http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(4): 410–418 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.839713
ORIGINAL ARTICLE
Antiulcer activity of Muntingia calabura leaves involves the modulation of endogenous nitric oxide and nonprotein sulfhydryl compounds Tavamani Balan1, Mohd. Hijaz Mohd. Sani1, Velan Suppaiah2, Norhafizah Mohtarrudin3, Zarizal Suhaili4, Zuraini Ahmad1, and Zainul Amiruddin Zakaria1 Department of Biomedical Sciences, 2Department of Medicine, and 3Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Serdang, Malaysia, and 4Department of Animal Science, Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu, Malaysia Abstract
Keywords
Context: Muntingia calabura L. (Muntingiaceae) is a native plant species of the American continent and is widely cultivated in warm areas in Asia, including Malaysia. The plant is traditionally used to relieve pain from gastric ulcers. Objective: This study was designed to determine the antiulcer activity of a methanol extract of M. calabura leaves (MEMC) and the possible mechanisms of action involved. Materials and methods: An acute toxicity study was conducted using a single oral dose of 2000 mg/kg MEMC. The antiulcer activity of MEMC was evaluated in absolute ethanol- and indomethacin-induced gastric ulcer rat models. MEMC was administered orally (dose range 25–500 mg/kg) to rats fasted for 24 h. The animals were pretreated with NG-nitro-L-arginine methyl esters (L-NAME) or N-ethylmaleimide (NEM) prior to MEMC treatment to assess the possible involvement of endogenous nitric oxide (NO) and nonprotein sulfhydryl (NP-SH) compounds in the gastroprotective effect of MEMC. Results: As the administered dose did not cause toxicity in the rats, the oral median lethal dose (LD50) of MEMC was42000 mg/kg in rats. MEMC exerted significant (p50.001) gastroprotective activity in the ethanol- and indomethacin-induced ulcer models dose-dependently. Histological evaluation supported the observed antiulcer activity of MEMC. L-NAME and NEM pretreatment significantly (p50.05) reversed and abolished the gastroprotective effect of MEMC, respectively. Discussion and conclusion: The results obtained indicate that MEMC has significant antiulcer activity that might involve the participation of endogenous NO and NP-SH compounds. These findings provide new pharmacological information regarding the potential use of M. calabura.
Gastroprotection, leaves, methanol extract, Muntingia calabura, nitric oxide, peptic ulcers, sulfhydryl group
Introduction Gastric ulcer is one of the most critical diseases of the modern world; it is associated with alcohol consumption, wide usage of nonsteroidal anti-inflammatory drugs (NSAIDs), inappropriate diet and a stressful lifestyle. Approximately 20% of the world population suffers from peptic ulcers (Abdelwahab et al., 2011) and there is a marked increase in the risk of developing gastric attacks from exposure to many noxious agents and chemicals (Chaturvedi et al., 2007). Peptic ulcers may result from an imbalance between the protective factors of the gastric mucosa, which include sufficient blood flow, mucus and bicarbonate secretion, prostaglandin E2, sulfhy-
Correspondence: Associate Professor Dr Zainul Amiruddin Zakaria, Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel: +603 89472654. Fax: +603 89436178. E-mail: [email protected]; [email protected]
History Received 4 February 2013 Revised 28 July 2013 Accepted 27 August 2013 Published online 5 November 2013
dryl (SH) compounds, nitric oxide (NO) and antioxidant enzymes, and aggressive factors such as acid and pepsin secretion (Hoogerwerf & Pasricha, 2006). Although many synthetic drugs are available, the discovery of plant-derived natural resources remains a major area of interest for many researchers. Medicinal plants are among the most attractive sources of new drugs and the range of chemicals isolated from these plants have produced promising results for the treatment of gastric ulcers (Borelli & Izzo, 2000). This study focused on Muntingia calabura L. (Muntingiaceae), commonly known as Jamaica cherry or kerukup siam. Muntingia calabura is native to southern Mexico, the Caribbean, Central America and western South America and is widely cultivated in warm areas in Asia, including Malaysia (Chin, 1989). There are several documented medicinal uses for various parts of this tree in both Southeast Asia and tropical America (Kaneda et al., 1991; Nshimo et al., 1993). Muntingia calabura is commonly used traditionally in Peru to treat various ailments (Jensen, 1999; Morton, 1987); the leaves are either boiled or steeped in water,
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DOI: 10.3109/13880209.2013.839713
which is consumed to obtain relief from gastric ulcers or to reduce prostate gland swelling (Jensen, 1999; Morton, 1987; Verheij & Coronel, 1992). In addition, its bark is used to reduce swelling in the lower extremities (Zakaria et al., 2007a). Several pharmacological assays have determined that M. calabura leaves possess antitumor (Kaneda et al., 1991; Su et al., 2003), antinociception (Zakaria et al., 2006a, 2007a,b,c), anti-inflammatory, antipyretic (Zakaria et al., 2007c), antibacterial (Zakaria et al., 2006b), antiproliferative and antioxidant (Zakaria et al., 2011) activities. Phytochemical screening of M. calabura leaves identified flavonoids, saponins, tannins, triterpenes and steroids (Zakaria et al., 2007c); phytochemical analysis of the methanol extract of M. calabura leaves (MEMC) detected flavonoids, saponins and tannins (Zakaria et al., 2011). Our recent study also reported the significant antinociception activity of MEMC (Mohd. Sani et al., 2012). Despite the claims regarding the medicinal uses of M. calabura, there has been no attempt to evaluate its gastroprotective effect scientifically. Therefore, this study aimed to investigate the antiulcer effect of MEMC using different ulcerogenic models.
Materials and methods Drugs and chemicals Absolute ethanol was purchased from Fisher Scientific (Pittsburgh, PA); the NSAID indomethacin (99%), ranitidine, carbenoxolone, L-arginine, NG-nitro-L-arginine methyl esters (L-NAME) and N-ethylmaleimide (NEM) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents used for the experiments were of analytical grade. All drugs and reagents were prepared immediately before use. Plant collection Muntingia calabura leaves were collected from their natural habitat in Shah Alam, Selangor, Malaysia, between May and August 2010. The plant was re-identified by a botanist from the Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), Serdang, Selangor, based on a voucher specimen (SK 964/04) deposited earlier at the Herbarium of Laboratory of Natural Products, IBS, UPM. Preparation of MEMC Methanol was used as the extraction solvent. The procedure was carried out according to Zakaria et al. (2011). Matured leaves (500 g) were air-dried at room temperature (27 2 C) for 1–2 weeks before they were ground into fine powder. The powder was soaked in methanol at a ratio of 1:20 (w/v) for 72 h. The mixture was filtered using a filter funnel, cotton and Whatman No. 1 filter paper. The residue underwent the same filtration procedure two times more. The filtrate collected from each extraction was pooled and evaporated in a rotary evaporator at 40 C under reduced pressure. Animals All experiments were performed with male Sprague–Dawley rats (180–200 g; 8–10 weeks old) obtained from the Animal Unit, Faculty of Medicine and Health Sciences, UPM.
