Abstract: Boric acid was evaluated for its potential to protect the gastric mucosa against the injuries caused by 80% ethanol, 25% NaCl, 0.2 M NaOH and indomethacin in male Wistar rats. The effects caused by ethanol on gastric wall mucus, gastric levels of non-protein sulfhydryl groups, protein, nucleic acids and malondialdehyde and histopathological effects were also investigated. Boric acid pretreatment at oral doses of 60, 120 and 240 mg kg-1 body weight provided a dose-dependent protection against the ulcerogenic effects of different necrotising agents used. Treatment of rats with 1 ml of 80% ethanol (gavage) was found to cause depletion of stomach-wall mucus, to lower the concentrations of proteins, nucleic acids and non-protein sulfhydryl groups (NP-SH) in the stomach wall and to cause histopathological lesions, including necrosis, corrosion, congestion and hemorrhage. Boric acid treatment showed a dose-dependent protection against all these effects. In the same manner it affected malondialdehyde concentrations altered by ethanol pretreatment. Boric acid also offered protection against mucosal damage caused by indomethacin. Present findings show that boric acid possesses antiulcerogenic effects. Further studies are required to determine its role in the prophylaxis and or the treatment of gastric ulcer disease.
INTRODUCTION
Compounds of boron have been used for a variety of application for thousands of years. The ancient Greeks and Romans used borates as a cleaning agent. The first recorded internal use of boron compounds as a medication was by Arabian Physicians in 875 AD[1]. Boron is an essential plant micronutrient and there is growing evidence of its essentiality in animals, as well[2-5]. The major sources of human explore to boron is diet (e.g. fruits, vegetables, nuts) and water[6]. Boric acid is fairly rapidly absorbed after its administration and appears to be rapidly distributed through the body water via passive diffusion. Boric acid is not metabolized and is excreted from the body via urine[7]. The half-life time (t½) of boric acid is approximately in the order of one day and it does not appear to accumulate in soft tissue, but it bones[8]. Normal levels of boron in soft tissue, urine and blood range from 0.05 to 10 ppm. In one study, Ku et al.[9] demonstrated an increase in boron levels in the tissue (testis, epidydymis, accessory sex organs, hypothalamus and rest of the brain) that appeared to reach steady-state boron levels (12-13 μg g-1) by three to four days after daily administration of 9000 ppm boric acid to adult Fischer rats for up to seven days.
Blech et al.[10,11] demonstrated that a 3% boric acid solution improved dramatically the healing of deep wounds. Patients who were treated with boric acid returned to a normal care unit about three times more rapidly (mean 20 days) than those have received conventional antiseptics, such as chlorhexidine or iodinated polyvinylpyrolidone (mean 55 days). In view of the vast medical utilization and other activities of boron compounds, the present study was designed to investigate boric acid for its effects on the gastric mucosa subjected to various noxious stimuli.
MATERIALS AND METHODS
Male Wistar albino rats (bred at the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia) and all roughly the same age, weighing 180-220 g, were used in the present study. All the animals were maintained under controlled condition of temperature, humidity and light and were provided with Purina chow and water ad lib. The rats were randomly assigned to control and treatment groups (6 rats in each group) and were fasted for 36 h with free access to drinking water. The necrotic agents, 80% ethanol, 25% NaCl and 0.2 M NaOH, were previously found to produce gastric lesions[12]. An arbitrary dose of 240 mg boric acid per kg body weight (WinLab, UK) provided significant protection against necrotic agents in an experimental trial and hence a dose range of 60 to 240 mg boric acid per kg was used. An aqueous solution of boric acid or distilled water alone was administered by gavage to the 36 h fasted rats 30 min before oral treatment by gavage with 1 ml 80% ethanol (25% NaCl and 0.2 M NaOH were used only in the cytoprotection studies). The animals were sacrificed under anesthesia, using ether 1 h after the treatment with the narcotizing agents.
Cytoprotection studies: The stomach of each of the animals was excised and opened along the greater curvature. After washing with normal saline the gastric lesions were quantified using a binocular magnifier. The ulcers were scored according to the method of Valcavi et al.[13]. The circular ulcers induced by indomethacin was assessed on the basis of their diameters: Deep circular ulcers more than 8 mm=10; 7-8 mm=8; 6-7 mm=7; 5-6 mm=6; 4-5 mm=5; 3-4 mm=4; 2-3 mm=3; 1-2 mm=2; 0-1 mm=1. The deep linear ulcers more than 10 mm in length=6 and less than 10 mm in length=3. The scores for each single lesion were then summed up for the determination of ulcer index.
Gastric-wall mucus determination: The modified procedure of Corne et al.[14] was used to determine gastric-wall mucus. The glandular segments from the stomachs were removed and weighed. Each segment was transferred immediately to 1% alcian blue solution (in sucrose solution, buffered with sodium acetate, pH 5) and the excess dye was removed by rinsing with sucrose solution. The dye complexed with the gastric wall mucus was extracted with magnesium chloride. A 4 ml sample of blue extract was then shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged and the absorbance of the aqueous layer was recorded at 580 nm. The quantity of alcian blue extracted/g (net) of glandular tissue was then calculated.
