appearance of normal gastric mucosa. The cells in the mucosa, gastric pits and
Fig. 1. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a control rat. H&E 400.
ARTICLE IN PRESS 176
A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
Fig. 2. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that received ethanol (45%). There are marked areas of congestion (arrowhead) in the mucosa. H&E 400.
gastric glands were intact and normal. There was no evidence of erosions or congestion in the mucosa of rats (Fig. 1). There was excessive congestion on the surface of epithelium and gastric glands in the stomachs exposed to ethanol (Fig. 2) or NEM (Fig. 3). In contrast, when honey was administered before ethanol was given, the mucosal cells were all normal (Fig. 4.). There was no evidence of congestion or erosions anywhere in the stomach. The histological structure of the mucosa looked like that of the control group stomach. The incidence and severity of congestion in the stomach of control and treatment groups are presented in Table 3. The incidence and severity of gastric mucosal congestion in the honey+ethanol group were significantly reduced when compared to the ethanol group.
Discussion The results show that oral administration of Anzer honey prevents ethanol-induced gastric injury. Generally, ethanol-induced vascular permeability is measured by extravasation of EB into the stomach. Szabo et al. (1985) published a paper on a similar experiment on the subject of the pathogenesis of ethanol-induced gastric mucosal injury. In their experiment, intragastric administration of 100% ethanol caused significant leakage of EB from mucosal micro vessels within minutes. They suggest that vascular injury
was an early pathogenic factor in the development of ethanol-induced gastric lesions. In this study, the mean amount of EB that leaked into the stomach of rats in ethanol groups was significantly higher than control values. These results are in agreement with those reported by Szabo et al. (1985). In contrast, pretreatment with Anzer honey significantly reduced the EB levels that leaked into the stomach. Furthermore, those results also confirm our histopathological finding. Pretreatment with Anzer honey nearly completely prevents the occurrence of ethanolinduced congestion in the gastric mucosa. In light of these findings, pretreatment with Anzer honey has been shown to afford protection against increased vascular permeability induced by ethanol. The pathogenesis of ethanol-induced gastric lesions is complex. Although the mechanism of ethanol causes gastric mucosal injury are unclear, several factors have been implicated: products of arachidonate metabolism, oxygen-derived free radicals (ODFRs), and mast cell secretory products (Kvietys et al., 1990). In addition, there is evidence to suggest that decreased levels of GSH and generation of ODFRs may initiate vascular damage and vascular permeability (Ali, 1995a, b). According to this report, ODFRs may be involved at some point in the pathogenesis of ethanol-induced vascular permeability observed in this study. Honey contains a number of components known to act as antioxidants. These include ascorbic acid, flavonoids, catalase, and glucose oxidase (Mckibben and Engeseth,
ARTICLE IN PRESS A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
177
Fig. 3. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that received NEM (0.050 g/kg). Congestion (arrowhead) areas are present. H&E 400.
Fig. 4. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that was pretreated with honey (0.275 g/kg/day) for 30 min, then received ethanol (45%). Gastric mucosal cells are normal. No evidence of congestion in the mucosa. H&E 400.
2002). Furthermore, the Anzer honey contains large amounts of ascorbic acid (Kolankaya et al., 1999). Nagai et al. (2001) determined that ascorbic acid displayed a high antioxidative activity. Thus, the protective effect of honey may also be due to its antioxidant properties.
In conclusion, this study demonstrates that Anzer honey prevents ethanol-induced increased vascular permeability. Although the mechanism of the protective effect of honey has not been fully elucidated, it may be mediated through contents of its ascorbic acid and other components in the honey.
ARTICLE IN PRESS 178
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Table 3. The incidence and severity of congestion in the stomach of control and treatment groups of rats Groups
Severity of congestion
Incidence of congestion
Control Ethanol Honey–ethanol NEM
+++ + ++
a 10b 1**a 9b
, no remarkable changes; +, slight changes; ++, moderate changes; +++, severe changes. n ¼ 10. Numbers indicate the numbers of animal with observed congestion in the stomach. **Significantly different from the ethanol group at P ¼ 0:008 by Fisher’ s exact test. Groups that are not significantly different share a common letter. a There is no significant difference between the groups. b There is no significant difference between the groups.
