8-phenyltheophylline or L-NAME, indicating that its effect is not mediated by ... KEY WORDS: 1,8-cineole; ethanol; gastric mucosal lesions; gastric mucus; ...
Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001), pp. 331–337
1,8-Cineol, a Food Flavoring Agent, Prevents Ethanol-Induced Gastric Injury in Rats F.A. SANTOS, PhD and V.S.N. RAO, PhD
This study investigated the gastroptrotective effect of 1,8-cineole (cineole) on ethanolinduced gastric mucosal damage in rats and the possible mechanisms involved. 1,8-Cineole (50 –200 mg/kg), given orally 1 hr before administration of 1 ml of absolute ethanol significantly attenuated the ethanol-induced gastric injury in a manner similar to nordihydroguairetic acid, a known lipoxygenase inhibitor. 1,8-Cineole showed a tendency to restore the ethanol-associated decreases in nonprotein sulfhydryls, suggesting a possible antioxidant effect. In gastric secretion studies, 1,8-cineole, similar to cimetidine, a known histamine-2 receptor antagonist, demonstrated significant inhibitions of both gastric juice volume as well as total acid output. The protection offered by 1,8-cineole was found to be unaltered by 8-phenyltheophylline or L-NAME, indicating that its effect is not mediated by endogenous adenosine or nitric oxide. These results, taken together with the earlier reports, suggest that the antioxidant and lipoxygenase inhibitory actions of 1,8-cineole are of prime importance in affording gastroprotection against ethanol injury in the rat. KEY WORDS: 1,8-cineole; ethanol; gastric mucosal lesions; gastric mucus; nonprotein sulfhydryls.
1,8-Cineole (cineole), also known as eucalyptol or cajeputol, is a terpene oxide found in many plant essential oils (1–3). It is often used as a flavoring agent for food products (4, 5). Traditionally, it has been used for treating symptoms of airway diseases exacerbated by infection, and a growth inhibitory effect of 1,8-cineole on several microorganisms has recently been documented (6). It is also frequently employed by the pharmaceutical industry in drug formulations as a percutaneous enhancer and for its decongestant and antitussive effects and in aromatherapy as a skin stimulant in the form of skin baths (7–10). Recent studies have shown its inhibitory effect on the production of inflammatory mediators such as cytokines, prostaglandins, and leukotrienes from lipopolysaccharide-stimulated human blood monoManuscript received March 31, 2000; accepted July 31, 2000. From the Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, C.P-3157, 60430-270 Fortaleza, Ceara´, Brazil. Address for reprint requests: Dr. V.S.N. Rao, Departamento de Fisiologia e Farmacologia, Rua Cel Nunes de Melo, 1127, C.P. 3157, Porangabussu, 60430-270 Fortaleza, CE, Brasil.
cytes both in vitro as well as ex vivo (11, 12). More recently, we provided evidence to show that 1,8cineole possesses antiinflammatory and analgesic properties (13). Ethanol-induced gastric mucosal damage in rats is often used as an experimental model when screening compounds for antiulcer activity because it represents the most common cause of gastric ulcer in man. Absolute ethanol rapidly promotes the formation of hyperemic blisters in the stomach mucosa, which is essentially an acute inflammatory reaction wherein increased vascular permeability is a common feature (14). Some plant substances, such as flavonoids, terpenoids and xanthines, known for their antiinflammatory activity have been shown to prevent gastric lesions induced by ethanol (15–17). The antiinflammatory effects of these compounds have been attributed to their ability to scavenge oxygen free radicals (5, 18, 19). On the other hand, nonsteroidal antiinflammatory drugs that inhibit prostaglandin biosynthesis actually promote gastric lesions (20, 21). Keeping in view the reputed usefulness of 1,8-
Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001) 0163-2116/01/0200-0331$19.50/0 © 2001 Plenum Publishing Corporation
