1,8Cineol, a Food Flavoring Agent, Prevents Ethanol-Induced Gastric Injury in Rats

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This study investigated the gastroptrotective effect of 1,8-cineole (cineole) on ethanol-induced 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
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  1,8-Cineol, a Food Flavoring Agent, PreventsEthanol-Induced Gastric Injury in Rats F.A. SANTOS, PhD and V.S.N. RAO, PhDThis study investigated the gastroptrotective effect of 1,8-cineole (cineole) on ethanol-induced 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 ethanolsignificantly attenuated the ethanol-induced gastric injury in a manner similar to nordihy-droguairetic acid, a known lipoxygenase inhibitor. 1,8-Cineole showed a tendency to restorethe ethanol-associated decreases in nonprotein sulfhydryls, suggesting a possible antioxidanteffect. In gastric secretion studies, 1,8-cineole, similar to cimetidine, a known histamine-2receptor antagonist, demonstrated significant inhibitions of both gastric juice volume as wellas total acid output. The protection offered by 1,8-cineole was found to be unaltered by8-phenyltheophylline or  L  -NAME, indicating that its effect is not mediated by endogenousadenosine or nitric oxide. These results, taken together with the earlier reports, suggest thatthe antioxidant and lipoxygenase inhibitory actions of 1,8-cineole are of prime importance inaffording 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 orcajeputol, is a terpene oxide found in many plantessential oils (1–3). It is often used as a flavoringagent for food products (4, 5). Traditionally, it hasbeen used for treating symptoms of airway diseasesexacerbated by infection, and a growth inhibitoryeffect of 1,8-cineole on several microorganisms hasrecently been documented (6). It is also frequentlyemployed by the pharmaceutical industry in drugformulations as a percutaneous enhancer and for itsdecongestant and antitussive effects and in aroma-therapy as a skin stimulant in the form of skin baths(7–10). Recent studies have shown its inhibitory effecton the production of inflammatory mediators such ascytokines, prostaglandins, and leukotrienes from li-popolysaccharide-stimulated human blood mono-cytes both  in vitro  as well as  ex vivo  (11, 12). Morerecently, we provided evidence to show that 1,8-cineole possesses antiinflammatory and analgesicproperties (13).Ethanol-induced gastric mucosal damage in rats isoften used as an experimental model when screeningcompounds for antiulcer activity because it representsthe most common cause of gastric ulcer in man. Absolute ethanol rapidly promotes the formation of hyperemic blisters in the stomach mucosa, which isessentially an acute inflammatory reaction whereinincreased vascular permeability is a common feature(14). Some plant substances, such as flavonoids, ter-penoids and xanthines, known for their antiinflamma-tory activity have been shown to prevent gastric le-sions induced by ethanol (15–17). Theantiinflammatory effects of these compounds havebeen attributed to their ability to scavenge oxygenfree radicals (5, 18, 19). On the other hand, nonste-roidal antiinflammatory drugs that inhibit prostaglan-din biosynthesis actually promote gastric lesions (20,21). Keeping in view the reputed usefulness of 1,8- Manuscript received March 31, 2000; accepted July 31, 2000.From the Department of Physiology and Pharmacology, Facultyof Medicine, Federal University of Ceara´, C.P-3157, 60430-270Fortaleza, Ceara´, Brazil. Address for reprint requests: Dr. V.S.N. Rao, Departamento deFisiologia e Farmacologia, Rua Cel Nunes de Melo, 1127, C.P.3157, Porangabussu, 60430-270 Fortaleza, CE, Brasil.  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001), pp. 331–337  331  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001) 0163-2116/01/0200-0331$19.50/0 © 2001 Plenum Publishing Corporation  cineole as a flavoring agent for foods, its widespreaduse by the pharmaceutical industry in drug formula-tions, and the established antiinflammatory potential,the present study was designed to verify whether ornot 1,8-cineole prevents ethanol-induced gastric dam-age in rats. MATERIALS AND METHODS  Animals.  Male Wistar rats weighing 180–200 g were usedin the study. They were housed in polypropylene cages andmaintained under normal laboratory conditions at 22  1°Con a 12-hr light–dark cycle and fed a standard diet (PurinaChow) and water  ad libitum . All the experimental protocols were approved by the University Committee for the Careand Use of Laboratory Animals. The animals were deprivedof food but had free access to tap water 24 hr beforeexperimentation. Drugs and Chemicals.  