Gastroprotective effect of Byrsonima sericea DC leaf extract against ethanol-induced gastric injury and its possible mechanisms of action

Byrsonima sericea leaves are extensively used in folk medicine in Brazil against gastric disorders. This study investigated the chemical constituents of B. sericea leaf ethanolic extract (BSLE) and its potential gastroprotective activity, with its possible mechanism of the action using ethanol to induce gastric mucosal damage in mice. The phytochemical analysis was carried out to identify the active constituents present in the extract, and the HPLC analysis was performed for the identification of flavonoids. BSLE at oral doses of 125, 250 and 500 mg/kg markedly attenuated the ethanol-evoked gastric lesions by 53.2, 84.9 and 87.6 %, respectively. The BSLE (250 mg/kg) prevented the depletion of gastric mucus and gastric mucosal nonproteic-sulfhydryl groups, SOD and CAT, as well as the increase in the MDA content promoted by absolute ethanol. Moreover, the effect of BSLE against ethanol damage was found to be significantly reduced in mice pretreated with Capsazepine (i.p.), L-NAME (i.p.) or glibenclamide (i.p.), the respective blockers/inhibitors of TRPV1, NO synthase and K+ATP channel. The phytochemical investigation on BSLE revealed the presence of flavonoids rutin, isoquercitrin, kaempferol 3-O-rutinoside and quercetin, which are compounds well known for their antioxidant and gastroprotective properties. These results suggest that BSLE affords gastroprotection through multiple mechanisms, which may be helpful in the treatment of pathologies associated with gastric dysfunctions.


INTRODUCTION
Plants of the genus Byrsonima (Malpighiaceae) are widely distributed in various parts of Brazil where local people call them murici.Leaves and trunk barks from various species the Byrsonima are popularly employed in folk medicine to treat fever, gastrointestinal dysfunction (diarrhea and gastric ulcer), asthma, skin infections, and snakebites (Lira et al. 2008, Mendes et al. 1999).While chemical investigations on various Byrsonima species have shown the presence of several bioactive compounds like flavonoids, triterpenes and tannins (Mendes et Anais da Academia Brasileira de Ciências ( 2012) 84(1): 113-122 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 www.scielo.br/aabcPATRÍCIA A. RODRIGUES et al. al. 1999, Martínez-Vázquez et al. 1999), bioactivity studies have demonstrated the gastroprotective, healing and antidiarrheal activities of B. fagifolia (Lima et al. 2008), the antimutagenic activity of B. basiloba (Lira et al. 2008), and the mutagenic and gastroprotective effects of Byrsonima crassa (Cardoso et al. 2006, Sannomiya et al. 2007).
The presence of phenolic compounds possibly explains the gastroprotective effect of the extracts of Byrsonima species.Byrsonima sericea DC (Fig. 1) grows abundantly in the Northeastern states of Brazil, and it is traditionally used to treat gastrointestinal dysfunction.An antioxidant activity of the methanol extract from B. sericea has recently been described, using the DPPH assay (Boscolo et al. 2007).

CHEMICALS
Capsaicin, capsazepine, glibenclamide, diazoxide, L-arginine and L-NAME were purchased from Sigma Chemical Co.(St Louis, MO, USA).Absolute ethanol was obtained from Synth (Brazil).All other chemicals used were of analytical grade.Quercetin, quercetin 3-O-glycoside (isoquercetin), quercetin 3-O-rutinoside (rutin) and kaempferol 3-O-rutinoside isolated from the flowers and leaves of Dimophandra gardneriana, were used as standards.(#39.451) has been deposited at Prisco Bezerra Herbarium.Fresh leaves (1.24 kg) were macerated with 70% ethanol at room temperature for 7 days.The resultant ethanolic solution was filtered with filter paper using a buchner funnel under vacuum and evaporated to dryness at 50 °C in a rotary evaporator to yield 153 g of crude ethanolic extract.This extract was then submitted to a chromatographic treatment process using silica gel column being eluted with solvents hexane, ethyl acetate and ethanol.With the evaporation of these solvents, the three correspondent extracts weighed 1.1, 5.6 and 126.2 g respectively.Only the ethanolic extract (BSLE) was utilized in the present work.