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The animals were housed in polypropylene cages containing wood shavings, fed a standard pellet diet with free access to water and housed at room temperature (27 2 C; 70–80% humidity; 12 h light/dark cycle) in the Animal Holding Unit, UPM. The rats were fasted prior to all assays and standard drugs and MEMC were administered orally (p.o.) by gavage with a vehicle (8% Tween 80, 10 mL/kg). The Animal Care and Use Committee of the Faculty of Medicine and Health Sciences, UPM approved the use of the animals in this study (Approval No: UPM/FPSK/PADS/BR-UUH/ 00474). Acute toxicity study Acute toxicity studies were performed as described in ‘‘Guideline for Testing of Chemicals – Acute Oral Toxicity – Fixed Dose Procedure’’ (OECD, 2002). Rats were fasted 24 h prior to MEMC administration. The treated group received a single dose of 2000 mg/kg MEMC, while one group each received either vehicle or distilled water (10 mL/kg) by gavage. Subsequently, the animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 h and daily thereafter for 14 d. Food and water were provided throughout the experiment. The mortality, body weight and behavioral screening were recorded daily for 14 d after treatment. The rats that survived were sacrificed and macroscopic analysis and the weight of vital organs such as the liver, kidney, heart, spleen and lung were recorded. These organs were fixed in 10% formalin for histological examination. Pharmacological assays Ethanol-induced gastric ulcers The experiment was performed according to the method of Noor et al. (2006) with some modifications. After 48 h fasting in total, MEMC (25, 50, 100, 250 or 500 mg/kg), ranitidine (100 mg/kg), or vehicle were administered p.o. to the rats (n ¼ 6). All rats received absolute ethanol (5 mL/kg) after 1 h of treatment to induce gastric ulcers. The animals were anesthetized using diethyl ether and euthanized by cervical dislocation 1 h after ulcer induction. The stomachs were removed and opened along the greater curvature to determine the lesion damage. Every stomach was photographed and the ulcer area (UA) was measured by superimposing transparent grid paper with minimum of squares equaling 1 mm2 (Bodhankar et al., 2006). The UA in millimeter squared was determined for each stomach. Protection percentage was calculated using the following formula: Protection ð%Þ ¼
ðUA control UA pretreated groupÞ 100% ðUA controlÞ
Indomethacin-induced gastric ulcers The experiment was carried out as described by Nwafor et al. (2000) with modifications. Indomethacin (100 mg/kg) was administered p.o. to 48 h fasted rats (n ¼ 6) to induce gastric ulcers. MEMC (100, 250 and 500 mg/kg), ranitidine (100 mg/kg) or vehicle were administered p.o. 1 h before ulcer induction. The animals were sacrificed 6 h after ulcer
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Histological observation of the stomach tissue from rats in the vehicle group in the ethanol-induced ulcer model revealed obvious lesions and severe damage to the gastric mucosa; 60
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The results were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test or the Newman–Keuls test and expressed as mean SEM. Results were considered significant when p50.05.
Effect of MEMC on ethanol-induced gastric ulcers
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Histopathological evaluation
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The involvement of SH compounds in the gastroprotective effects of MEMC against ethanol-induced ulcers was determined using the method of Andreo et al. (2006). Rats were randomly assigned six per group and fasted for 24 h before i.p. pretreatment with NEM, which is a SH blocker or saline. Animals received vehicle, MEMC (100, 250 and 500 mg/kg), or carbenoxolone (100 mg/kg) after 30 min. Sixty minutes later, gastric damage was induced by oral administration of absolute ethanol (5 mL/kg). Animals were anesthetized using diethyl ether and sacrificed by cervical dislocation, 1 h later, the stomachs were excised and the UA determined as described above.
Indomethacin (100 mg/kg) produced gastric mucosal ulceration in the rats. As shown in Figure 2, pretreatment with 100, 250 and 500 mg/kg MEMC inhibited ulcer formation dosedependently, with protection percentage of 47.8, 51.8 and 68.9%, respectively. MEMC led to significant (p50.001) reduction in the total UA, which were 22.17, 20.5 and 13.2 mm2, respectively, when compared to the vehicle group. In contrast, 100 mg/kg ranitidine exerted 78% protection, with the mean UA being 9.3 mm2.
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Ethanol-induced gastric mucosal lesion in NEM pretreated rats
Indomethacin-induced gastric ulcers
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In order to evaluate the role of endogenous NO in the gastroprotective effect of MEMC, a modified method of Andreo et al. (2006) was performed. Six male rats were assigned per group and were fasted for 24 h. They were pretreated intraperitoneally (i.p.) with saline or L-NAME (70 mg/kg), and vehicle, carbenoxolone (100 mg/kg), or MEMC (100, 250 and 500 mg/kg) were administered p.o. 30 min thereafter. Sixty minutes later, all groups received absolute ethanol (5 mL/kg) by gavage to induce gastric ulcers. L-Arginine (200 mg/kg) was administered 30 min after saline or L-NAME treatment and 30 min before ethanol administration. The animals were sacrificed 1 h after ethanol administration by diethyl ether anesthesia and cervical dislocation. The stomach was removed and gastric damage was determined as described above.
Macroscopic analysis of the stomach removed from ethanolinduced animals pretreated with the vehicle (negative control group) revealed complete ulceration. In contrast, pretreatment with 25, 50, 100, 250 and 500 mg/kg MEMC led to significant (p50.001) and dose-dependent reduction of gastric lesions, with 63.2, 79.3, 96.7, 94.3 and 95% protection (Figure 1), respectively, when compared to the vehicle group. All but the 25 mg/kg MEMC dose resulted in smaller UA, which were 10.3, 1.7, 2.8 and 2.5 mm2, respectively, demonstrating a better protective effect than the ranitidine group, in which there was 70% reduction with a mean UA of 14.8 mm2.
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Ethanol-induced gastric mucosal lesion in L -NAME pretreated rats
Ethanol-induced gastric ulcers
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Gastric tissue samples were collected and fixed in 10% formalin. The specimens were embedded in paraffin and then sectioned (3–5 mm) and stained with hematoxylin and eosin. The sections were viewed and analyzed using light microscopy and photographed. This method was applied for both induced gastric ulcer models.
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Histopathology evaluation
There was no mortality or behavioral pattern alteration. Animals showed no changes in food and water intake behavior. The relative weight of the vital organs and the respective histological examinations revealed no signs of toxicity (data not shown). The single oral administration of 2000 mg/kg MEMC did not produce any symptom or sign of toxicity in the treated animals after 14 days. Based on this finding, we concluded that the LD50 of MEMC is greater than 2000 mg/kg body weight.
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induction by diethyl ether anesthesia and cervical dislocation. The stomachs were removed and opened along the greater curvature to determine lesion damage. Macroscopic analysis was performed, the UA determined and protection percentage was calculated as described earlier.
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Results Acute toxicity study All animals in the treatment and control groups had increased body weight at weeks 1 and 2 as compared with day 0.
Figure 1. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg), MEMC 25, 50, 100, 250 and 500 mg/kg on absolute ethanol-induced ulcer. The ulcerated area (millimeter squared) was expressed as mean SEM for six animals. One-way ANOVA was done, followed by Dunnett’s post hoc test, ***p50.001 versus vehicle.
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there was severe hemorrhage, edema, necrosis and erosion (Figure 3). Interestingly, the mucosal architecture in the stomach tissue of the rats pretreated with 100, 250 and 500 mg/kg MEMC was almost normal, albeit with mild edema (Table 1). Overall, the groups that received MEMC had comparatively better gastroprotection, as indicated by the reduction of the UA on the stomach wall. In the ranitidine
Ulcer area (mm2)
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Figure 2. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg), MEMC 100, 250 and 500 mg/kg on indomethacin-induced ulceration. The ulcerated area (millimeter squared) was expressed as mean SEM for six animals. One-way ANOVA was done, followed by Dunnett’s post hoc test, ***p50.001 versus vehicle.