References Ali ATMM. Natural honey, exert ıts protective effects against ethanol-induced gastric lesions in rats by preventing depletion of glandular nonproteinsulfhydryls. Tropical Gastroenterol 1995a;16(1):18–26. Ali ATMM. Natural honey accelerates healing of indomethacin-induced antral ulcers in rats. Saudi Med J 1995b; 16:161–6. Ali ATMM, Al-Swayeh OA. Natural honey prevents ethanolinduced gastric lesions in rats. Ethnopharmacology 1997;55:231–8. Ali ATMM, Al-Humayyd S, Madan BR. Natural honey prevents indomethecin- and ethanol-induced gastric lesions in rats. Saudi Med J 1990;11(4):275–9. Gharzouli K, Gharzouli A, Amira S, Khennouf S. Protective effect of mannitol, glucose–fructose–sucrose–maltose mix-
ture, and natural honey hyperosmolar solutions against ethanol-induced gastric mucosal damage in rats. Exp Toxic Pathol 2001;53:175–80. Gheldof N, Wang X, Engeseth NJ. Identification and quantification of antioxidant components of honeys from various floral sources. J Agric Food Chem 2002;50: 5870–7. Kolankaya, D, U¨lger C, Sorkun K, U¨nal H, 1999. II. National Congress of Society of Free Radicals and Antioxidant Research, Antioxidant properties of Rize-Anzer honey, the Eskis- ehir, the Turkey 19–22 Eylu¨l. Kvietys PR, Twohig B, Danzell J Specian D. Ethanol-induced injury to the rat gastric mucosa. Gatroenterology 1990;98:909–20. Lange S, Delbro DS, Jennisch E. Evans Blue permeation of intestinal mucosa in the rat. Scand J Gastroenterol 1994;29: 38–46. Mckibben J, Engeseth NJ. Honey as aprotective agent against lipid oxidation in ground Turkey. J Agric Food Chem 2002;50:592–5. Nagai T, Sakai M, Inoue R, Inoue H, Suzuki N. Antioxidative activities of some commercially honeys royal jelly and propolis. Food Chem 2001;75:237–40. National Research Council. Guide for the care and use of laboratory animals. Washington, DC: National Academy; 1996. Saria A, Lundberg JM. Evans blue fluorescence: morphological evaluation of vascular permeability in animal tissue. J Neurosci Methods 1983;8:41–9. Szabo S, Trier JS, Brown A, Schonoor J. Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 1985;88:228–36. Woods KL, Smith L, Graham DY. Intragastric accumulation of Evan’s Blue as a method for assessing aspirin – induced acute gastric mucosal injury in humans. Dig Dis Sci 1988;33(7):733–69.
Experimental and Toxicologic Pathology 57 (2005) 173–178
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Protective effect of Anzer honey against ethanol-induced increased vascular permeability in the rat stomach Aslı Dog˘an, Du¨rdane Kolankaya Department of Biology, Faculty of Science, Hacettepe University, 06532 Beytepe Campus, Ankara, Turkey Received 9 December 2004; accepted 5 April 2005
Abstract The purpose of this study was to determine the protective effect of Anzer honey on ethanol-induced increased vascular permeability in rats. Evan’s Blue (EB) dye, administered intracardiacly and extravasation of EB into the stomach, served as an indicator of vascular permeability following exposure to alcohol. Ethanol was given orally to the ethanol group for 90 days, and N-etylmaleimide (NEM) was given subcutaneously to the NEM group, and we observed increased extravasation of EB in the stomach in both groups. For this reason, we used NEM as a positive control for ethanol. Anzer honey, which contains 25.44 mg/g ascorbic acid, was given to the honey+ethanol group orally 30 min before beginning the 90-day ethanol administration. The mean amount of EB that leaked into the stomach of rats in the ethanol group and the NEM group was higher than that of the control group. Furthermore, if compared to the control, EB values in the stomachs were significantly reduced when receiving honey before administration of ethanol in rats. Histopathologically, the incidence and severity of gastric mucosal congestion were significantly reduced in the honey+ethanol group when compared to the ethanol group. These result indicate that Anzer honey is able to protect the stomach of the rat against ethanol-induced increased vascular permeability, which may be correlated with the ascorbic acid content. r 2005 Elsevier GmbH. All rights reserved. Keywords: Anzer honey; Ethanol; Ascorbic acid; Vascular permeability; Rat stomach; Evans blue
Introduction Honey is produced by honey bees from sugars present in the nectar of various plants. Honey is made up mostly of carbohydrates (70–80%), while it also contains preservative substances such as a-tocopherol, ascorbic acid and other phenolics and enzymes such as glucose
Corresponding author. Fax: +90 312 299 2028.