PHYTOCHEMICAL ANALYSIS
BSLE was analyzed for the presence of phenols, tannins, leucoantocianidins, flavonoids, steroids, triterpenes and alkaloids by the qualitative chemical analysis (Matos 1997).To our knowledge, there were no scientific studies in literature on B. sericea extracts in relation to the regarding its gastrointestinal activity.Since antioxidants can afford gastroprotection both in clinical and experimental settings, the present study aimed at evaluating the B. sericea leaf extract (BSLE) for potential gastroprotection and its underlying mechanism of the action using the ethanol to induce gastric damage in mice as a model system.In addition, the phytochemical analysis of the extract was carried out to identify the chemical compounds that were probably responsible for protecting the plants.GASTROPROTECTIVE EFFECT OF Byrsonima sericea EXTRACT HPLC ANALYSIS The HPLC system (Waters 2690 Alliance) was connected to a Waters 486 tunable absorbance, column detector (Waters C18, 3.9 X 150mm X 4um).The analysis was run under isocratic conditions using the solvent mixture: 80:20 (ACN: H 2 O, pH 2.8 phosphoric acid), flow rate: 1 mL/min, 20 µL; and temperature: 21 o C. The concentrations of extract and standards were 1 mg/mL, the injection volume was 30 µL and the eluted was monitored at 350 nm.

ANIMALS
Female Swiss mice (20-25 g) obtained from the Central Animal House of this university were used.Experimental groups consisted of 8 animals per group.They were housed at 24 ± 2°C under a 12-h light/12-h dark cycle and had free access to standard pellet diet (Purina chow) and tap water.The animals were deprived of food for 15 h before the experimentation, but had free access to drinking water.The Institutional Ethics Committee on the Care and Use of Animals for experimentation approved the experimental protocols (Nº 07520831-8), and all experiments were performed in accordance with the guidelines of the National Institute of Health, Bethesda, USA.

GASTRIC DAMAGE INDUCED BY ETHANOL
Groups of mice (n=8), were pre-treated with the vehicle (0.5% of DMSO in saline, 10 mL/kg), N-acetylcysteine (NAC, 150 mg/kg, i.p.) or BSLE (125, 250 and 500 mg/kg, v.o.) one hour before the induction of gastric damage by oral administration of absolute ethanol (96%, 0.2 mL/animal) (Robert et al. 1979).After 30 min the animals were sacrificed and their stomachs excised, opened along the greater curvature and rinsed with saline (0.9%).The mucosal lesion area (mm 2 ) was measured by planimetry using a transparent grid (area: 1mm 2 ) placed on the glandular mucosal surface and expressed in percentage (%) in relation to the total area of the corpus.After the evaluation of the lesions induced by ethanol, the glandular parts of the stomachs were frozen and stored at −70 o C for further biochemical analyses.
DETERMINATION OF GASTRIC MUCUS Gastric mucus content was measured according to the methodology described by Corne et al. (1974).The gastric tissues were transferred to 0.1% Alcian Blue solution prepared in 16 mM sucrose and 50 mM sodium acetate (pH 5.8) and kept for 2 h at room temperature.The segments were then rinsed twice with 25 mM sucrose solution for 15 and 45 min, and the dye solution, together with the gastric mucus with 5 mM of magnesium chloride solution, was extracted for 2 h.The extract was then mixed with an equal volume of diethyl ether and centrifuged (1000g) for 10 min.The absorbance was determined at 598 nm.The amount of mucus was calculated using a standard curve of Alcian Blue.
ESTIMATION OF ANTI-OXIDANT PARAMETERS Gastric strips were cut into small pieces and then homogenized in ice-cold 50 mM Phosphate buffer pH 7.4 to obtain a 10% homogenate.The homogenate was then made into aliquots and used for the assessment of anti-oxidant parameters.
DETERMINATION OF NONPROTEIC SULFHYDRYL GROUPS (NP-SH) NP-SH was determined according to the method described by Sedlak and Lindsay (1968).Aliquots (2 mL) of 10% tissue homogenate were mixed with 1.6 mL of distilled water and 0.4 mL of trichloroacetic acid (TCA) 50% (w/v) and then centrifuged at 3000 g for 15 min.The supernatants PATRÍCIA A. RODRIGUES et al.
(2 mL) were then mixed with 4mL of tris buffer (40 mM, pH 8.9), and 5,5'-ditiobis (2-nitrobenzoic acid) (DTNB, 10 mM) was added.The absorbance was measured within 5 min after the addition of DTNB at 412 nm against a reagent blank with no homogenate.The absorbance values were extrapolated from a glutathione standard curve and expressed as µg/g of stomach tissue.