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pretreated rats, there was moderate protection of the mucosa, which exhibited moderate edema and mild hemorrhage. Effect of MEMC on indomethacin-induced gastric ulcers In the indomethacin-induced ulcer model, histological evaluation of stomach tissue from the vehicle group animals revealed severe edema and moderate necrosis that may have led to mild erosion (Figure 4). Conversely, the stomach tissue of rats pretreated with 100, 250 and 500 mg/kg MEMC had almost normal mucosal architecture with mild edema (Table 1). Overall, the groups that received MEMC had gastroprotection when compared to the vehicle group, as the macroscopic analysis revealed a reduction in UA on the stomach wall in the MEMC-treated rats. Rats pretreated with ranitidine also had similar mucosal protection when compared to the vehicle group. Ethanol-induced gastric lesions in rats pretreated with L -NAME In groups pretreated with saline, animals that received MEMC prior to ethanol induction showed a significant reduction (p50.001) of the UA as compared to the vehicle group (Figure 5). The group treated with the standard antiulcer drug carbenoxolone (100 mg/kg) or L-arginine (200 mg/kg) also had significantly reduced UA (p50.001) as compared to the vehicle group (Figure 5). However, pretreatment with
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Figure 3. Histological evaluation of antiulcer activity of MEMC against ethanol-induced gastric lesions in rats. (A) Stomach of a negative control rat; (B) stomach of a rat pretreated with 100 mg/kg ranitidine; (C) stomach of a rat pretreated with 100 mg/kg MEMC; (D) stomach of a rat pretreated with 250 mg/kg MEMC; (E) stomach of a rat pretreated with 500 mg/kg MEMC. Respective histopathological sections are shown together; (Ai) stomach of the control animal showing a severe effect on mucosa with hemorrhagic erosion, edema, necrosis and erosion; (Bi) stomach of 100 mg/ kg ranitidine-treated animals showing moderate hemorrhage and edema; (Ci) stomach of 100 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Di) stomach of 250 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Ei) stomach of 500 mg/kg MEMC-treated animals show almost normal mucosa. H, hemorrhage; ED, edema; ER, erosion; N, normal architecture.
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Table 1. Histopathological evaluation of MEMC on ethanol-induced gastric lesions in rats. Ulcer model
Ethanol-induced Indomethacin-induced
Pretreatment group
Edema
Hemorrhage
Necrosis
Erosion
Vehicle 100 mg/kg ranitidine 100 mg/kg MEMC 250 mg/kg MEMC 500 mg/kg MEMC Vehicle 100 mg/kg ranitidine 100 mg/kg MEMC 250 mg/kg MEMC 500 mg/kg MEMC
þþþ þþ þ þ þ þþþ þ þ þ þ
þþþ þþ þ
þþ þþ
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() normal; (þ) mild effect; (þþ) moderate effect; (þþþ) severe effect. (A)
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Figure 4. Histological evaluation of antiulcer activity of MEMC against indomethacin-induced gastric lesions in rats. (A) Stomach of a negative control rat; (B) stomach of a rat pretreated with 100 mg/kg ranitidine; (C) stomach of a rat pretreated with 100 mg/kg MEMC; (D) stomach of a rat pretreated with 250 mg/kg MEMC; (E) stomach of a rat pretreated with 500 mg/kg MEMC. Respective histopathological sections are shown together: (Ai) stomach of the control animal showing a severe effect on mucosa with necrosis and erosion; (Bi) stomach of 100 mg/kg ranitidine-treated animals showing moderate edema; (Ci) stomach of 100 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Di) stomach of 250 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Ei) stomach of 500 mg/kg MEMC-treated animals showing almost normal mucosa. H, hemorrhage; ED, edema; ER, erosion; N, normal architecture. L-NAME
(70 mg/kg) prior to ethanol administration aggravated the gastric lesions significantly (p50.001) as compared to the vehicle in the saline group. L-NAME pretreatment also worsened gastric ulcers significantly in all treated groups (p50.05), as compared with their saline pretreated counterparts. Treatment with L-arginine (200 mg/kg) significantly (p50.01) reversed the deleterious effect of L-NAME on the gastric mucosa. Therefore, these findings indicate NO participation in the gastroprotection exerted by MEMC.
(p50.001) of gastric lesions as compared to the vehicle group (Figure 6). Pretreatment with NEM (10 mg/kg) markedly increased gastric lesions (p50.001) when compared to that of the vehicle in the saline group. There was a significant reduction of UA in animals treated with MEMC in the saline group (p50.01, 250 and 500 mg/kg; p50.05, 100 mg/kg) when compared with the NEM pretreated animals. This indicates the participation of endogenous SH compounds in the gastroprotective effect demonstrated by MEMC.
Ethanol-induced gastric lesions in rats pretreated with NEM
Discussion
Animals that received carbenoxolone and MEMC in the groups pretreated with saline exhibited significant reduction
Gastric ulcer is a common disease that occurs in the human population worldwide. Gastric ulcers arise due to an
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Figure 5. Effect of vehicle (Tween 80, 8%, p.o.), MEMC (100, 250 and 500 mg/kg, p.o), carbenoxolone (CBX, 100 mg/kg, p.o.) and L-arginine (L-ARG, 200 mg/kg, i.p.) on gastric lesions induced by absolute ethanol in rats pretreated with saline i.p. and/or L-NAME (70 mg/kg, i.p.). Each column represents the mean SEM of six animals. Oneway ANOVA was done, followed by Newman–Keuls test, ***p50.001 versus saline vehicle; yyyp50.001 versus vehicle L-NAME; #p50.05, ##p50.01 saline pretreatment versus L-NAME pretreatment. 100 *** Ulcer area (mm2)
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Figure 6. Effect of vehicle (Tween 80, 8%, p.o.), MEMC (100, 250 and 500 mg/kg, p.o.) and carbenoxolone (CBX, 100 mg/kg, p.o.) on gastric lesions induced by absolute ethanol in rats pretreated with saline i.p. and/ or NEM (10 mg/kg, i.p.). Each column represents the mean SEM of six animals. One-way ANOVA was done, followed by Newman–Keuls test, ***p50.001 versus saline vehicle; yyyp50.001 versus vehicle NEM; #p50.05, ##p50.01 saline pretreatment versus NEM pretreatment.
imbalance in mucosal aggressive and defensive factors such as acid–pepsin secretion, parietal cells, the mucosal barrier, mucus secretion, blood flow, cellular regeneration and endogenous protective agents (prostaglandins and epidermal growth factor) (Gilman et al., 2001). Gastric ulcers are commonly associated with damage to the mucosal layer of the stomach that occurs due to excess generation of exogenous and endogenous active oxygen and free radicals. Some of the main causes of gastric ulcers include chronic use of alcohol and anti-inflammatory drugs, stress and Helicobacter pylori infection (Barocelli et al., 1997).
