Anti-inflammatory and non ulcerogenic activities of acetylbergenin

1 Programa de Pós-graduação em Biotecnologia, Rede Bionorte, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil. 2 Programa de Pós-Graduação em Agroenergia, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil. 3 Laboratório de Química, Universidade Federal do Pará, Rua Augusto Corrêa, 01-Setor Básico, 66075-110 Belém, Pará, Brazil. 4 Laboratório de Farmacodinâmica, Faculdade de Farmácia, Universidade Federal do Pará, Belém, Brazil. 5 Laboratório de Pesquisa em Fármacos, Departamento de Ciências Biológicas e da Saúde, Universidade Federal do Amapá, Macapá, Amapá, Brazil.

. E. uchi is widely used by the people to combat myoma and arthritis, although only few biological studies are available on the properties of its extract.Bergenin (1) is a C-glucoside of 4-O-methyl gallic acid that has been isolated as the major component from the cortex of E. uchi and is used for treating gastrointestinal diseases such as gastritis, gastric ulcer, diarrhea, and constipation (Okada et al., 1973;Abe et al., 1980).In addition, studies report that bergenin reveals anti-inflammatory (Swarnalakshmi et al., 1984;Nunomura et al., 2009), antiarthritic (Nazir et al., 2007) and hypolipidemic effects (Jahromi et al., 1992).In our laboratory, bergenin (1) was isolated as the principal component from the aqueous extract of the E. uchi cortex and acetylbergenin (2) was obtained by acetylation of bergenin in order to increase its lipophilic and physiological activities.In the present study, the antiinflammatory and ulcerogenic activities of acetylbergenin in various experimental models in vivo were investigated.

Acetylation of bergenin
The method described by Borges et al. (2011) was employed in this assay.In a round-bottomed flask of 125 mL, 700 mg of bergenin (1), 17.5 mL of acetic anhydride (Ac2O, 100% degree of purity, Synth, Brazil) and 6.5 mL of anhydrous pyridine (100% degree of purity, Synth, Brazil) were added.After agitation, the mixture was maintained for 24 h at room temperature and then transferred to a separator funnel of 125 and 25 mL of distilled water was added and the mixture was extracted with ethyl acetate (3 × 40 mL).The organic phases were collected, washed with distilled water (2 × 40 mL), 5% hydrochloric acid solution (1 × 40 mL) and then with distilled water (2 × 40 mL) until neutral pH was obtained.The organic phase was dried with anhydrous Na2SO4 and after filtration, the solvent was evaporated at room temperature in a chapel.The solid material obtained in the form of white crystals was recrystallized in methanol resulting in 1.13 g (yield 99%) of crystals of acetylbergenin (2) with 99% degree of purity.

Animals
Swiss albino mice (Mus musculus) male adults, weighing between Borges et al. 403 20 and 25 g and male Mac Coy rats (

Drugs, chemicals and dose used
For the accomplishment of the experiments, the dose of acetylbergenin was based on the ED50 of 6.8 mg/kg previously determined by Borges et al. (2011).Acetylbergenin was dissolved in 0.2 mL of 2% DMSO (Sigma Chemical Co., USA) and 5% Tween-80 solution (Merck, Brazil).Negative control groups received the same solution used to solubilize the substance acetylbergenin.The drugs used in the experiments were: dexamethasone (0.5 mg/kg, MSD Co., Brazil), indomethacin (5 mg/kg, MSD Co., Brazil), acetylsalicylic acid (100 mg/kg, Bayer, Brazil), and cyproheptadine hydrochloride (Chemical Co., USA) were dissolved and diluted in 0.9% physiological solution.Croton oil (2.5%, Sigma Chemical Co., USA) was solubilized in acetone (Synth, Brazil).The total volume of solution administered orally was 0.25 mL for mice and 0.5 mL for rats.