E-mail address: [email protected] (A. Dog˘an). 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.04.004
oxidase, catalase and peroxidase. Many of these substances have antioxidant properties (Gharzouli et al., 2001; Mckibben and Engeseth, 2002). Furthermore, honey has an in vitro antioxidant capacity similar to those of many fruits and vegetables on a fresh weight basis, as measured by the oxygen radical absorbance capacity (ORAC) assay (Gheldof et al., 2002). Honey is considered an antioxidant because of the presence of ascorbic acid, flavonoid and a-tocopherol. Rize-I˙kizdere-Anzer honey contains 6267 mg/g ascorbate and can be used as an alternative source of dietary
ARTICLE IN PRESS 174
A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
antioxidant (Kolankaya et al., 1999). This amount of ascorbic acid contains the recommended daily allowance of vitamin C for a 70 kg man. This is the reason we used Anzer honey in this study. It has been reported that natural honey is able to protect the rat stomach against acute ethanol-induced lesions (Ali et al, 1990; Ali and Al-Swayeh, 1997). Similarly, as an antioxidant drug, sucralfate, has also been shown to afford protection against ethanolinduced gastric lesions. Following the oral administration of ethanol, there was a marked increase in vascular permeability that preceded the development of visible mucosal damage (congestion or hemorrhage). It has been postulated that such an increase is a precursor of ethanol-induced macroscopic mucosal damage (Woods et al., 1988). Although the mechanisms by which luminal ethanol causes gastric mucosal injury are unclear, several factors have been implicated: products of arachidonate metabolism, oxygen-derived radicals, and mast cell secretory products (Kvietys et al., 1990). The present study was undertaken to examine the protective effect of Anzer honey against ethanol-induced increased vascular permeability and gastric lesion in rats. We used Evan’s Blue (EB) to demonstrate increased vascular permeability in the rat stomach. The azo dye EB is widely used to demonstrate pathologically increased microvascular permeability (Saria and Lundberg, 1983). When injected into the circulation, the dye binds within seconds to serum proteins (mainly albumin), forming a dissociable complex. An increase in local capillary permeability to macromolecules, due to inflammation or other types of damage, will therefore be detected by the extravasation and deposition in the interstitial tissue of protein–EB complex (Lange et al., 1994).
Materials and methods
Choosing Anzer honey The Anzer honey was obtained from the RizeI˙kizdere-Anzer region of Turkey. We conducted a preliminary study with five different Anzer honey samples that contain various quantities of ascorbic acid according to spectrophotometric measurement. The different samples of Anzer honey were administered 30 min before ethanol (45%) treatment on five groups of rats. Then 1 ml of EB was injected (intracardiacly) 15 min before killing the animals. As a result of this study, we found that the EB accumulation was the highest for the animals, which were fed with the least ascorbic acid-containing honey. Consistent with this value, as the ascorbic acid quantity in those honey samples increased, EB accumulation in the stomach decreased (Table 1). According to this result, we chose the honey containing the highest quantity of ascorbic acid for the 90-day experiment.
Animal treatment The first group of animals served as a control. The ethanol (45%), at a dose of 2 ml/kg body weight/day, was administrated orally each day for 90 days to the second group of rats. The third group received 0.275 g/ kg of honey 30 min before administration of the same value of ethanol (45%). The last group was administered 0.050 g/kg NEM at the very beginning of the 90-day period. In each group, 1 ml of EB (0.5%) was injected intracardiacly 30 min before killing the animals, and the EB that had accumulated in the gastric contents and in the mucosa was quantified by the method described by Lange et al. (1994). At the end of the treatment, the animals were killed by cervical dislocation. After removing the stomachs, we opened each along its greater curvature. The corpus mucosa was scraped off using a glass slide and placed
Animals and experimental design Female Wistar-albino rats (Rattus rattus) 2.3–3 months of age and weighing 200–250 g were obtained from Production Center of Experimental Animals in Hacettepe University, Anakara, Turkey. The rats were fed a commercial pelleted feed and tap water throughout the experiment. After one week of acclimation, the rats were randomly divided into four groups of ten animals each and were housed five per cage. Laboratory conditions were kept at 20 1C and 50% relative humidity. Procedures were performed in the following four groups of rats; first: control, second: ethanol, third: Anzer honey and ethanol, fourth: N-ethylmaleimide (NEM). The study conforms the National Research Council guidelines for animal experimentation (National Research Council, 1996).