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In the acute toxicity study, 2000 mg/kg MEMC led to no signs of toxicity or mortality in the treated rats. In accordance with the OECD guidelines, the LD50 of MEMC is 42000 mg/kg. Thus, we may conclude that administration of M. calabura extract in a single high dose is nontoxic and safe for use in oral formulations. The highest dose used for the antiulcer assays (500 mg/kg) was based on the toxicity study, being derived from a 4-fold reduction of the dose used in that toxicity study. At 25–500 mg/kg, MEMC had dose-dependent gastroprotective activity in both gastric ulcer models. Ethanol-induced ulcer is a classic ulcer model that is commonly used to evaluate the antiulcerogenic activity of drugs. The resulting gastric lesions appear along the glandular stomach as multiple hemorrhagic red bands of different sizes. The mechanism of ethanol-induced gastric lesions varies. Kinoshita et al. (1995) reported that the gastric mucosal lesions caused by ethanol might depress the gastric defense mechanism. This may include the depletion of gastric mucus content, mucosal cell injury and damaged mucosal blood flow (Galani et al., 2010). Ethanol damage to the gastrointestinal mucosa begins with microvascular injury, including edema, epithelial lifting and disruption of the vascular endothelium that leads to increased vascular permeability, where bicarbonate secretion is reduced while mucus production is increased, causing increased sodium and potassium flow, pepsin secretion and the loss of hydrogen ions and histamine into the lumen (Szabo, 1987). According to Kumar et al. (2011), ethanol acts by rapidly penetrating the gastric mucosa, damaging the cell and plasma membranes. This may lead to increase intracellular membrane permeability to sodium and water, causing the massive accumulation of calcium that clarifies the pathogenesis of gastric mucosal injury. Ethanol was also reported to enhance the production of reactive oxygen species (ROS), including superoxide anion and hydroxyl radicals (Bagchi et al., 1998), apart from other mechanisms such as damage to the capillary endothelium, release of arachidonate metabolites, leukotriene C4/D4 and platelet-activating factor (PAF) (Rachchh & Jain, 2008). In this study, the significant gastroprotective effect exerted by MEMC may have been due to a decrease in gastric motility, as flattening of the mucosal folds was observed. Changes in gastric motility may also contribute to the development and prevention of gastric lesions. Abdelwahab et al. (2011) reported that relaxation of the circular muscles might contribute to gastric mucosa protection via flattening of the folds, increasing the mucosal area exposure to necrotizing agents while reducing the volume of gastric irritants on the rugal crest. Contraction of the circular muscles of the rat fundus strip caused by ethanol intake may lead to compression of the mucosa at the crest of the mucosal folds, causing ulceration and necrosis (Jamal et al., 2006). In addition, the protection conferred by MEMC can be linked to its antioxidant properties. MEMC has been reported to contain flavonoids (Zakaria et al., 2011) that may act as antioxidant agents. Flavonoids can activate the mucosal defense system by stimulating gastric mucus secretion and scavenging the ROS and free radicals produced by ethanol (Abdelwahab et al., 2011). An endogenous antioxidant defense mechanism may constantly remove the ROS produced continuously during normal metabolism (Lemos et al., 2012). This indicates that
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the modulation of hydroxyl radical production from superoxide radicals or the scavenger activity demonstrated by MEMC may be due to its antioxidant properties. NSAIDs induce gastric ulceration via their ability to suppress prostaglandin synthesis (Wallace, 2001) that inhibits cyclooxygenase-1 (COX-1). Indomethacin is a preferential COX-1 inhibitor. COX-1 is constitutively expressed in large amounts in the gastrointestinal tract and has been reported to maintain mucosal integrity through the continuous generation of prostaglandins (Hatler et al., 2001). Prostaglandin suppression may stimulate bicarbonate and mucus secretion, maintaining mucosal blood flow and regulating mucosal cell turnover and repair that leads to increase susceptibility to mucosal injury and gastroduodenal ulceration (Hayllar & Bjarnason, 1995). Moreover, NSAIDs are also reported to be involved in the generation of ROS that initiate lipid peroxidation, leukocyte infiltration and apoptosis induction (Beck et al., 2000). Significant inhibition of ulceration was observed in the MEMC pretreated indomethacin-induced ulcer model. The ability of MEMC to reduce indomethacininduced gastric ulceration suggests the possible involvement of prostaglandins or mucus in the MEMC antiulcer effect, where MEMC can regulate prostaglandin synthesis or stimulate mucus or bicarbonate secretion. As MEMC exerted significant gastroprotective effects in both ethanol and indomethacin-induced ulcer models, we decided to investigate the possible pharmacological mechanism involved. For this purpose, ethanol-induced gastric mucosal lesions in rats pretreated with L-NAME and NEM were used to determine the involvement of NO and nonprotein SH (NP-SH) compounds in the protective effect of MEMC. NO is considered one of the most important defensive endogenous agents in the gastric mucosa (Brzozowski et al., 2006). It is synthesized by NO synthase (NOS) from L-arginine (Takahashi et al., 1998). NO is involved in the modulation of gastric mucosal integrity and is important in the regulation of acid and alkaline secretion, mucus secretion and gastric mucosal blood flow (Chandranath et al., 2002). In ethanol-induced gastric injury, the endothelium is the main and most preferred target of ethanol damage (de-Faria et al., 2012). This is because systemic administration of L-NAME that inhibits NO may cause an increase in systemic blood pressure and the vasoconstriction of several vascular beds that damage the gastric mucosa and its endothelium (Sikiric et al., 1997). Carbenoxolone was used as the standard model drug because other researchers have reported the partial participation of prostaglandins, SH compounds and NO in the gastroprotective mechanism conferred by carbenoxolone (Navarete et al., 2005; Cha´vez-Pin˜a et al., 2011). L-NAME pretreatment reversed the gastroprotective effects exerted by MEMC against ethanol-induced damage. These findings indicate the possible participation of the NOS pathway in the gastroprotection exerted by MEMC, supporting the premise of the free radical scavenging effect of this extract. The possible involvement of endogenous NP-SH compounds in the gastroprotective effect of MEMC was also investigated. The synthesis of mucus is one of the important functions in gastroprotection, as mucus aids in strengthening the mucosal barrier against harmful agents. The continuous
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adherence of the mucus layer is a barrier to luminal pepsin and creates a stable, undisturbed layer to support the surface neutralization of acid, protecting the underlying mucosa from proteolytic digestion (Allen & Flemstrom, 2005). Disulfide bridges connect the mucus subunits; if reduced, the mucus may become water-soluble (Avila et al., 2005). Szabo and Vattay (1990) reported that the endogenous NP-SH compound is a key compound in the mucosal protection against ethanolinduced gastric injury. In addition to NO, SH compounds are important for maintenance of the gastric mucosa because they confer protection against pro-oxidant agents (de-Faria et al., 2012), binding the free radicals caused by the ingestion of noxious agents. As presented in Figure 6, NEM increased the UA in ethanol-induced animals. The gastroprotection exerted by MEMC was abolished by co-administration of NEM, suggesting that the gastroprotective effect showed by MEMC may depend on the participation of mucosal SH compound levels. The pathogenesis of gastric ulcers involves oxidative stress; antioxidants play a very important role in protecting the gastric mucosa against necrotic agents (Trivedi & Rawal, 2001). The ingestion of noxious agents may lead to the formation of free radicals and ROS that contribute to gastric mucosal damage. Antioxidants act as radical scavengers that may protect the gastric mucosa from oxidative damage. MEMC was reported to contain high phenolic content that exhibited high antioxidant activity when tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and superoxide scavenging assays (Zakaria et al., 2011). Therefore, it is suggested that the antioxidant and free radical scavenging properties of MEMC may have contributed to the observed gastroprotection ability. In terms of phytochemical constituents, MEMC was reported to contain flavonoids, tannins and saponins (Zakaria et al., 2011). A number of phenolic substances are known to act as antioxidants, such as flavonoids, coumarins, tannins, xanthines and more recently, procyanidins, which can scavenge radicals in a dose-dependent manner (Czinner et al., 2001). In addition to free radical scavenging, several mechanisms may be involved in the antioxidant activity of flavonoids, including oxidant enzymes inhibition, chelating of transition metal ion and regeneration of a-tocopherol from a-tocopheroxyl radicals (Repetto & Llesuy, 2002). Flavonoids promote formation of the gastric mucosa, inhibit pepsinogen production, decrease acid mucosal secretion and reduce ulcerogenic lesions (La Casa et al., 2000). Tannins may prevent ulcer development by promoting protein precipitation on the ulcer site that forms a protective pellicle. This may help to prevent the absorption of toxic substances and withstand the effects of proteolytic enzymes (John & Onabanjo, 1990; Nwafor et al., 1996). Therefore, the presence of these phytochemical substances may protect against gastric lesions by enhancing the antioxidant activity of the extract.
Conclusions This study demonstrated that MEMC has significant antiulcer activity that involves the participation of endogenous NO and SH compounds. This gastroprotective activity could have been due to the antioxidant activity and the presence
DOI: 10.3109/13880209.2013.839713
of flavonoids, tannins and phenolic compounds in the extract. Further studies are in progress to identify the bioactive compound(s) that may be present, which could be responsible for the antiulcer properties of M. calabura.
Acknowledgements The authors thanked the Faculty of Medicine and Health Sciences, UPM, Malaysia for providing the facilities and supporting this study.