Croton oil-induced dermatitis
The method described by Tubaro et al. (1985) was used in this experiment.Cutaneous inflammation was induced in several groups of mice by applying 0.1 mL (1 mg/ear) of croton oil solution in acetone on the surface of the right ear.The same volume of acetone was applied to the left ear.One hour before the application, three groups of mice (n=10/group) were orally treated with acetylbergenin (6.8 mg/kg, 0.25 mL), vehicle (0.25 mL, 2% dimethyl sulfoxide (DMSO) and 2% Tween-80 in distilled water, control group, or acetylsalicylic acid (100 mg/kg, 0.25 mL).Six hours later, the mice were submitted to euthanasia and the antiinflammatory effect was evaluated.Samples of 6 mm in diameter were extracted using a punch biopsy, and the weight difference between the samples of the control ear (left) and the croton oiltreated ear (right) was calculated.The results obtained are represented in weight (mg).

Carrageenan-induced paw edema in rats assay
Edema was induced by intraplantar injection of 1% carrageenan (100 µg/paw, 0.1 mL, Sigma Co., USA) into the right paw of Mac Coy rats (n = 5/group).A saline sample of equal volume was injected in the left paw (0.1 mL).The distinct experimental groups were treated with vehicle (2% DMSO and 2% Tween-80 in distilled water, negative control, 0.5 mL), acetylbergenin (6.8 mg/kg, 0.5 mL), or indomethacin (positive control, 10 mg/kg, 0.5 mL), and after60 min they received intraplantar injections of carrageenan in the right hind paw and saline in the left hind paw.A digital pachymeter (Zaas Precision, Mitutoyo Co., Japan) was used to determinate the paw diameter at 1 h intervals after stimulus application over 6 h.The amount of edema was calculated by subtracting the measured volume of the paw injected with saline from the measured volume of the paw injected with carrageenan.

Dextran-induced paw edema in rats assay
The paw edema was induced by dextran in rats, following the method described by Carvalho et al. (1999).The Mac Coy rats were randomly divided into three groups (n = 5/group).A volume of 0.1 mL of 1% dextran (100 µg/paw, 0.1 mL, Sigma Co., USA) solution was injected on the plantar surface of the right hind paw in rats pretreated 60 min earlier with vehicle (2% DMSO and 2% Tween-80 in distilled water, 0.5 mL, control group, p.o.), acetylbergenin (6.8 mg/kg, 0.5 mL, p.o.) or the reference drug cyproheptadine (10 mg/kg, 0.5 mL, p.o.).The inflammation was quantified by measuring the volume (mL) displaced by the paw using a digital pachymeter (Zaas Precision, Mitutoyo Co., Japan) at 0, 30, 60, 90, and 120 min after dextran injection.Results were expressed as variation in volume (mL) between the right and left paws at each time.
The cell migration analysis was based on the methods described by Carvalho et al. (1999).The results obtained in the differential count were expressed as the number of neutrophils per milliliter of exudates.

Stress-induced acute gastric ulcer
Ulcers were induced according to the method described by Basile et al. (1990).Wistar rats were fasted with free access to water for 24 h and were further treated with vehicle (2% DMSO and 2% Tween-80 in distilled water 0.5 mL, p.o), acetylbergenin (6.8 mg/kg, 0.5 mL, p.o), and indomethacin (10 mg/kg, 0.5 mL, p.o). Groups of five animals each were treated and 30 min later, each animal was kept for 17 h in a contender tube, which was immersed vertically until the water reaching the neck region of the animal in a tank with current water at 25°C.Furthermore, the rats were submitted to euthanasia by CO2 inhalation.Their stomachs were immediately excised, opened by cutting along the greater curvature, and the inner wall was examined for lesions using a binocular stereomicroscope with a magnification of 10× (Nikon SMZ-10).The number and the severity of the acute lesions were enumerated and graded as follows: light (1+) = presence of hyperemia and single mucosal punctiform hemorrhages (petechiae); moderate (2+) = presence of submucosal hemorrhagic lesions with small erosions; severe (3+) = presence of hemorrhagic edges with severe erosions and some invasive lesions.A lesion index was determined following the formula reported by Basile et al. (1990).

Statistical analysis
Results were expressed as mean ± standard error of mean (SEM).Statistical analysis was performed using Student's t-test and analysis of variance (ANOVA), followed by Student-Newman-Keuls.
A value of p<0.05 was considered as statistically significant.The analysis was performed using a GraphPad Prism 5.0 program.