SUPEROXIDE DISMUTASE (SOD) ACTIVITY
The SOD activity was assessed through the measurement of the enzyme capacity for the photochemical inhibition of nitroblue-tetrazolium (NBT) (Beauchamp and Fridovich 1971).The reduction of NBT by O 2 − was utilized as the basis of assays for superoxide dismutase, which shows its presence by inhibiting the reduction of NBT and producing formazan that is absorbed at 560 nm.Aliquots of tissue homogenates were centrifuged 12,000 g for 20 min.In a dark room 40 μl of phosphate buffer or supernatants were added to glass test tubes containing 1mL of reaction mixture (Phosphate buffer 50 mM, EDTA 100 nM and L-methionine 19.5 μM pH 7.8).Then 150 μL of NBT 750 μM and 300 μL riboflavin 10 μM were added.After shacking, the tubes were exposed to light (20 Watt) for 15min.The absorbance was measured at 560 nm.The results were expressed in enzyme units, which represent the amount of SOD necessary to inhibit the NBT reduction by 50%.The enzymatic activity was expressed as U/µg of protein.
DETERMINATION OF CATALASE (CAT) ACTIVITY The CAT activity was measured by the method that employs hydrogen peroxide to generate H2O and O2 (Maehly and Chance 1954).The activity was measured by the degree of this reaction.In short, the supernatant (20 μL) was added to a quartz cuvette containing 980 μL of H 2 O 2 800 μM, EDTA 25 μM and tris buffer HCl (50 mM, pH 8.0).The change in absorbance was monitored at 240 nm over a 6-min period using a spectrophotometer.The CAT activity was expressed as U/µg of protein.

DETERMINATION PROTEIN CONTENT
The protein concentration was determined by the method of Bradford 1976, using bovine serum albumin as a standard.
DETERMINATION OF TOTAL THIOBARBITURIC ACID-REACTIVE

SUBSTANCES (TBARS)
Total thiobarbituric acid-reactive substances (TBARS) was determined according to the method of Ohkawa et al. (1979).Gastric strips were cut into small pieces and then homogenized in ice-cold phosphate buffer (50 mM pH 7.4) to give a 10% homogenate.The homogenates were transferred to test tubes and incubated in a water bath at 37ºC for 60 min.After this period, 35% of perchloric acid was added.The mixture was centrifuged at 15000 g for 10 min.Thiobarbituric acid at 0.6% was then added to the upper layer.The mixture was submitted to a water bath at 100ºC for 30 min, after which the absorbance was measured at 532 nm.The standard curve was obtained using several concentrations of MDA solutions, expressed in nmol/g of wet tissue.Mice (n=8/per group) were pretreated with vehicle (0.9% saline in a 0.5% of DMSO, 10 mL/kg), BSLE (250 mg/kg, v.o.) and L-arginine (600 mg/ kg, i.p.), alone or in their combinations with L-NAME (20 mg/kg, i.p.) prior to the induction of gastric damage with ethanol (0.2 ml of ethanol, 96%).While BSLE was administered 1h before, L-NAME and L-arginine were given 30 min prior to ethanol (Arrieta et al. 2003).

STATISTICAL ANALYSIS
The results are presented as the mean ± S.E.M. of 8 animals per group.Statistical analysis was carried out using the one way analysis of variance (ANOVA) followed by Student Newman Keul´s post hoc test for multiple comparisons.P-values less than 0.05 (P<0.05) were considered as indicative of statistical significance.