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cineole as a flavoring agent for foods, its widespread use by the pharmaceutical industry in drug formulations, and the established antiinflammatory potential, the present study was designed to verify whether or not 1,8-cineole prevents ethanol-induced gastric damage in rats. MATERIALS AND METHODS Animals. Male Wistar rats weighing 180 –200 g were used in the study. They were housed in polypropylene cages and maintained under normal laboratory conditions at 22 ⫾ 1°C on a 12-hr light– dark cycle and fed a standard diet (Purina Chow) and water ad libitum. All the experimental protocols were approved by the University Committee for the Care and Use of Laboratory Animals. The animals were deprived of food but had free access to tap water 24 hr before experimentation. Drugs and Chemicals. The following drugs were used: 1,8-cineole, nordihydroguiaretic acid (NDGA), 8-phenyltheophylline (8-PT), and L-NAME (Sigma Chemical Co., St. Louis, Missouri, USA); cimetidine (Tagamet, SmithKline), and Tween 80 (Synth). The chemicals used were all of analytical grade. Gastric Mucosal Damage. Gastric mucosal damage was induced in conscious rats by intragastric instillation of 1.0 ml/rat of absolute ethanol (99.5%) as previously described (22). The test drug 1,8-cineole was emulsified in Tween 80 (5% in water) and administered at doses of 25, 50, 100, and 200 mg/kg body weight by oral gavage 45 min before ethanol. Nordihydroguairetic acid (75 mg/kg) was used as a reference drug for comparison. Control rats received only the vehicle (5% Tween 80). The animals were killed 60 min after ethanol and their stomachs removed and opened along the greater curvature to observe the lesions macroscopically. Lesion severity was determined by measuring the area of lesions with a transparent grid (consisting of 1-mm squares) placed on the glandular mucosal surface (23), which was expressed in percentage of the total area of gladular stomach. In a few experiments, rats were simultaneously treated with 8-phenyltheophylline (8 mg/kg, intraperitoneally) or L-NAME (20 mg/kg) to verify a possible influence of endogenous adenosine or nitric oxide (NO) on 1,8-cineole effect. Gastric Wall Mucus. The ethanol-induced changes on gastric mucus was indirectly analyzed by determining the amount of Alcian blue bound to the gastric wall (24). The glandular segments from the stomachs of rats treated with 1,8-cineole (100 and 200 mg/kg) or vehicle were collected and weighed. Each segment was transferred to 1% Alcian blue solution (0.1 M sucrose in 0.05 M sodium acetate, pH 5.8). Excess dye was removed by washing the segments with 0.25 M sucrose solution. The mucus– dye complex was extracted by placing the segments in 10 ml of 0.5 M magnesium chloride for 2 hr. Four milliliters of dye extract was mixed with an equal volume of diethyl ether, centrifuged at 3000 rpm for 10 min, and the absorbance of supernatent was measured at 598 nm. The quantity of Alcian blue extracted per gram of glandular tissue was then calculated. Gastric Secretary Studies. The pylorus of each rat was ligated under light ether anesthesia to study the basal
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gastric secretion (25). Cineole (100 and 200 mg/kg) and cimetidine (60 mg/kg) were administered intraduodenally immediately after pylorus ligation. Control animals were given the vehicle instead of test drugs. The animals were killed after 4 hr. The stomachs were removed and the volume of gastric juice was determined after centrifugation (3000g for 10 min). Total acidity was assessed by titration against 0.01 N NaOH to pH 7.0, and expressed as microequivalents per hour. Gastric Mucosal Nonprotein Sulfhydryls (NP-SH). Gastric mucosal NP-SH was measured according to the method described earlier (26). The glandular stomachs from rats treated with 1,8-cineole (100 and 200 mg/kg) or vehicle were removed and homogenized in ice-cold 0.02 M ethylenediaminetetraacetic acid. The homogenate was mixed with distilled water and 50% trichloroacetic acid and then centrifuged. The supernatents 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. Statistical Analysis. All results were expressed as the mean ⫾ SEM. Data were analyzed using analysis of variance followed by Fisher’s or Student-Newman-Keuls multiple comparison tests. Values of P ⬍ 0.05 were taken as significant.