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Declaration of interest The authors report no declarations of interest. This research was supported by the Research University Grant Scheme (Reference no. 04-02-10-0925RU) awarded by the UPM
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ORIGINAL ARTICLE
Antiulcer activity of Muntingia calabura leaves involves the modulation of endogenous nitric oxide and nonprotein sulfhydryl compounds Tavamani Balan1, Mohd. Hijaz Mohd. Sani1, Velan Suppaiah2, Norhafizah Mohtarrudin3, Zarizal Suhaili4, Zuraini Ahmad1, and Zainul Amiruddin Zakaria1 Department of Biomedical Sciences, 2Department of Medicine, and 3Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Serdang, Malaysia, and 4Department of Animal Science, Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu, Malaysia Abstract
Keywords
Context: Muntingia calabura L. (Muntingiaceae) is a native plant species of the American continent and is widely cultivated in warm areas in Asia, including Malaysia. The plant is traditionally used to relieve pain from gastric ulcers. Objective: This study was designed to determine the antiulcer activity of a methanol extract of M. calabura leaves (MEMC) and the possible mechanisms of action involved. Materials and methods: An acute toxicity study was conducted using a single oral dose of 2000 mg/kg MEMC. The antiulcer activity of MEMC was evaluated in absolute ethanol- and indomethacin-induced gastric ulcer rat models. MEMC was administered orally (dose range 25–500 mg/kg) to rats fasted for 24 h. The animals were pretreated with NG-nitro-L-arginine methyl esters (L-NAME) or N-ethylmaleimide (NEM) prior to MEMC treatment to assess the possible involvement of endogenous nitric oxide (NO) and nonprotein sulfhydryl (NP-SH) compounds in the gastroprotective effect of MEMC. Results: As the administered dose did not cause toxicity in the rats, the oral median lethal dose (LD50) of MEMC was42000 mg/kg in rats. MEMC exerted significant (p50.001) gastroprotective activity in the ethanol- and indomethacin-induced ulcer models dose-dependently. Histological evaluation supported the observed antiulcer activity of MEMC. L-NAME and NEM pretreatment significantly (p50.05) reversed and abolished the gastroprotective effect of MEMC, respectively. Discussion and conclusion: The results obtained indicate that MEMC has significant antiulcer activity that might involve the participation of endogenous NO and NP-SH compounds. These findings provide new pharmacological information regarding the potential use of M. calabura.
Gastroprotection, leaves, methanol extract, Muntingia calabura, nitric oxide, peptic ulcers, sulfhydryl group
Introduction Gastric ulcer is one of the most critical diseases of the modern world; it is associated with alcohol consumption, wide usage of nonsteroidal anti-inflammatory drugs (NSAIDs), inappropriate diet and a stressful lifestyle. Approximately 20% of the world population suffers from peptic ulcers (Abdelwahab et al., 2011) and there is a marked increase in the risk of developing gastric attacks from exposure to many noxious agents and chemicals (Chaturvedi et al., 2007). Peptic ulcers may result from an imbalance between the protective factors of the gastric mucosa, which include sufficient blood flow, mucus and bicarbonate secretion, prostaglandin E2, sulfhy-
Correspondence: Associate Professor Dr Zainul Amiruddin Zakaria, Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel: +603 89472654. Fax: +603 89436178. E-mail: [email protected]; [email protected]
History Received 4 February 2013 Revised 28 July 2013 Accepted 27 August 2013 Published online 5 November 2013
dryl (SH) compounds, nitric oxide (NO) and antioxidant enzymes, and aggressive factors such as acid and pepsin secretion (Hoogerwerf & Pasricha, 2006). Although many synthetic drugs are available, the discovery of plant-derived natural resources remains a major area of interest for many researchers. Medicinal plants are among the most attractive sources of new drugs and the range of chemicals isolated from these plants have produced promising results for the treatment of gastric ulcers (Borelli & Izzo, 2000). This study focused on Muntingia calabura L. (Muntingiaceae), commonly known as Jamaica cherry or kerukup siam. Muntingia calabura is native to southern Mexico, the Caribbean, Central America and western South America and is widely cultivated in warm areas in Asia, including Malaysia (Chin, 1989). There are several documented medicinal uses for various parts of this tree in both Southeast Asia and tropical America (Kaneda et al., 1991; Nshimo et al., 1993). Muntingia calabura is commonly used traditionally in Peru to treat various ailments (Jensen, 1999; Morton, 1987); the leaves are either boiled or steeped in water,
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DOI: 10.3109/13880209.2013.839713
which is consumed to obtain relief from gastric ulcers or to reduce prostate gland swelling (Jensen, 1999; Morton, 1987; Verheij & Coronel, 1992). In addition, its bark is used to reduce swelling in the lower extremities (Zakaria et al., 2007a). Several pharmacological assays have determined that M. calabura leaves possess antitumor (Kaneda et al., 1991; Su et al., 2003), antinociception (Zakaria et al., 2006a, 2007a,b,c), anti-inflammatory, antipyretic (Zakaria et al., 2007c), antibacterial (Zakaria et al., 2006b), antiproliferative and antioxidant (Zakaria et al., 2011) activities. Phytochemical screening of M. calabura leaves identified flavonoids, saponins, tannins, triterpenes and steroids (Zakaria et al., 2007c); phytochemical analysis of the methanol extract of M. calabura leaves (MEMC) detected flavonoids, saponins and tannins (Zakaria et al., 2011). Our recent study also reported the significant antinociception activity of MEMC (Mohd. Sani et al., 2012). Despite the claims regarding the medicinal uses of M. calabura, there has been no attempt to evaluate its gastroprotective effect scientifically. Therefore, this study aimed to investigate the antiulcer effect of MEMC using different ulcerogenic models.
Materials and methods Drugs and chemicals Absolute ethanol was purchased from Fisher Scientific (Pittsburgh, PA); the NSAID indomethacin (99%), ranitidine, carbenoxolone, L-arginine, NG-nitro-L-arginine methyl esters (L-NAME) and N-ethylmaleimide (NEM) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents used for the experiments were of analytical grade. All drugs and reagents were prepared immediately before use. Plant collection Muntingia calabura leaves were collected from their natural habitat in Shah Alam, Selangor, Malaysia, between May and August 2010. The plant was re-identified by a botanist from the Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), Serdang, Selangor, based on a voucher specimen (SK 964/04) deposited earlier at the Herbarium of Laboratory of Natural Products, IBS, UPM. Preparation of MEMC Methanol was used as the extraction solvent. The procedure was carried out according to Zakaria et al. (2011). Matured leaves (500 g) were air-dried at room temperature (27 2 C) for 1–2 weeks before they were ground into fine powder. The powder was soaked in methanol at a ratio of 1:20 (w/v) for 72 h. The mixture was filtered using a filter funnel, cotton and Whatman No. 1 filter paper. The residue underwent the same filtration procedure two times more. The filtrate collected from each extraction was pooled and evaporated in a rotary evaporator at 40 C under reduced pressure. Animals All experiments were performed with male Sprague–Dawley rats (180–200 g; 8–10 weeks old) obtained from the Animal Unit, Faculty of Medicine and Health Sciences, UPM.
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The animals were housed in polypropylene cages containing wood shavings, fed a standard pellet diet with free access to water and housed at room temperature (27 2 C; 70–80% humidity; 12 h light/dark cycle) in the Animal Holding Unit, UPM. The rats were fasted prior to all assays and standard drugs and MEMC were administered orally (p.o.) by gavage with a vehicle (8% Tween 80, 10 mL/kg). The Animal Care and Use Committee of the Faculty of Medicine and Health Sciences, UPM approved the use of the animals in this study (Approval No: UPM/FPSK/PADS/BR-UUH/ 00474). Acute toxicity study Acute toxicity studies were performed as described in ‘‘Guideline for Testing of Chemicals – Acute Oral Toxicity – Fixed Dose Procedure’’ (OECD, 2002). Rats were fasted 24 h prior to MEMC administration. The treated group received a single dose of 2000 mg/kg MEMC, while one group each received either vehicle or distilled water (10 mL/kg) by gavage. Subsequently, the animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 h and daily thereafter for 14 d. Food and water were provided throughout the experiment. The mortality, body weight and behavioral screening were recorded daily for 14 d after treatment. The rats that survived were sacrificed and macroscopic analysis and the weight of vital organs such as the liver, kidney, heart, spleen and lung were recorded. These organs were fixed in 10% formalin for histological examination. Pharmacological assays Ethanol-induced gastric ulcers The experiment was performed according to the method of Noor et al. (2006) with some modifications. After 48 h fasting in total, MEMC (25, 50, 100, 250 or 500 mg/kg), ranitidine (100 mg/kg), or vehicle were administered p.o. to the rats (n ¼ 6). All rats received absolute ethanol (5 mL/kg) after 1 h of treatment to induce gastric ulcers. The animals were anesthetized using diethyl ether and euthanized by cervical dislocation 1 h after ulcer induction. The stomachs were removed and opened along the greater curvature to determine the lesion damage. Every stomach was photographed and the ulcer area (UA) was measured by superimposing transparent grid paper with minimum of squares equaling 1 mm2 (Bodhankar et al., 2006). The UA in millimeter squared was determined for each stomach. Protection percentage was calculated using the following formula: Protection ð%Þ ¼
ðUA control UA pretreated groupÞ 100% ðUA controlÞ
Indomethacin-induced gastric ulcers The experiment was carried out as described by Nwafor et al. (2000) with modifications. Indomethacin (100 mg/kg) was administered p.o. to 48 h fasted rats (n ¼ 6) to induce gastric ulcers. MEMC (100, 250 and 500 mg/kg), ranitidine (100 mg/kg) or vehicle were administered p.o. 1 h before ulcer induction. The animals were sacrificed 6 h after ulcer