The following drugs were used:1,8-cineole, nordihydroguiaretic acid (NDGA), 8-phenyl-theophylline (8-PT), and  L  -NAME (Sigma Chemical Co.,St. Louis, Missouri, USA); cimetidine (Tagamet, Smith-Kline), and Tween 80 (Synth). The chemicals used were allof analytical grade. Gastric Mucosal Damage.  Gastric mucosal damage wasinduced in conscious rats by intragastric instillation of 1.0ml/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, and200 mg/kg body weight by oral gavage 45 min before etha-nol. Nordihydroguairetic acid (75 mg/kg) was used as areference drug for comparison. Control rats received onlythe vehicle (5% Tween 80). The animals were killed 60 minafter ethanol and their stomachs removed and openedalong the greater curvature to observe the lesions macro-scopically. Lesion severity was determined by measuring thearea of lesions with a transparent grid (consisting of 1-mmsquares) 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 simulta-neously treated with 8-phenyltheophylline (8 mg/kg, intra-peritoneally) or  L  -NAME (20 mg/kg) to verify a possibleinfluence of endogenous adenosine or nitric oxide (NO) on1,8-cineole effect. Gastric Wall Mucus.  The ethanol-induced changes ongastric mucus was indirectly analyzed by determining theamount of Alcian blue bound to the gastric wall (24). Theglandular segments from the stomachs of rats treated with1,8-cineole (100 and 200 mg/kg) or vehicle were collectedand weighed. Each segment was transferred to 1% Alcianblue solution (0.1 M sucrose in 0.05 M sodium acetate, pH5.8). Excess dye was removed by washing the segments with0.25 M sucrose solution. The mucus–dye complex wasextracted by placing the segments in 10 ml of 0.5 M mag-nesium chloride for 2 hr. Four milliliters of dye extract wasmixed with an equal volume of diethyl ether, centrifuged at3000 rpm for 10 min, and the absorbance of supernatent was measured at 598 nm. The quantity of Alcian blueextracted per gram of glandular tissue was then calculated. Gastric Secretary Studies.  The pylorus of each rat wasligated under light ether anesthesia to study the basalgastric secretion (25). Cineole (100 and 200 mg/kg) andcimetidine (60 mg/kg) were administered intraduodenallyimmediately after pylorus ligation. Control animals weregiven the vehicle instead of test drugs. The animals werekilled after 4 hr. The stomachs were removed and the volume of gastric juice was determined after centrifugation(3000  g   for 10 min). Total acidity was assessed by titrationagainst 0.01 N NaOH to pH 7.0, and expressed as micro-equivalents per hour. Gastric Mucosal Nonprotein Sulfhydryls (NP-SH).  Gas-tric mucosal NP-SH was measured according to the methoddescribed earlier (26). The glandular stomachs from ratstreated with 1,8-cineole (100 and 200 mg/kg) or vehicle were removed and homogenized in ice-cold 0.02 M ethyl-enediaminetetraacetic acid. The homogenate was mixed with distilled water and 50% trichloroacetic acid and thencentrifuged. 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 areagent blank with no homogenate. Statistical Analysis.  All results were expressed as themean  SEM . Data were analyzed using analysis of variancefollowed by Fisher’s or Student-Newman-Keuls multiplecomparison tests. Values of   P     0.05 were taken as signif-icant. RESULTSGastric Lesions.  Intragastric administration of ab-solute ethanol to the control group of rats treated with vehicle (5% Tween 80) produced large bandlikehemorrhagic erosions in the glandular stomach. Thelesion area in control rats was 16.91    2.56 mm 2 .1,8-Cineole at oral doses of 25, 50, 100, and 200 mg/kgprevented the gastric mucosal injury by 21, 74, 90, and92%, respectively (Figure 1). NDGA at an oral doseof 75 mg/kg also prevented the mucosal damage by84%. The gastroprotective effect of 1,8-cineole (200mg/kg,  per os ) against ethanol injury was not signifi-cantly altered in rats treated with 8-PT (8 mg/kg,intraperitoneally) or L-NAME (20 mg/kg, intraperi-toneally) (Figure 2). Gastric Wall Mucus and Mucosal Sulfhydryls.  Thecontent of gastric mucus as measured by Alcian bluebinding was slightly, but not significantly, elevated inthe ethanol-treated animals (53.91  7.05   g/g) com-pared to that in the mucosa of normal rats (31.39   5.42   g/g) (Table 1). 1,8-Cineole at 50 mg/kg signifi-cantly enhanced the gastric mucus content (91.06   16.56   g/g), but at higher doses it showed no signifi-cant effect. Treatment of animals with 1 ml of abso-lute ethanol significantly lowered the gastric NP-SHconcentration (187.66  16.46   mol/g) by 37% com-pared to vehicle-treated rats (296.77  16.46  mol/g).The group of animals treated with absolute ethanol  SANTOS AND RAO 332  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)  1,8-cineole (50, 100, and 200 mg/kg) showed lesserreductions (31, 22, and 5%, respectively) than thegroup treated with ethanol alone. In rats that receivedethanol    1,8-cineole (200 mg/kg), the levels of NP-SH were almost similar to those found in vehicle-treated control. Gastric Secretion.  