Table 1. A preliminary study on effect of pretreatment with Anzer honey on the ethanol-induced increased vascular permeability in the rat stomach Groups
Ascorbic acid values in honey (mg/g)
Evans Blue values in stomach (mg/g)
Control Ethanol Honey 1–ethanol Honey 2–ethanol Honey 3–ethanol Honey 4–ethanol Honey 5–ethanol
— — 6.66 8.63 13.03 13.49 25.44
0.725 3.00 1.388 0.925 0.413 0.362 0.0625
ARTICLE IN PRESS A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
into tubes containing 5 ml of distilled water. The extraction of EB from the gastric contents and mucosa was performed separately by adding 5 ml of formamide to each tube. The tubes were kept in a shaking water bath at a temperature of 50 1C for 24 h. After incubation of the samples, they were centrifuged at 3000 rpm for 15 min, and then supernatant was separated for analysis. The absorbance of supernatant was measured against a blank at 612 nm on a spectrophotometer. The amount of EB in the samples was calculated from a previously created standard curve and expressed as microgram per stomach.
Histopathological studies For histopathological examinations, all stomach samples were fixed in Bouin’s solution. These tissues
Table 2. Effects of exposures to NEM and ethanol (45%) with or without pretreatment with Anzer honey (0.275g/kg) on the vascular permeabiltiy in the rat stomach Groups
Amount of EB (mg/stomach)
% decrease () or increase (+)
Control Ethanol Honey–ethanol NEM
0.5170.27a 1.2970.34b,c,d 0.2670.15a,d 0.7370.11a,c
— +152 49 +43
a
Significantly different from second group P ¼ 0:05. Significantly different from first group P ¼ 0:05. c Significantly different from third group P ¼ 0:05. d Significantly different from fourth group P ¼ 0:05. b
175
were embedded in paraffin wax and sectioned (6 mm). The tissue sections were then stained with Harris Haematoxylin & eosin to evaluate gastric morphology.
Statistical analysis We studied EB results using one way analysis of variance (ANOVA) followed by Levene’s test. All values were expressed as the mean7standard deviation. Comparisons were made between the control and experimental groups. Values of Pp0:05 were regarded as statistically significant. Results of the incidence and severity of congestion in the stomach of control and treatment groups were analysed with the Fisher’s exact test. Results were considered significant at a P-value of less than 0.05.
Results The quantitative values of EB in gastric mucosa of four groups of rats that were given ethanol, Anzer honey+ethanol, NEM and control are presented in Table 2. The mean amount of EB that leaked into the stomach of rats in the ethanol group and the NEM group was higher than that of the control group. On the other hand, if compared to the control, EB values in the stomachs were significantly reduced when receiving honey before administration of ethanol in rats. Histologicaly, the stomach of the animals in the control group has a characteristic appearance of normal gastric mucosa. The cells in the mucosa, gastric pits and
Fig. 1. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a control rat. H&E 400.
ARTICLE IN PRESS 176
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Fig. 2. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that received ethanol (45%). There are marked areas of congestion (arrowhead) in the mucosa. H&E 400.
gastric glands were intact and normal. There was no evidence of erosions or congestion in the mucosa of rats (Fig. 1). There was excessive congestion on the surface of epithelium and gastric glands in the stomachs exposed to ethanol (Fig. 2) or NEM (Fig. 3). In contrast, when honey was administered before ethanol was given, the mucosal cells were all normal (Fig. 4.). There was no evidence of congestion or erosions anywhere in the stomach. The histological structure of the mucosa looked like that of the control group stomach. The incidence and severity of congestion in the stomach of control and treatment groups are presented in Table 3. The incidence and severity of gastric mucosal congestion in the honey+ethanol group were significantly reduced when compared to the ethanol group.