PHYTOCHEMICAL ANALYSIS
The phytochemical analysis of BSLE revealed the presence of flavones, flavonols, flavanones, xanthones and hydrolizable tannins.The amounts of phenolic compounds present in the extract were: total phenols 0.37 mg/g and flavonoids 0.17 mg/g.For the HPLC analysis of ethanol extract of B. sericea (Fig. 2), rutin, isoquercitrin, kaempferol 3-O-rutinoside and quercetin were used as standards.The structural characteristics of each identified peak in Fig. 2 was determined by comparing its correspondent UV spectrum with those UV spectra available at the HPLC computer library.For dried leaves, the percentage concentration for each flavonoid was evaluated in relation to a quercetin standard curve as: isoquercitrin 0.13%, quercetin 0.003%, rutin 0.007% and kaempferol 3-O-ritinoside 0.07%.
The oral administration of BSLE (125, 250 and 500 mg/kg) exhibited a protective effect against ethanol-induced gastric lesions in comparison to the vehicle group.The inhibition percentages for the respective doses employed were 53.2, 84.9 and 87.6 %.NAC, which was the positive control included for the study, also offered a significant protection (Table I).The animals that received only ethanol showed a significant decrease in gastric catalase (CAT), superoxide dismutase (SOD) and NP-SH levels, and a significant TBARS level increase (Table II).Animals pretreated with BSLE (250 mg/kg) effectively decreased TBARS levels almost to a similar extent to NAC (150 mg/kg).Animals treated with BSLE showed a significant enhanced activity of the antioxidant enzyme SOD.The NP-SH level in gastric mucosa of normal control mice (280.1 ± 12.7 µg/g) was significantly lowered by the ethanol treatment (102.3 ± 10.4 µg/g) (Table II).
EFFECT OF BSLE ON GASTRIC MUCUS The amount of gastric mucus was significantly (p < 0.01) less in the gastric tissues collected from ethanol-treated animals, as compared to non-damaged tissues from the control.The pretreatment of animals with BSLE greatly enhanced the gastric mucus when compared to animals with ethanol injury (Fig. 3).
EFFECT OF TRPV1 AND K + ATP-CHANNELS AND THE ROLE OF NITRIC OXIDE ON THE GASTROPROTECTIVE EFFECT OF BSLE In mice pretreated with vanilloid antagonist capsazepine, the gastroprotective effect of BSLE (250 mg/kg) and capsaicin (0.3 mg/kg, v.o.) was significantly reduced (Fig. 4A).These data indicate that the gastroprotective effect of BSLE is mediated by the activation of capsaicin-sensitive primary afferents.L-NAME (20 mg/kg) pretreatment significantly blocked the gastroprotection produced by BSLE and L-arginine (400 mg/kg, i.p.), suggesting the participation of NO (Fig. 4B) on the BSLE gastroprotection.The pretreatment with K+ATP channel blocker, glibenclamide (5 mg/kg, i.p.), also significantly reduced the gastroprotection produced by BSLE and diazoxide (3 mg/kg, i.p.) (Fig. 4C), which indicates a role for K + ATP channels in the gastroprotection.

DISCUSSION
The phytochemical analysis of BSLE indicated the presence of flavonoids and tannins, and the HPLC analysis confirmed the presence of rutin (quercetin 3-O-rutinoside), isoquercitrin (quercetin 3-O-glucoside), kaempferol 3-O-rutinoside and quercetin as the main secondary metabolites.Several flavonoids and their structurally related compounds have been shown to inhibit lipid peroxidation (Katsube et al. 2006).
The results of this study show that BSLE at doses of 125, 250 and 500 mg/kg affords a pronounced gastroprotection against ethanol that induced lesions, which is probably due to the presence of strong antioxidant flavonoids in the extract.The gastroprotective and antioxidant effects of quercetin and rutin are well known (La Casa et al. 2000, De la Lastra et al. 1994), and there are many studies showing the antiulcerogenic properties of flavonoids (Gonzalez and Di Stasi 2002).The role of reactive oxygen species in the pathogenesis of acute ethanol that induced gastric mucosal lesions and the effects of quercetin have been evaluated in a few studies.It has been confirmed that the quercetin treatment significantly inhibits the gastric erosions induced by ethanol (Gracioso et al. 2002).Galati et al. (2003) have also stated that there is a correlation between the antioxidant and the antiulcer activities of flavonoids.
The oxidative stress and impaired prostaglandin synthesis contribute to gastric mucosal damage in experimental models of gastric lesions induced by ethanol (Chattopadhyay et al. 2006).The administration of BSLE (250 mg/kg) to animals increased the levels of gastric NP-SH (GSH) and the amount of mucus.Baggio et al. (2007) demonstrated that the oral administration of the Maytenus ilicifoli leaf extract that is rich in flavonoids can increase the mucus production in the ethanol model of gastric damage in mice, which is in agreement with our data.Both gastric mucus and glutathione serve as protective agents against mucosal injury (Chen et al. 2005, Cnubben et al. 2001).The plant-derived natural antioxidants are extremely useful to combat the oxidative stress, and antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), in a preventive way, act as the first line of defense against the reactive oxygen species (Repetto and LLesuy 2002).Acute treatment with ethanol promotes oxidative stress, enhancing consequently the lipid peroxidation and malonaldehyde formation (Peskar et al. 2002).The present study clearly shows that BSLE inhibits lipid peroxidation and lowers TBARS formation, which indicates a cytoprotective function.
Several studies have reported that NO is involved in the preservation of mucous membrane integrity in experimental models of gastric ulceration (Ancha et al. 2003, Cho 2001, Kwiecień et al. 2002).In this study, L-arginine (400 mg/kg) and BSLE (250 mg/kg) induced gastroprotection, but it was reversed by L-NAME, a non-selective NOS inhibitor, suggesting that the gastroprotective effect of BSLE is mediated, in part, by NO.
It is known that potassium channels are involved in a variety of stomach physiological functions and that gastroprotection can be inhibited by several agents such as indomethacin, K+ATP blockers and glibenclamide (Peskar et al. 2002).Glibenclamide, a K + ATP channels blocker, significantly antagonized the protective effect of BSLE and diazoxide.These results support the hypothesis that the K + ATP channel opening is involved in the gastroprotective activity of BSLE.Since this protection is also sensitive to indomethacin, it is suggested that endogen prostaglandins activate the K + ATP channels and that this mechanism is responsible, in part, for the BSLE gastroprotective action (Campos et al. 2008).The results of this study indicate that BSLE has a cytoprotective role in the gastroprotection against gastric damage induced by ethanol, which is presumably mediated, in part, by the nitric oxide release and K + ATP channel opening.The extract does not seem to be toxic as consecutive daily oral administrations of BSLE (500 or 1000 mg/kg) over a period of 7 days to mice did not produce any signs of overt toxicity.In conclusion, these findings suggest that the ethanolic extract of B. sericea is a safe gastroprotectant to obliterate gastric dysfunction associated with pathologies.