Bergenin and acetylbergenin
Compounds 1 and 2 (Figure 1) were identified as bergenin and acetylbergenin, respectively, by the aid of

Croton oil-induced dermatitis
Pretreatment with acetylbergenin (6.8 mg/kg, p.o.) inhibited 75.42% of the ear edema formation induced by croton oil injection (p<0.001) when compared with the control group.This inhibition of the edematogenic process was similar to that observed with the group treated with acetylsalicylic acid 100 mg/kg (positive control) decreasing the inflammatory process by 78.53% (Figure 2).

Carrageenan-induced paw edema in rat
Carrageenan injection in the animal paws produced a visible and measurable edema, with maximum inflammation observed 4 h after the injection of the inflammatory agent.The group treated with acetylbergenin at a dose of 6.8 mg/kg inhibited the edema formation over the 6 h of the experiment (Figure 3).The maximum edema inhibition was 35.09% (p<0.05,Student's test, and ANOVA).

Dextran-induced paw edema in rats assay
Dextran 1%-induced intense paw edema in rats, an effect that reached a maximum level at 1 h after administration and decreased over the subsequent hours.The oral administration with 6.8 mg/kg of acetylbergenin inhibited the dextran-induced edema by 33% (p<0.05).The reference drug cyproheptadine (10 mg/kg, p.o.) significantly (p<0.05)inhibited the dextran-induced paw edema at 30, 60, 90, and 120 min after administration when compared with the control (Figure 4).

Carrageenan-induced peritonitis in rats
In this model of carrageenan-induced leukocyte migration, it was possible to observe an acute inflammatory response in the peritoneal cavity of rats by neutrophil concentration of 3893.25 ×10 6 cells/mL after 4 h.The acetylbergenin (6.8 mg/kg, p.o.) was able to significantly reduce the carrageenan-induced neutrophil count (70%), when compared with the control group treated with distilled water (Figure 5).Treatment of the animals with dexamethasone (0.5 mg/kg, p.o.) 1 h before the experiment, used as a positive control, significantly reduced (94.23%) the cell migration.

Stress-induced acute gastric ulcer
In the stress ulcer experiment, the animals treated with indomethacin (10 mg/kg, p. o) produced more lesions when compared with those treated with acetylbergenin at a dose of 6.8 mg/kg (p.o) (Table 1).Acetylbergenin at a dose of 6.8 mg/kg has revealed significant effect with a ulcer index of 10.08 and protection of 78.55% when Table 1.Effect of oral administration of control (2% DMSO and 2% Tween-80 in distilled water), acetylbergenin (6.8 mg/kg) and indomethacin (10 mg/kg) on the incidence of gastric lesions of rats produced by stress.compared with indomethacin group (p<0.001).