Figure 3 shows the effects of 1,8-cineole on gastric secretory volume and total acidoutput in 4-hr pylorus-ligated rats. The gastric secre-tory volume and total acid output in rats that receivedthe vehicle were 1.80  0.09 ml/hr and 229.92  20.17  eq/hr, respectively. 1,8-Cineole at 50 mg/kg caused aslight increase in the above parameters; higher doses(100 and 200 mg/kg) significantly reduced the gastricsecretory volume (41 and 52%) as well as the totalacid 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 gas-troprotective property. It was found to be active, atoral doses ranging from 50 to 200 mg/kg, in theprevention of ethanol-induced gastric damage. At 200mg/kg, its potency was almost the same as that pro-duced by NDGA (75 mg/kg), a known lipoxygenaseinhibitor (27). Studies have shown a role for leuko-triene C 4  (LTC 4 ), a lipoxygenase-derived metaboliteof arachidonic acid (28), mast cell-derived histamine(29), and oxygen-derived free radicals (30, 31) in thepathogenic mechanism of gastric injury induced byethanol. Ethanol causes constriction of submucosal venules with subsequent stasis of blood flow in mu-cosal microcirculation as well as arteriolar dilatationand plasma leakage from the vascular bed (32–34),events that could lead to the formation of bandlikeblisters in the gastric mucosa.1,8-Cineole was shown to inhibit arachidonic acidmetabolism and the generation of prostaglandins andleukotrienes (11, 12, 35). Our previous results (un-published) demonstrate that 1,8-cineole has the abil-ity to protect gastric mucosa against indomethacin-induced gastric ulcers also, but only at a high dose(400 mg/kg). Since 1,8-cineole prevented ethanol-induced gastric injury at relatively smaller doses (50– 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 AND ETHANOL-INDUCED GASTRIC DAMAGE 333  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)  200 mg/kg), it might be a preferential inhibitor of thelipoxygenase enzyme.The gastric mucosa possesses a high basal concen-tration of glutathione, which functions as an antioxi-dant (36). Glutathione accounts for the majority of soluble reduced sulfhydryl compounds (37) and eth-anol-induced gastric damage has been shown to beassociated with a significant decrease in mucosal lev-els of sulfhydryl compounds (38). Therefore we inves-tigated whether 1,8-cineole can mitgate the ethanol-induced decreases in gastric NP-SH. The resultsobtained reveal that 1,8-cineole abrogates ethanol-induced depletion of NP-SH in a dose-related man-ner, suggesting a possible antioxidant activity.Studies in the past have shown that adenosine, apurine metabolite, inhibits gastric acid secretion (39), Fig 2.  Effects of 8-phenyltheophylline (8-PT, 8 mg/kg, intraperitoneally) and  L  -NAME (20 mg/kg, intraperitoneally) treatments on1,8-cineole (200 mg/kg,  per os )-induced gastroprotection against ethanol in rats. Drugs and vehicle were administered 1 hr beforeabsolute ethanol (1 ml/rat). Each column denotes   SEM  for six rats. ***  P     0.001 compared to control.T  ABLE  1. E FFECT OF  1,8-C INEOLE ON  A  BSOLUTE  E THANOL  -I NDUCED  C HANGESIN  G  ASTRIC  W  ALL   M UCUS AND  N ONPROTEIN  S ULFHYDRYLS (NP-SH) C ONCENTRATION * Treatment and dose(mg/kg)Gastric wall mucus(    g Alcian blue/g)Gastric NP-SH concentration(    mol/g) Vehicle control 31.39  5.42 296.77  16.46Ethanol control 53.91  7.05 187.66  7.30†1,8-Cineole50 91.06  16.56† 204.98  18.86†100 58.81  9.06 231.40  14.18200 50.38  16.87 282.81  21.27‡*Values denote mean  SEM  from six rats.†  P     0.05 vs vehicle control.‡  P     0.05 vs ethanol control. SANTOS AND RAO 334  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)  increases gastric mucosal blood flow and stimulatesantioxidant enzymes (40), and protects against etha-nol-induced gastric damage in rats (41). Adenosinemay also stimulate vascular endothelium to producenitric oxide (NO) and play a role in the maintenanceof gastric mucosal integrity (42). The possibility that1,8-cineole-mediated gastroprotection is a result of itsability to release endogenous adenosine and NO wastested by treating rats with the adenosine antagonist,8-PT (8 mg/kg) and the calcium-dependent nitric Fig 3.  Effects of 1,8-cineole and cimetidine on gastric secretory volume (A) and total acid output (B) in 4-hr pylorus-ligatedrats. Drugs and vehicle (control) were administered intraduodenally, immediately after pyloric ligation. Each columndenotes the mean    SEM  for six rats. **  P     0.01; ***  P     0.001 compared to control. 1,8-CINEOLE AND ETHANOL-INDUCED GASTRIC DAMAGE 335  Digestive Diseases and Sciences, Vol. 46, No. 2 (February 2001)
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