RESULTS Gastric Lesions. Intragastric administration of absolute ethanol to the control group of rats treated with vehicle (5% Tween 80) produced large bandlike hemorrhagic erosions in the glandular stomach. The lesion area in control rats was 16.91 ⫾ 2.56 mm2. 1,8-Cineole at oral doses of 25, 50, 100, and 200 mg/kg prevented the gastric mucosal injury by 21, 74, 90, and 92%, respectively (Figure 1). NDGA at an oral dose of 75 mg/kg also prevented the mucosal damage by 84%. The gastroprotective effect of 1,8-cineole (200 mg/kg, per os) against ethanol injury was not significantly altered in rats treated with 8-PT (8 mg/kg, intraperitoneally) or L-NAME (20 mg/kg, intraperitoneally) (Figure 2). Gastric Wall Mucus and Mucosal Sulfhydryls. The content of gastric mucus as measured by Alcian blue binding was slightly, but not significantly, elevated in the ethanol-treated animals (53.91 ⫾ 7.05 g/g) compared to that in the mucosa of normal rats (31.39 ⫾ 5.42 g/g) (Table 1). 1,8-Cineole at 50 mg/kg significantly enhanced the gastric mucus content (91.06 ⫾ 16.56 g/g), but at higher doses it showed no significant effect. Treatment of animals with 1 ml of absolute ethanol significantly lowered the gastric NP-SH concentration (187.66 ⫾ 16.46 mol/g) by 37% compared to vehicle-treated rats (296.77 ⫾ 16.46 mol/g). The group of animals treated with absolute ethanol ⫹ Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)
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Fig 1. Effects of 1,8-cineole and nordehydroguiaretic acid (NDGA) on gastric lesions induced by absolute ethanol in rats. Drugs or vehicle (control) were administered orally 1 hr before absolute ethanol (1 ml/rat). Each column denotes mean ⫾ SEM for six rats. ***P ⬍ 0.001 compared to control.
1,8-cineole (50, 100, and 200 mg/kg) showed lesser reductions (31, 22, and 5%, respectively) than the group treated with ethanol alone. In rats that received ethanol ⫾ 1,8-cineole (200 mg/kg), the levels of NP-SH were almost similar to those found in vehicletreated control. Gastric Secretion. Figure 3 shows the effects of 1,8-cineole on gastric secretory volume and total acid output in 4-hr pylorus-ligated rats. The gastric secretory volume and total acid output in rats that received the vehicle were 1.80 ⫾ 0.09 ml/hr and 229.92 ⫾ 20.17 eq/hr, respectively. 1,8-Cineole at 50 mg/kg caused a slight increase in the above parameters; higher doses (100 and 200 mg/kg) significantly reduced the gastric secretory volume (41 and 52%) as well as the total acid output (40 and 84%), respectively. Cimetidine (100 mg/kg), the reference standard used in the study, also showed significant inhibition of gastric secretory volume (85%) and total acid output (81%). DISCUSSION This study demonstrates that 1,8-cineole has a gastroprotective property. It was found to be active, at Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)
oral doses ranging from 50 to 200 mg/kg, in the prevention of ethanol-induced gastric damage. At 200 mg/kg, its potency was almost the same as that produced by NDGA (75 mg/kg), a known lipoxygenase inhibitor (27). Studies have shown a role for leukotriene C4 (LTC4), a lipoxygenase-derived metabolite of arachidonic acid (28), mast cell-derived histamine (29), and oxygen-derived free radicals (30, 31) in the pathogenic mechanism of gastric injury induced by ethanol. Ethanol causes constriction of submucosal venules with subsequent stasis of blood flow in mucosal microcirculation as well as arteriolar dilatation and plasma leakage from the vascular bed (32–34), events that could lead to the formation of bandlike blisters in the gastric mucosa. 1,8-Cineole was shown to inhibit arachidonic acid metabolism and the generation of prostaglandins and leukotrienes (11, 12, 35). Our previous results (unpublished) demonstrate that 1,8-cineole has the ability to protect gastric mucosa against indomethacininduced gastric ulcers also, but only at a high dose (400 mg/kg). Since 1,8-cineole prevented ethanolinduced gastric injury at relatively smaller doses (50 –
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Fig 2. Effects of 8-phenyltheophylline (8-PT, 8 mg/kg, intraperitoneally) and L-NAME (20 mg/kg, intraperitoneally) treatments on 1,8-cineole (200 mg/kg, per os)-induced gastroprotection against ethanol in rats. Drugs and vehicle were administered 1 hr before absolute ethanol (1 ml/rat). Each column denotes ⫾SEM for six rats. ***P ⬍ 0.001 compared to control.