DISCUSSION
This study aimed to investigate the anti-inflammatory and antiulcerogenic activities of acetylbergenin using various experimental models in vivo.Acetylbergenin was extracted by acetylation of bergenin, according to Borges et al. (2011).It is well known that several plants in nature comprise a huge reservoir of bioactive molecules that can be developed as new chemical entities, analogs, derivatives, and synthetic compounds to form a natural product (Rastogi and Rawat, 2008).Experimental models using carrageenan as an inflammatory agent are widely used to investigate the pathophysiology of the inflammatory response, as well as to characterize the novel anti-inflammatory drugs (Tobacman, 2001).Carrageenan induces a measurable local inflammatory response.This model of paw edema is most frequently used to evaluate the effects of antiinflammatory drugs.This model presents two inflammatory phases and a third, uncharacteristic one.In the first hour after the carrageenan injection, an increase in vascular permeability mediated by histamine and serotonin is observed.In the second hour, the permeability increase is caused by kinines.In the third hour, the increase of vascular permeability occurs due to prostaglandin action (Perazzo et al., 2005).
The data presented in this study revealed that pretreatment of the animals with acetylbergenin at a dose of 6.8 mg/kg, 1 h prior to the intraplantar injection of carrageenan, inhibited the development of paw edema at all evaluation times; however, this inhibition was higher during the second and third peaks (2 and 3 h), revealing the participation of prostaglandins in the third hour.Indomethacin (10 mg/kg, p.o.), considered as a nonspecific cyclooxygenase inhibitor, also significantly reduced the volume of carrageenan-induced paw edema at all evaluation times, compared to the respective negative control used.In order to demonstrate the participation of prostaglandins in carrageenan-induced edema, some authors have confirmed that pretreatment with aspirin, a nonsteroidal anti-inflammatory drug, promotes endogenous prostaglandin and edema reduction, whereas administration of high doses of prostaglandin E2 (PGE2) or prostacyclin elicited an increase in paw edema (Lewis et al., 1975;Vane and Botting, 1987).
Another phlogistic agent used in the present study was dextran.This substance is a high molecular weight polysaccharide that induces an anaphylactic reaction, characterized by extravasation and formation of edema with little protein and few neutrophils, besides the degranulation of mast cells and subsequent release of histamine and serotonin (Vinegar et al., 1969).Thus, it was possible to observe that in the dextran-induced edema, oral administration of 6.8 mg/kg of acetylbergenin inhibited the edema at the initial stage (1 and 2 h), where the degranulation of inflammatory cells occurred with a release of biogenic amines, serotonin, and histamine.Swarnalakshmi et al. (1984) reported the antiedematogenic activity of bergenin, with a dose dependent inhibition in the third hour, with 44.1, 53.6, and 65.5% inhibition at oral doses of 60, 120, and 240 mg/kg, respectively; however, considering this data, it is possible to verify that acetylbergenin was more effective at anoral dose of 6.8 mg/kg, since at this dose it was able to cause 47% edema inhibition induced by carrageenan in the third hour besides being significant in other instances.
Acetylbergenin activity on leukocyte migration was studied in the carrageenan-induced peritonitis model in rats.Oral administration of acetylbergenin (6.8 mg/kg) inhibited neutrophil cell migration by 70%, following the profile similar to that of dexamethasone which caused 94.23% inhibition.This model of acute inflammation allows the quantification of leukocytes that migrate to the peritoneal cavity under the action of chemotactic agents, mainly leukotrienes LTB 4 (Bastos et al., 2001).
Therefore, in general, the initial process of the acute inflammatory response is characterized by the increase of neutrophils in the circulating blood, being the first line of physiological defense, followed by lymphocytes and monocytes (Male, 2003).The mode of action of carrageenan in inducing the leukocyte migration may be a result of synergism between PGE2, LTB4, and other potent chemotactic agents such as complement (C5a) and interleukins (IL-8), promoting vasodilation, plasma exudation, and leukocyte accumulation in the lesion sites (Thomazzi et al., 2009).Hence, it is possible that nonsteroidal anti-inflammatory drugs (NSAIDs), by inhibiting the synthesis of vasodilatory PGE2, promote blood flow reduction by compromising the leukocyte migration to the area of inflammatory reaction (Almeida et al., 1980).The results suggest that the anti-inflammatory activity of acetylbergenin is related to the biosynthesis of prostaglandins and lipoxygenase products.
While testing dermatitis induced by the topical application of croton oil, the activation of phospholipase A 2 , releasing arachidonic acid with consequent biosynthesis of leukotrienes and prostaglandins, by cyclooxygenase and lipoxygenase pathways, respectively, was observed.This dermatitis is sensitive to the action of topical anti-inflammatories, also responding to the systemic administration of steroidal antiinflammatory drugs (Tubaro, 1986).Acetylbergenin at a dose of 6.8 mg/kg significantly inhibited (p<0.001) the dermatitis and presented a similar result to the nonsteroidal anti-inflammatory acetylsalicylic acid (100 mg/kg).
On the evaluation of the ulcerogenic effect of 6.8 mg/kg acetylbergenin, the substance produced 78.55% less ulcerative damages.In addition, 1+ and 3+ (hemorrhagic) gastric lesions in animals submitted to stress were significantly reduced by treatment with 6.8 mg/kg of acetylbergenin when compared with indomethacin.These results suggest that, despite having performed significantly on carrageenan edema, however, did not show an ulcerogenic effect such as indomethacin, which is a nonspecific cyclooxygenase inhibitor.This effect was like that found for bergenin, at a dose of 30 mg/kg, i.p., when it exerted a non-ulcerogenic effect in rats submitted to the stress-induced gastric ulcer test (Abe et al., 1979).
Gastric cytoprotection is conferred by certain substances, such as prostaglandins.The clinical use of traditional NSAIDs for treating inflammation and pain is often accompanied by adverse gastrointestinal effects.The pharmacological effects of NSAIDs are due to the inhibition of a membrane enzyme called cyclooxygenase (COX), which is involved in the prostaglandin biosynthesis.There are two isoforms, COX-1 and COX-2, which share the same substrates, produce the same products, and catalyze the same reaction using identical catalytic mechanisms, but differ in inhibitor selectivity.The isoform, COX-1, chiefly plays a physiological role in the kidneys and the stomach, whereas COX-2, induces inflammatory conditions and is involved in the production of prostaglandins that mediate pain.Inhibition of COX-1 is responsible for the adverse gastrointestinal and renal effects of NSAIDs, while the inhibition of COX-2 accounts for NSAIDs therapeutic effects.All classical NSAIDs, such as aspirin and indomethacin are nonselective inhibitors of both COX-1 and COX-2, but bind more tightly to COX-1 (Alanazi et al., 2015).Chung et al. (2001) reported that acetylbergenin, presented greater activity in vivo when compared with bergenin, against the hepatotoxicity in rats.Notably, acetylbergenin is more readily absorbed due to its ability to cross the bilayer of intestinal cell membranes, resulting in an increase in the protective activity after being hydrolyzed into a hydrophilic polyphenol such as norbergenin and bergenin.
Other studies have demonstrated the importance of adding an acetyl radical to a molecule for antiedematogenic activity (Carvalho et al., 1999).This may be partially explained by the high specificity of acetylated anti-inflammatory compounds (Souza et al., 2004), such as aspirin, which inhibit prostaglandin synthesis by inactivating COX.Aspirin selectively acetylates the hydroxyl group of a serine residue (Ser 53), among the terminal 70 amino acids of the PGEs enzyme chains.Acetylation leads non-selectively to the irreversible inhibition of isoenzymes (COX-1 and COX-2) (Cerella et al., 2010).