Estimation of total protein and nucleic acids: The levels of proteins and nucleic acids in the stomach were determined according to the following procedure: the stomachs were rapidly dissected from the animals, frozen in liquid nitrogen and stored at -20°C until they were analyzed for total proteins and nucleic acids (DNA, RNA). Total protein was determined by the method of Lowry et al.[15]. The method described by Bregman[16] was used to determine the levels of nucleic acids. Tissues were homogenized and the homogenate was suspended in ice-cold trichloroacetic acid (TCA). After centrifugation, the pellet was extracted with ethanol. DNA was determined by treating the nucleic acid extract with diphenylamine reagent and measuring the intensity of the blue colour at 600 nm. For quantification of RNA, the nucleic acid extract was treated with orcinol and the green colour was read at 660 nm. Standard curves were used to determine the amounts of nucleic acids present.
Estimation of non-protein sulfhydryl groups (NP-SH): Gastric mucosal NP-SH was measured according to the method of Sedlak and Linday[17]. The glandular stomach removed and homogenized in ice-cold 0.02 M ethylenediaminetetraacetic acid (EDTA). The homogenate was mixed with distilled water and 50% TCA and centrifuged; the supernatants were mixed with Tris buffer, 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) was added and the sample was shaken. The absorbance was read within 5 min of addition of DTNB, at 412 nm, against a reagent blank with no homogenate.
Malondialdehyde estimation: The method described by Fong et al.[18] was used. Tissues were homogenized in TCA and the homogenates were suspended in thiobarbituric acid. After centrifugation, the optical density of the clear pink supernatants was read at 532 nm. Malondialdehyde bis (dimethyl acetal) was used as standard.
Indomethacin-induced gastric ulcers: Indomethacin was suspended in 1% caroboxymethylcellulose in water (6 mg ml-1) and administered to the fasted rats in a dose of 30 mg kg-1 (0.5 ml/100g). Control rats were similarly treated with an equivalent amount of the vehicle[19]. The stomachs of the animals were removed, rinsed with normal saline and studied according to the procedure of Szabo et al.[20].
Histopathological procedures: Tissue samples were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Using an American Optical Rotary Microtone, sections of thickness about 5 μm were cut and stained with haematoxylin and eosin. These were examined under the microscope for histopathological changes such as congestion, corrosion, hemorrhage and necrosis by an observer who was blind with respect to the treatment groups. The severity of histopathological changes was expressed according to an arbitary scale[21].
Statistical analysis: One-way analysis of variance was carried out in order to compare the two groups. Results are presented as mean±standard error (S.E.). Statistical analysis was carried out using SPSS 10.0.
RESULTS
Effects on gross gastric lesions: Treatment with boric acid did not induce any ulcers in the gastric mucosa (pilot study, results not shown). Ethanol and other necrotizing agents were found to cause extensive damage to the gastric mucosa. Boric acid pretreatment provided the gastric mucosa with a significant dose dependent protection against ulceration caused by 80% ethanol, hypertonic saline (25% NaCl) and 0.2 M NaOH (Table 1).
Effects on stomach wall mucus: Treatment with ethanol significantly reduced the amount of stomach wall mucus. Pretreatment with boric acid inhibited the ethanol-induced depletion of gastric wall mucus in a dose-dependent manner (Table 2).
Effects on protein and nucleic acid concentrations: The treatment with ethanol significantly reduced the protein and nucleic acid concentrations of the stomachs of treated animals (Table 3). Boric acid pretreatment provided dose-dependent protection against the action of ethanol on protein and nucleic acid concentrations.
Effects on gastric mucosal NP-SH concentrations: The NP-SH concentrations in gastric mucosa were significantly decreased after treatment with ethanol. Pretreatment with boric acid prevented the NP-SH depletion caused by ethanol (Table 4).
Effects on malondialdehyde concentrations: Ethanol treatment caused considerable increase in the gastric mucosal malondialdehyde concentration. Pretreatment with boric acid caused a dose-dependent reduction in malondialdehyde concentration previously increased by ethanol treatment (Table 5).
Effects on indomethacin-induced gastric mucosal damage: Pretreatment with boric acid was found to protect the gastric mucosal damage induced by indomethacin in dose-dependent manner (Table 6).