T. Balan et al.
Histological observation of the stomach tissue from rats in the vehicle group in the ethanol-induced ulcer model revealed obvious lesions and severe damage to the gastric mucosa; 60
40
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0
*** 25
10
0
0
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*** 0
***
50
20
50
The results were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test or the Newman–Keuls test and expressed as mean SEM. Results were considered significant when p50.05.
Effect of MEMC on ethanol-induced gastric ulcers
25
Statistical analysis
Histopathological evaluation
ne
The involvement of SH compounds in the gastroprotective effects of MEMC against ethanol-induced ulcers was determined using the method of Andreo et al. (2006). Rats were randomly assigned six per group and fasted for 24 h before i.p. pretreatment with NEM, which is a SH blocker or saline. Animals received vehicle, MEMC (100, 250 and 500 mg/kg), or carbenoxolone (100 mg/kg) after 30 min. Sixty minutes later, gastric damage was induced by oral administration of absolute ethanol (5 mL/kg). Animals were anesthetized using diethyl ether and sacrificed by cervical dislocation, 1 h later, the stomachs were excised and the UA determined as described above.
Indomethacin (100 mg/kg) produced gastric mucosal ulceration in the rats. As shown in Figure 2, pretreatment with 100, 250 and 500 mg/kg MEMC inhibited ulcer formation dosedependently, with protection percentage of 47.8, 51.8 and 68.9%, respectively. MEMC led to significant (p50.001) reduction in the total UA, which were 22.17, 20.5 and 13.2 mm2, respectively, when compared to the vehicle group. In contrast, 100 mg/kg ranitidine exerted 78% protection, with the mean UA being 9.3 mm2.
di
Ethanol-induced gastric mucosal lesion in NEM pretreated rats
Indomethacin-induced gastric ulcers
iti
In order to evaluate the role of endogenous NO in the gastroprotective effect of MEMC, a modified method of Andreo et al. (2006) was performed. Six male rats were assigned per group and were fasted for 24 h. They were pretreated intraperitoneally (i.p.) with saline or L-NAME (70 mg/kg), and vehicle, carbenoxolone (100 mg/kg), or MEMC (100, 250 and 500 mg/kg) were administered p.o. 30 min thereafter. Sixty minutes later, all groups received absolute ethanol (5 mL/kg) by gavage to induce gastric ulcers. L-Arginine (200 mg/kg) was administered 30 min after saline or L-NAME treatment and 30 min before ethanol administration. The animals were sacrificed 1 h after ethanol administration by diethyl ether anesthesia and cervical dislocation. The stomach was removed and gastric damage was determined as described above.
Macroscopic analysis of the stomach removed from ethanolinduced animals pretreated with the vehicle (negative control group) revealed complete ulceration. In contrast, pretreatment with 25, 50, 100, 250 and 500 mg/kg MEMC led to significant (p50.001) and dose-dependent reduction of gastric lesions, with 63.2, 79.3, 96.7, 94.3 and 95% protection (Figure 1), respectively, when compared to the vehicle group. All but the 25 mg/kg MEMC dose resulted in smaller UA, which were 10.3, 1.7, 2.8 and 2.5 mm2, respectively, demonstrating a better protective effect than the ranitidine group, in which there was 70% reduction with a mean UA of 14.8 mm2.
hi cl e
Ethanol-induced gastric mucosal lesion in L -NAME pretreated rats
Ethanol-induced gastric ulcers
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Gastric tissue samples were collected and fixed in 10% formalin. The specimens were embedded in paraffin and then sectioned (3–5 mm) and stained with hematoxylin and eosin. The sections were viewed and analyzed using light microscopy and photographed. This method was applied for both induced gastric ulcer models.
R
Histopathology evaluation
There was no mortality or behavioral pattern alteration. Animals showed no changes in food and water intake behavior. The relative weight of the vital organs and the respective histological examinations revealed no signs of toxicity (data not shown). The single oral administration of 2000 mg/kg MEMC did not produce any symptom or sign of toxicity in the treated animals after 14 days. Based on this finding, we concluded that the LD50 of MEMC is greater than 2000 mg/kg body weight.
Ve
induction by diethyl ether anesthesia and cervical dislocation. The stomachs were removed and opened along the greater curvature to determine lesion damage. Macroscopic analysis was performed, the UA determined and protection percentage was calculated as described earlier.
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Ulcer area (mm2)
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MEMC (mg/kg)
Results Acute toxicity study All animals in the treatment and control groups had increased body weight at weeks 1 and 2 as compared with day 0.
Figure 1. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg), MEMC 25, 50, 100, 250 and 500 mg/kg on absolute ethanol-induced ulcer. The ulcerated area (millimeter squared) was expressed as mean SEM for six animals. One-way ANOVA was done, followed by Dunnett’s post hoc test, ***p50.001 versus vehicle.
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there was severe hemorrhage, edema, necrosis and erosion (Figure 3). Interestingly, the mucosal architecture in the stomach tissue of the rats pretreated with 100, 250 and 500 mg/kg MEMC was almost normal, albeit with mild edema (Table 1). Overall, the groups that received MEMC had comparatively better gastroprotection, as indicated by the reduction of the UA on the stomach wall. In the ranitidine
Ulcer area (mm2)
40 30
***
***
20
***
***
10
0 50
25
0
0 10
R
an iti
di
ic
ne
le
0
Ve h
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Figure 2. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg), MEMC 100, 250 and 500 mg/kg on indomethacin-induced ulceration. The ulcerated area (millimeter squared) was expressed as mean SEM for six animals. One-way ANOVA was done, followed by Dunnett’s post hoc test, ***p50.001 versus vehicle.
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pretreated rats, there was moderate protection of the mucosa, which exhibited moderate edema and mild hemorrhage. Effect of MEMC on indomethacin-induced gastric ulcers In the indomethacin-induced ulcer model, histological evaluation of stomach tissue from the vehicle group animals revealed severe edema and moderate necrosis that may have led to mild erosion (Figure 4). Conversely, the stomach tissue of rats pretreated with 100, 250 and 500 mg/kg MEMC had almost normal mucosal architecture with mild edema (Table 1). Overall, the groups that received MEMC had gastroprotection when compared to the vehicle group, as the macroscopic analysis revealed a reduction in UA on the stomach wall in the MEMC-treated rats. Rats pretreated with ranitidine also had similar mucosal protection when compared to the vehicle group. Ethanol-induced gastric lesions in rats pretreated with L -NAME In groups pretreated with saline, animals that received MEMC prior to ethanol induction showed a significant reduction (p50.001) of the UA as compared to the vehicle group (Figure 5). The group treated with the standard antiulcer drug carbenoxolone (100 mg/kg) or L-arginine (200 mg/kg) also had significantly reduced UA (p50.001) as compared to the vehicle group (Figure 5). However, pretreatment with
(A)
(B)
(C)
(D)
(Ai)
(Bi)
(Ci)
(Di)
(E)
(Ei)
Figure 3. Histological evaluation of antiulcer activity of MEMC against ethanol-induced gastric lesions in rats. (A) Stomach of a negative control rat; (B) stomach of a rat pretreated with 100 mg/kg ranitidine; (C) stomach of a rat pretreated with 100 mg/kg MEMC; (D) stomach of a rat pretreated with 250 mg/kg MEMC; (E) stomach of a rat pretreated with 500 mg/kg MEMC. Respective histopathological sections are shown together; (Ai) stomach of the control animal showing a severe effect on mucosa with hemorrhagic erosion, edema, necrosis and erosion; (Bi) stomach of 100 mg/ kg ranitidine-treated animals showing moderate hemorrhage and edema; (Ci) stomach of 100 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Di) stomach of 250 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Ei) stomach of 500 mg/kg MEMC-treated animals show almost normal mucosa. H, hemorrhage; ED, edema; ER, erosion; N, normal architecture.