Conclusion
The anti-inflammatory action of acetylbergenin appears to be dependent on COX-2 inhibition.Furthermore, although the anti-inflammatory activity of acetylbergenin is a characteristic of nonsteroidal compounds, it causes little deleterious interference in the gastric mucosa.Based on these results, it was concluded that acetylbergenin has a potential anti-inflammatory activity.The addition of five acetyl groups, from the natural prototype, increased the anti-inflammatory properties of acetylbergenin compared to the original isocoumarin bergenin, as changes in the molecule were associated with changes in the antiinflammatory properties.Nevertheless, detailed investigations are still necessary in order to study the relationship between the structure and pharmacological activity of acetylbergenin, since its results were quite promising.
DEPT, and HMBC spectra, and by comparison of its NMR spectral data with those related to the literature(Ramaiah et al., 1979;Borges et al., 2011).

Figure 2 .
Figure2.Effect of the administration (p.o.) of acetylbergenin (6.8 mg/kg) and acetylsalicylic acid (ASA, 100 mg/kg) on dermatitis induced by croton oil in mice.Each column represents the mean ± SEM of ten animals.***p<0.00when compared to control; a p>0.05 when compared with ASA, Student-Newman-Keuls test ANOVA.
180 to 200 g), from the Evandro Chagas Animal Hospital of Belém, PA, Brazil, were used in this study.Male albino Wistar rats weighing between 180 and 200 g from the Multidisciplinary Center for Biological Research in the Laboratory Animal Science Area (Multidisciplinary Center for Biological Investigation in the Area of Science in Laboratory Animals) of the Faculty of Medical Sciences of Unicamp, Campinas, SP, were used in several experiments.