Effects on histopathological gastric lesions: Ethanol treatment caused considerable damage in the form of necrosis, corrosion, congestion and haemorrhagic mucosal lesions in the stomach walls of treated animals; however, no neutrophilic component was evident (Table 7). The area involved was mainly the glandular segments. There was also evidence of interluminal bleeding in these animals. Pretreatment with boric acid provided significant and dose-dependent protection against the action of ethanol (Fig. 1-4).
| Table 1: | Effect of boric acid on the induction of gastric ulcers by various necrotic agents in rats |
| Six rats were used in each group. Groups 2,3 and 4 were statistically
compared with group 1 *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 2: | Effect of boric acid on the induction of changes in gastric-wall mucus by 80% ethanol |
| Groups 2, was statistically compared with group 1. Groups
3, 4 and 5 were statistically compared with group 5 *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 3: | Effect of boric acid on the nucleic acids and protein concentrations in the stomach wall of rats treated with 80% ethanol |
| Groups 2 was statistically compared with group 1. Groups 3,
4 and 5 were statistically compared with group 2 *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 4: | Effect of boric acid on glutathione (NP-SH) concentrations in the glandular stomach of rats treated with 80% ethanol |
| Groups 2 was statistically compared with group 1. Groups 3,
4 and 5 were statistically compared with group 2 *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 5: | Effect of boric acid on Malondialdehyde concentrations in the glandular stomach of rats treated with 80% ethanol |
| Groups 2 was statistically compared with group 1. Groups 3,
4 and 5 were statistically compared with group 2 *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 6: | Effect of boric acid on indomethacin-induced gastric mucosal damage in rats |
| Groups 2, 3 and 4 were statistically compared with group 1. *P<0.05, **P<0.01 and ***P<0.001 by analysis of variance |
|
| Table 7: | Effect of boric acid on the induction of histopathological lesions by treatment with 80% ethanol in stomach wall of rats. |
| Fig. 1: | The stomach wall of a control rat showing its normal appearance. Haemotoxylin and eosin, x 40 |
| Fig. 2: | The stomach wall of a rat after treatment with 1 ml 80% ethanol, by gavage. Haemotoxylin and eosin, x 40 |
DISCUSSION
The results of present study clearly demonstrate that boric acid confers on the gastric mucosa a dose-dependent protection against the gross damaging action of ethanol and other necrotizing agents. These findings were verified and substantiated by histopathological investigations which showed marked reduction in congestion, necrosis, corrosion and haemorrhage of the mucosa.
In earlier studies it was shown that ethanol treatment decreased gastric wall mucus thickness[21] and this effect was similarly observed during our present experiments. Boric acid was found to inhibit the ethanol-induced depletion of stomach-wall mucus. The role of mucus in the prevention of gastric mucosal injury is controversial[22-,24]. However, the gastric mucus coat is considered important both in preventing damage to the gastric epithelium and in facilitating its repair[25]. The results of our present study demonstrate that boric acid is effective against ethanol-induced gastric mucus depletion.
Ethanol is known to be metabolized in the body and to produce free radicals[26]. The alcohol-induced reduction in protein and nucleic acid contents of the rat stomach observed during the present study may be the result of the accumulation of toxic free radicals in the mucosa[27].
| Fig. 3: | The stomach wall of a rat after treatment with boric acid (120 mg kg-1) and 80% ethanol by gavage. Haemotoxylin and eosin, x 40 |
| Fig. 4: | The stomach wall of a rat after treatment with boric acid (240 mg kg-1) and 80% ethanol by gavage. Haemotoxylin and eosin, x 40 |
Antioxidants are known to protect cellular damage by scavenging free radical formation may be due to its possible antioxidant nature. The antioxidant nature of boric acid is suggested in present study as it significantly reduced malondialdehyde concentration increased by ethanol treatment.
The administration of ethanol significantly decreased the gastric mucosal NP-SH concentration. Tissue damage by different chemical agents has been associated with reduced levels of endogenous sulfhydryls[28,29]. NP-SH are known to be involved in the cytoprotective effects of various drugs[30,31]. Pretreatment with boric acid was found to prevent NP-SH depletion caused by ethanol. This effect suggests the possible involvement of NP-SH in the cytoprotective of boric acid.
Several non-steroidal anti-inflammatory drugs (NSAID) including indomethacin are known to induce gastric damage[13] and this effect was verified for indomethacin during the present study. Boric acid conferred protection against indomethacin-induced gastric mucosal damage in dose-dependent manner. This finding suggest the potential antiulcer activity of boric acid. It has shown that indomethacin and other NSAIDs cause mucosal damage, leading to the formation of peptic ulcer by the suppression of mucosal generation of prostaglandins[32]. Various compounds with potential to generate prostaglandins have been reported to protect gastric mucosa against various ulcerogenic agents[33-35]. Although the effect of boric acid on prostaglandin generation is not known, our present findings suggest that boric acid may promote prostaglandin synthesis.
The results of the present investigations clearly demonstrate the protective effect of boric acid against gastric mucosal damage induced by ethanol. Although at this time it is difficult to explain the exact mechanism of action of boric acid, the effects of boric acid on gastric mucus production, NP-SH and malondialdehyde concentrations observed in the present study suggest a multifactorial mechanism. Further studies are warranted to evaluate the toxicity and safety of boric acid before clinical trials can be considered.