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Table 1. Histopathological evaluation of MEMC on ethanol-induced gastric lesions in rats. Ulcer model
Ethanol-induced Indomethacin-induced
Pretreatment group
Edema
Hemorrhage
Necrosis
Erosion
Vehicle 100 mg/kg ranitidine 100 mg/kg MEMC 250 mg/kg MEMC 500 mg/kg MEMC Vehicle 100 mg/kg ranitidine 100 mg/kg MEMC 250 mg/kg MEMC 500 mg/kg MEMC
þþþ þþ þ þ þ þþþ þ þ þ þ
þþþ þþ þ
þþ þþ
þ þ
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() normal; (þ) mild effect; (þþ) moderate effect; (þþþ) severe effect. (A)
(B)
(C)
(D)
(Ai)
(Bi)
(Ci)
(Di)
(E)
(Ei)
Figure 4. Histological evaluation of antiulcer activity of MEMC against indomethacin-induced gastric lesions in rats. (A) Stomach of a negative control rat; (B) stomach of a rat pretreated with 100 mg/kg ranitidine; (C) stomach of a rat pretreated with 100 mg/kg MEMC; (D) stomach of a rat pretreated with 250 mg/kg MEMC; (E) stomach of a rat pretreated with 500 mg/kg MEMC. Respective histopathological sections are shown together: (Ai) stomach of the control animal showing a severe effect on mucosa with necrosis and erosion; (Bi) stomach of 100 mg/kg ranitidine-treated animals showing moderate edema; (Ci) stomach of 100 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Di) stomach of 250 mg/kg MEMC-treated animals showing almost normal mucosa with mild edema; (Ei) stomach of 500 mg/kg MEMC-treated animals showing almost normal mucosa. H, hemorrhage; ED, edema; ER, erosion; N, normal architecture. L-NAME
(70 mg/kg) prior to ethanol administration aggravated the gastric lesions significantly (p50.001) as compared to the vehicle in the saline group. L-NAME pretreatment also worsened gastric ulcers significantly in all treated groups (p50.05), as compared with their saline pretreated counterparts. Treatment with L-arginine (200 mg/kg) significantly (p50.01) reversed the deleterious effect of L-NAME on the gastric mucosa. Therefore, these findings indicate NO participation in the gastroprotection exerted by MEMC.
(p50.001) of gastric lesions as compared to the vehicle group (Figure 6). Pretreatment with NEM (10 mg/kg) markedly increased gastric lesions (p50.001) when compared to that of the vehicle in the saline group. There was a significant reduction of UA in animals treated with MEMC in the saline group (p50.01, 250 and 500 mg/kg; p50.05, 100 mg/kg) when compared with the NEM pretreated animals. This indicates the participation of endogenous SH compounds in the gastroprotective effect demonstrated by MEMC.
Ethanol-induced gastric lesions in rats pretreated with NEM
Discussion
Animals that received carbenoxolone and MEMC in the groups pretreated with saline exhibited significant reduction
Gastric ulcer is a common disease that occurs in the human population worldwide. Gastric ulcers arise due to an
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150
Ulcer area (mm2)
*** 100 # ††† # # ††† †††
50
## †††
*** ***
*** *** ***
†††
200
100 250 500
mg/kg
CBX MEMC L-ARG
L-NAME
SALINE
Figure 5. Effect of vehicle (Tween 80, 8%, p.o.), MEMC (100, 250 and 500 mg/kg, p.o), carbenoxolone (CBX, 100 mg/kg, p.o.) and L-arginine (L-ARG, 200 mg/kg, i.p.) on gastric lesions induced by absolute ethanol in rats pretreated with saline i.p. and/or L-NAME (70 mg/kg, i.p.). Each column represents the mean SEM of six animals. Oneway ANOVA was done, followed by Newman–Keuls test, ***p50.001 versus saline vehicle; yyyp50.001 versus vehicle L-NAME; #p50.05, ##p50.01 saline pretreatment versus L-NAME pretreatment. 100 *** Ulcer area (mm2)
80 60 # ## ## ††† ††† †††
40 20 ***
*** *** ***
†††
mg/kg
hi
cl e 10 0 10 0 25 0 50 Ve 0 hi cl e 10 0 10 0 25 0 50 0
0
Ve
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CBX MEMC L-ARG
100
Vehicle
200
100 250 500
100
Vehicle
0
CBX
MEMC
SALINE
CBX
MEMC NEM
Figure 6. Effect of vehicle (Tween 80, 8%, p.o.), MEMC (100, 250 and 500 mg/kg, p.o.) and carbenoxolone (CBX, 100 mg/kg, p.o.) on gastric lesions induced by absolute ethanol in rats pretreated with saline i.p. and/ or NEM (10 mg/kg, i.p.). Each column represents the mean SEM of six animals. One-way ANOVA was done, followed by Newman–Keuls test, ***p50.001 versus saline vehicle; yyyp50.001 versus vehicle NEM; #p50.05, ##p50.01 saline pretreatment versus NEM pretreatment.
imbalance in mucosal aggressive and defensive factors such as acid–pepsin secretion, parietal cells, the mucosal barrier, mucus secretion, blood flow, cellular regeneration and endogenous protective agents (prostaglandins and epidermal growth factor) (Gilman et al., 2001). Gastric ulcers are commonly associated with damage to the mucosal layer of the stomach that occurs due to excess generation of exogenous and endogenous active oxygen and free radicals. Some of the main causes of gastric ulcers include chronic use of alcohol and anti-inflammatory drugs, stress and Helicobacter pylori infection (Barocelli et al., 1997).