Fig. 1 -
Fig. 1 -View of fruits and flowers and an adult individual of Byrsonima sericea (by Pablito Augusto Travassos Ferreira) .
Effect of B. sericea extract (BSLE) on gastric damage induced by absolute ethanol in mice.Effects of B. sericea leaf extract on the contents of CAT, SOD and MDA on gastric damage induced by absolute ethanol in mice.Treatment Treatment Data are mean ± S.E.M of 8 animals in each group.***p < 0.001 vs. Vehicle (ANOVA followed by Student Newman Keul's test).Data are mean ± S.E.M of 8 animals in each group.a p < 0.01 vs.No lesion; b p < 0.01 vs. Vehicle (ANOVA followed by Student Newman Keul's test).

Fig. 3 -
Fig. 3 -Effect of the B. sericea extract (BSLE) treatment on the amount of mucus after induction of gastric lesions by ethanol in mice.The results are expressed as mean ± S.E.M. * p < 0.01 vs. no lesion; ** p < 0.01 vs. vehicle (ANOVA followed by Student Newman Keul's test).

Fig. 4 -
Fig. 4 -(A) The involvement of TRPV1 channel in the gastroprotective effect of BSLE against ethanol that induced gastric damage in mice -(B) The involvement of nitric oxide in the gastroprotective effect of BSLE against ethanol that induced gastric damage in mice -(C) The role of K+ATP channels in the gastroprotective effect of BSLE against ethanol-induced gastric damage in mice.The results are expressed as mean ± S.E.M. a p < 0.001 vs vehicle; b p < 0.001 vs BSLE alone; c p < 0.001 vs L-arginine (L-arg) or diazoxide alone or capsaicin alone (ANOVA followed by Student Newman Keul's test).
PLANT MATERIAL AND EXTRACT PREPARATION B. sericea leaves were collected from the State University of Ceará campus, Ceará State, Brazil, in April 2008, after their identification by Dr. Afrânio G. Fernandes, botanist of the Federal University of Ceará.A voucher specimen When given alone, BSLE and capsaicin were administered 1h before the ethanol.Capsazepine was administered 30 min prior to ethanol.GASTROPROTECTIVE EFFECT OF Byrsonima sericea EXTRACT ROLE OF NITRIC OXIDE IN THE GASTROPROTECTIVE EFFECT OF BSLE

Table I Table II GASTROPROTECTIVE
EFFECT OF Byrsonima sericea EXTRACT