200 mg/kg), it might be a preferential inhibitor of the lipoxygenase enzyme. The gastric mucosa possesses a high basal concentration of glutathione, which functions as an antioxidant (36). Glutathione accounts for the majority of soluble reduced sulfhydryl compounds (37) and ethanol-induced gastric damage has been shown to be associated with a significant decrease in mucosal lev-
els of sulfhydryl compounds (38). Therefore we investigated whether 1,8-cineole can mitgate the ethanolinduced decreases in gastric NP-SH. The results obtained reveal that 1,8-cineole abrogates ethanolinduced depletion of NP-SH in a dose-related manner, suggesting a possible antioxidant activity. Studies in the past have shown that adenosine, a purine metabolite, inhibits gastric acid secretion (39),
TABLE 1. EFFECT OF 1,8-CINEOLE ON ABSOLUTE ETHANOL-INDUCED CHANGES IN GASTRIC WALL MUCUS AND NONPROTEIN SULFHYDRYLS (NP-SH) CONCENTRATION* Treatment and dose (mg/kg) Vehicle control Ethanol control 1,8-Cineole 50 100 200
Gastric wall mucus (g Alcian blue/g)
Gastric NP-SH concentration (mol/g)
31.39 ⫾ 5.42 53.91 ⫾ 7.05
296.77 ⫾ 16.46 187.66 ⫾ 7.30†
91.06 ⫾ 16.56† 58.81 ⫾ 9.06 50.38 ⫾ 16.87
204.98 ⫾ 18.86† 231.40 ⫾ 14.18 282.81 ⫾ 21.27‡
*Values denote mean ⫾ SEM from six rats. †P ⬍ 0.05 vs vehicle control. ‡P ⬍ 0.05 vs ethanol control.
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Fig 3. Effects of 1,8-cineole and cimetidine on gastric secretory volume (A) and total acid output (B) in 4-hr pylorus-ligated rats. Drugs and vehicle (control) were administered intraduodenally, immediately after pyloric ligation. Each column denotes the mean ⫾ SEM for six rats. **P ⬍ 0.01; ***P ⬍ 0.001 compared to control.
increases gastric mucosal blood flow and stimulates antioxidant enzymes (40), and protects against ethanol-induced gastric damage in rats (41). Adenosine may also stimulate vascular endothelium to produce nitric oxide (NO) and play a role in the maintenance Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)
of gastric mucosal integrity (42). The possibility that 1,8-cineole-mediated gastroprotection is a result of its ability to release endogenous adenosine and NO was tested by treating rats with the adenosine antagonist, 8-PT (8 mg/kg) and the calcium-dependent nitric
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oxide synthase inhibitor L-NAME (20 mg/kg), respectively. The results show that both these agents were unable to modify the response of 1,8-cineole on ethanol injury, suggesting that endogenous adenosine and NO do not participate in its gastroprotective effect. Ethanol-induced gastric mucosal injury has been also suggested to be due to impairments in defensive factors such as gastric mucus and mucosal blood flow (43). When 1,8-cineole was tested on pylorus-ligated rats, the smaller dose (50 mg/kg) produced an increase in gastric mucus content, gastric secretory volume, and total acidity and higher doses (100 and 200 mg/kg) resulted in significant inhibition of gastric secretory volume and total acidity, suggesting its modulatory influence on gastric secretion. The effect of 1,8-cineole on ethanol-induced gastric injury, however, seems to be independent of its effect on gastric secretion and gastric mucus since gastroprotection was observed at almost all doses employed. Interestingly, a recent study has observed a global increase in cerebral blood flow after prolonged inhalation of 1,8-cineole in human subjects (44). It remains to be verified whether such an effect of 1,8-cineole could account for the mitigation of ethanol-induced gastric mucosal injury through an increase in gastric mucosal blood flow. In conclusion, these data demonstrate that 1,8cineole offers protection against ethanol damage in the rat at nontoxic doses. Considering that an oral LD50 of 3.5 g/kg was observed in mice (13), the doses utilized in this study are safe and demonstrated no untoward behavioral effects in rats. However, 1,8cineole administered to rats at doses above 600 mg/kg has been shown to be associated with decreased body weight gain, even more so after prolonged administration, but it showed no signs of nephrotoxicity or hepatotoxicity (4). This suggests that 1,8-cineole is safe as a gastroprotective agent and supports its use in drug formulations. It also implies that the gastroprotection afforded by 1,8-cineole, in part, involves its antioxidant action, preventing ethanol-induced depletion of NP-SH, and also, partly due to lipoxygenase inhibition, blocking leukotrienes formation. However, further studies are needed to clarify the exact mechanism of gastroprotective effect of 1,8-cineole. ACKNOWLEDGEMENTS This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´ gico (CNPq), Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de
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Ensino Superior (CAPES) and Financiadora de Estudos e Projetos (FINEP) of Brasil.
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