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In the acute toxicity study, 2000 mg/kg MEMC led to no signs of toxicity or mortality in the treated rats. In accordance with the OECD guidelines, the LD50 of MEMC is 42000 mg/kg. Thus, we may conclude that administration of M. calabura extract in a single high dose is nontoxic and safe for use in oral formulations. The highest dose used for the antiulcer assays (500 mg/kg) was based on the toxicity study, being derived from a 4-fold reduction of the dose used in that toxicity study. At 25–500 mg/kg, MEMC had dose-dependent gastroprotective activity in both gastric ulcer models. Ethanol-induced ulcer is a classic ulcer model that is commonly used to evaluate the antiulcerogenic activity of drugs. The resulting gastric lesions appear along the glandular stomach as multiple hemorrhagic red bands of different sizes. The mechanism of ethanol-induced gastric lesions varies. Kinoshita et al. (1995) reported that the gastric mucosal lesions caused by ethanol might depress the gastric defense mechanism. This may include the depletion of gastric mucus content, mucosal cell injury and damaged mucosal blood flow (Galani et al., 2010). Ethanol damage to the gastrointestinal mucosa begins with microvascular injury, including edema, epithelial lifting and disruption of the vascular endothelium that leads to increased vascular permeability, where bicarbonate secretion is reduced while mucus production is increased, causing increased sodium and potassium flow, pepsin secretion and the loss of hydrogen ions and histamine into the lumen (Szabo, 1987). According to Kumar et al. (2011), ethanol acts by rapidly penetrating the gastric mucosa, damaging the cell and plasma membranes. This may lead to increase intracellular membrane permeability to sodium and water, causing the massive accumulation of calcium that clarifies the pathogenesis of gastric mucosal injury. Ethanol was also reported to enhance the production of reactive oxygen species (ROS), including superoxide anion and hydroxyl radicals (Bagchi et al., 1998), apart from other mechanisms such as damage to the capillary endothelium, release of arachidonate metabolites, leukotriene C4/D4 and platelet-activating factor (PAF) (Rachchh & Jain, 2008). In this study, the significant gastroprotective effect exerted by MEMC may have been due to a decrease in gastric motility, as flattening of the mucosal folds was observed. Changes in gastric motility may also contribute to the development and prevention of gastric lesions. Abdelwahab et al. (2011) reported that relaxation of the circular muscles might contribute to gastric mucosa protection via flattening of the folds, increasing the mucosal area exposure to necrotizing agents while reducing the volume of gastric irritants on the rugal crest. Contraction of the circular muscles of the rat fundus strip caused by ethanol intake may lead to compression of the mucosa at the crest of the mucosal folds, causing ulceration and necrosis (Jamal et al., 2006). In addition, the protection conferred by MEMC can be linked to its antioxidant properties. MEMC has been reported to contain flavonoids (Zakaria et al., 2011) that may act as antioxidant agents. Flavonoids can activate the mucosal defense system by stimulating gastric mucus secretion and scavenging the ROS and free radicals produced by ethanol (Abdelwahab et al., 2011). An endogenous antioxidant defense mechanism may constantly remove the ROS produced continuously during normal metabolism (Lemos et al., 2012). This indicates that
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the modulation of hydroxyl radical production from superoxide radicals or the scavenger activity demonstrated by MEMC may be due to its antioxidant properties. NSAIDs induce gastric ulceration via their ability to suppress prostaglandin synthesis (Wallace, 2001) that inhibits cyclooxygenase-1 (COX-1). Indomethacin is a preferential COX-1 inhibitor. COX-1 is constitutively expressed in large amounts in the gastrointestinal tract and has been reported to maintain mucosal integrity through the continuous generation of prostaglandins (Hatler et al., 2001). Prostaglandin suppression may stimulate bicarbonate and mucus secretion, maintaining mucosal blood flow and regulating mucosal cell turnover and repair that leads to increase susceptibility to mucosal injury and gastroduodenal ulceration (Hayllar & Bjarnason, 1995). Moreover, NSAIDs are also reported to be involved in the generation of ROS that initiate lipid peroxidation, leukocyte infiltration and apoptosis induction (Beck et al., 2000). Significant inhibition of ulceration was observed in the MEMC pretreated indomethacin-induced ulcer model. The ability of MEMC to reduce indomethacininduced gastric ulceration suggests the possible involvement of prostaglandins or mucus in the MEMC antiulcer effect, where MEMC can regulate prostaglandin synthesis or stimulate mucus or bicarbonate secretion. As MEMC exerted significant gastroprotective effects in both ethanol and indomethacin-induced ulcer models, we decided to investigate the possible pharmacological mechanism involved. For this purpose, ethanol-induced gastric mucosal lesions in rats pretreated with L-NAME and NEM were used to determine the involvement of NO and nonprotein SH (NP-SH) compounds in the protective effect of MEMC. NO is considered one of the most important defensive endogenous agents in the gastric mucosa (Brzozowski et al., 2006). It is synthesized by NO synthase (NOS) from L-arginine (Takahashi et al., 1998). NO is involved in the modulation of gastric mucosal integrity and is important in the regulation of acid and alkaline secretion, mucus secretion and gastric mucosal blood flow (Chandranath et al., 2002). In ethanol-induced gastric injury, the endothelium is the main and most preferred target of ethanol damage (de-Faria et al., 2012). This is because systemic administration of L-NAME that inhibits NO may cause an increase in systemic blood pressure and the vasoconstriction of several vascular beds that damage the gastric mucosa and its endothelium (Sikiric et al., 1997). Carbenoxolone was used as the standard model drug because other researchers have reported the partial participation of prostaglandins, SH compounds and NO in the gastroprotective mechanism conferred by carbenoxolone (Navarete et al., 2005; Cha´vez-Pin˜a et al., 2011). L-NAME pretreatment reversed the gastroprotective effects exerted by MEMC against ethanol-induced damage. These findings indicate the possible participation of the NOS pathway in the gastroprotection exerted by MEMC, supporting the premise of the free radical scavenging effect of this extract. The possible involvement of endogenous NP-SH compounds in the gastroprotective effect of MEMC was also investigated. The synthesis of mucus is one of the important functions in gastroprotection, as mucus aids in strengthening the mucosal barrier against harmful agents. The continuous
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adherence of the mucus layer is a barrier to luminal pepsin and creates a stable, undisturbed layer to support the surface neutralization of acid, protecting the underlying mucosa from proteolytic digestion (Allen & Flemstrom, 2005). Disulfide bridges connect the mucus subunits; if reduced, the mucus may become water-soluble (Avila et al., 2005). Szabo and Vattay (1990) reported that the endogenous NP-SH compound is a key compound in the mucosal protection against ethanolinduced gastric injury. In addition to NO, SH compounds are important for maintenance of the gastric mucosa because they confer protection against pro-oxidant agents (de-Faria et al., 2012), binding the free radicals caused by the ingestion of noxious agents. As presented in Figure 6, NEM increased the UA in ethanol-induced animals. The gastroprotection exerted by MEMC was abolished by co-administration of NEM, suggesting that the gastroprotective effect showed by MEMC may depend on the participation of mucosal SH compound levels. The pathogenesis of gastric ulcers involves oxidative stress; antioxidants play a very important role in protecting the gastric mucosa against necrotic agents (Trivedi & Rawal, 2001). The ingestion of noxious agents may lead to the formation of free radicals and ROS that contribute to gastric mucosal damage. Antioxidants act as radical scavengers that may protect the gastric mucosa from oxidative damage. MEMC was reported to contain high phenolic content that exhibited high antioxidant activity when tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and superoxide scavenging assays (Zakaria et al., 2011). Therefore, it is suggested that the antioxidant and free radical scavenging properties of MEMC may have contributed to the observed gastroprotection ability. In terms of phytochemical constituents, MEMC was reported to contain flavonoids, tannins and saponins (Zakaria et al., 2011). A number of phenolic substances are known to act as antioxidants, such as flavonoids, coumarins, tannins, xanthines and more recently, procyanidins, which can scavenge radicals in a dose-dependent manner (Czinner et al., 2001). In addition to free radical scavenging, several mechanisms may be involved in the antioxidant activity of flavonoids, including oxidant enzymes inhibition, chelating of transition metal ion and regeneration of a-tocopherol from a-tocopheroxyl radicals (Repetto & Llesuy, 2002). Flavonoids promote formation of the gastric mucosa, inhibit pepsinogen production, decrease acid mucosal secretion and reduce ulcerogenic lesions (La Casa et al., 2000). Tannins may prevent ulcer development by promoting protein precipitation on the ulcer site that forms a protective pellicle. This may help to prevent the absorption of toxic substances and withstand the effects of proteolytic enzymes (John & Onabanjo, 1990; Nwafor et al., 1996). Therefore, the presence of these phytochemical substances may protect against gastric lesions by enhancing the antioxidant activity of the extract.
Conclusions This study demonstrated that MEMC has significant antiulcer activity that involves the participation of endogenous NO and SH compounds. This gastroprotective activity could have been due to the antioxidant activity and the presence
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of flavonoids, tannins and phenolic compounds in the extract. Further studies are in progress to identify the bioactive compound(s) that may be present, which could be responsible for the antiulcer properties of M. calabura.
Acknowledgements The authors thanked the Faculty of Medicine and Health Sciences, UPM, Malaysia for providing the facilities and supporting this study.
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Declaration of interest The authors report no declarations of interest. This research was supported by the Research University Grant Scheme (Reference no. 04-02-10-0925RU) awarded by the UPM
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