Discussion The results show that oral administration of Anzer honey prevents ethanol-induced gastric injury. Generally, ethanol-induced vascular permeability is measured by extravasation of EB into the stomach. Szabo et al. (1985) published a paper on a similar experiment on the subject of the pathogenesis of ethanol-induced gastric mucosal injury. In their experiment, intragastric administration of 100% ethanol caused significant leakage of EB from mucosal micro vessels within minutes. They suggest that vascular injury
was an early pathogenic factor in the development of ethanol-induced gastric lesions. In this study, the mean amount of EB that leaked into the stomach of rats in ethanol groups was significantly higher than control values. These results are in agreement with those reported by Szabo et al. (1985). In contrast, pretreatment with Anzer honey significantly reduced the EB levels that leaked into the stomach. Furthermore, those results also confirm our histopathological finding. Pretreatment with Anzer honey nearly completely prevents the occurrence of ethanolinduced congestion in the gastric mucosa. In light of these findings, pretreatment with Anzer honey has been shown to afford protection against increased vascular permeability induced by ethanol. The pathogenesis of ethanol-induced gastric lesions is complex. Although the mechanism of ethanol causes gastric mucosal injury are unclear, several factors have been implicated: products of arachidonate metabolism, oxygen-derived free radicals (ODFRs), and mast cell secretory products (Kvietys et al., 1990). In addition, there is evidence to suggest that decreased levels of GSH and generation of ODFRs may initiate vascular damage and vascular permeability (Ali, 1995a, b). According to this report, ODFRs may be involved at some point in the pathogenesis of ethanol-induced vascular permeability observed in this study. Honey contains a number of components known to act as antioxidants. These include ascorbic acid, flavonoids, catalase, and glucose oxidase (Mckibben and Engeseth,
ARTICLE IN PRESS A. Dog˘an, D. Kolankaya / Experimental and Toxicologic Pathology 57 (2005) 173–178
177
Fig. 3. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that received NEM (0.050 g/kg). Congestion (arrowhead) areas are present. H&E 400.
Fig. 4. Photomicrograph of haematoxylin–eosin stained section of gastric mucosa from a rat that was pretreated with honey (0.275 g/kg/day) for 30 min, then received ethanol (45%). Gastric mucosal cells are normal. No evidence of congestion in the mucosa. H&E 400.
2002). Furthermore, the Anzer honey contains large amounts of ascorbic acid (Kolankaya et al., 1999). Nagai et al. (2001) determined that ascorbic acid displayed a high antioxidative activity. Thus, the protective effect of honey may also be due to its antioxidant properties.
In conclusion, this study demonstrates that Anzer honey prevents ethanol-induced increased vascular permeability. Although the mechanism of the protective effect of honey has not been fully elucidated, it may be mediated through contents of its ascorbic acid and other components in the honey.
ARTICLE IN PRESS 178
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Table 3. The incidence and severity of congestion in the stomach of control and treatment groups of rats Groups
Severity of congestion
Incidence of congestion
Control Ethanol Honey–ethanol NEM
+++ + ++
a 10b 1**a 9b
, no remarkable changes; +, slight changes; ++, moderate changes; +++, severe changes. n ¼ 10. Numbers indicate the numbers of animal with observed congestion in the stomach. **Significantly different from the ethanol group at P ¼ 0:008 by Fisher’ s exact test. Groups that are not significantly different share a common letter. a There is no significant difference between the groups. b There is no significant difference between the groups.
References Ali ATMM. Natural honey, exert ıts protective effects against ethanol-induced gastric lesions in rats by preventing depletion of glandular nonproteinsulfhydryls. Tropical Gastroenterol 1995a;16(1):18–26. Ali ATMM. Natural honey accelerates healing of indomethacin-induced antral ulcers in rats. Saudi Med J 1995b; 16:161–6. Ali ATMM, Al-Swayeh OA. Natural honey prevents ethanolinduced gastric lesions in rats. Ethnopharmacology 1997;55:231–8. Ali ATMM, Al-Humayyd S, Madan BR. Natural honey prevents indomethecin- and ethanol-induced gastric lesions in rats. Saudi Med J 1990;11(4):275–9. Gharzouli K, Gharzouli A, Amira S, Khennouf S. Protective effect of mannitol, glucose–fructose–sucrose–maltose mix-
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