Mechanism(s) of action involved in the gastroprotective activity of Muntingia calabura

Journal of Ethnopharmacology 151 (2014) 1184–1193

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Research Paper

Mechanism(s) of action involved in the gastroprotective activity of Muntingia calabura$ Zainul Amiruddin Zakaria a,n, Tavamani Balan a, Velan Suppaiah b, Syahida Ahmad c, Fadzureena Jamaludin d a

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia c Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia d Natural Products Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor, Malaysia b

art ic l e i nf o

a b s t r a c t

Article history: Received 4 October 2013 Received in revised form 4 December 2013 Accepted 20 December 2013 Available online 28 December 2013

Ethnopharmacological relevance: Muntingia calabura L. (Muntingiaceae) is locally known as kerukup siam. Its leaves, flowers, barks and roots have been used traditionally in East Asia and South America to treat various diseases including ulcer-related diseases. The present study aimed to investigate the mechanism (s) of gastroprotective effect of methanol extract of Muntingia calabura leaves (MEMC) using the pylorus ligation induced gastric ulceration in rats. Materials and methods: Five groups of rats (n ¼ 6) were administered orally once daily for 7 days with 8% Tween 80 (negative control), 100 mg/kg ranitidine (positive control), or MEMC (100, 250 or 500 mg/kg), followed by the ulcer induction via ligation of the pyloric part of the rat’s stomach. This was followed by the macroscopic analysis of the stomach, evaluation of gastric content parameters, and quantification of mucus content. The antioxidant (measured using the superoxide anion and 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical scavenging, oxygen radical absorbance capacity (ORAC) and total phenolic content (TPC) assays), anti-inflammatory (evaluated using the in vitro lipoxygenase and xanthine oxidase assays), phytoconstituents and HPLC analysis of MEMC were also carried out. Results: The MEMC significantly (po0.05) reduced gastric lesion in this model. Furthermore, the extract also significantly (po0.01) reduced the volume of gastric content whereas the total acidity was significantly (po0.05) reduced in the doses of 100 and 500 mg/kg MEMC. Moreover, the mucus content increased significantly (po0.01) in MEMC-treated rats. The extract also showed high antioxidant and antiinflammatory activities in all assays tested, and demonstrated the presence of high tannins and saponins followed by flavonoids. Conclusion: The MEMC exerted gastroprotective effect via several mechanisms including the anti-secretory, antioxidant and anti-inflammatory activities. These activities could be attributed to the presence of tannins, saponins and flavonoids (e.g. rutin, quercitrin, fisetin and dihydroquercetin). & 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Muntingia calabura Muntingiaceae Gastric ulcer Anti-secretory Antioxidant Anti-inflammation

1. Introduction Peptic ulcers have become one of the major human illness affecting nearly 8–10% of the global population (Calam and Baron, 2001), and of these number, 5% suffer from gastric ulcers (Bandyopadhyay et al., 2001). Diverse factors such as alcohol consumption, a stressful lifestyle, use of steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) and drugs which stimulate gastric

☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. n Corresponding author. Tel.: þ 60 19 2117090; fax: þ 60 3 89472537. E-mail address: [email protected] (Z.A. Zakaria).

acid and pepsin secretion, Helicobacter pylori infections and smoking contribute to the pathogenesis of gastric ulcers (Rang et al., 2012). An imbalance between the aggressive factors such as acid and pepsin secretion, Helicobacter pylori, refluxed bile, release of leukotrienes and reactive oxygen species (ROS) and mucosal defensive factors that include bicarbonate secretion, mucus-bicarbonate barrier, surface active phospholipids, prostaglandins (PGs), mucosal blood flow, cell renewal and migration, non-enzymatic and enzymatic antioxidants and some growth factors (Mota et al., 2009) leads to gastric damages. The prevention or cure of peptic ulcers has become an important challenge in the current medicine world. Although gastric ulcer is linked to many causative factors, secretion of gastric acid is still believed to remain as the central component of this disease (Mota et al., 2009). Thus, inhibition of gastric acid secretion is the key therapeutic target for ulcer diseases (Jain et al., 2007). Therefore,

0378-8741/$ - see front matter & 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.045

Z.A. Zakaria et al. / Journal of Ethnopharmacology 151 (2014) 1184–1193

current medicinal treatment of gastric ulcers available is generally based on the inhibition of gastric acid secretion by histamine H2-antagonists, proton pump inhibitors, and antimuscarinics, as well as on acid-independent therapy provided by sucralfate and bismuth cholinergics (Bighetti et al., 2005). However, gastric ulcer therapy faces a major drawback nowadays as most of the drugs available in the market are often associated with side effects (Bandyopadhyay et al., 2002; Rang et al., 2012). In this context, the use of medicinal plants has gained interest of many researchers. Natural product is in continuous expansion all over the world and became the most attractive source of new drug for the treatment and prevention of many diseases. Diverse range of bioactive molecules isolated from plant natural product have been shown to produce promising results for the treatment of gastric ulcer (Borelli and Izzo, 2000). One of the plants that is currently under investigation for its potential pharmacological activities in our laboratory is Muntingia calabura L. (family Muntingiaceae), commonly known as Jamaican cherry or kerukup siam in Malaysia. Muntingia calabura is widely cultivated in warm areas of Asian region, including Malaysia (Chin, 1989) and has become one of the most common roadside trees in Malaysia. Several medicinal uses have been documented on various parts of this tree in Asia and tropical America. Muntingia calabura’s leaves, barks, flowers, and roots have been used as a folk remedy to treat incipient cold, fever, headaches, employed as an emmenagogue and abortifacient, as well as an antiseptic, antispasmodic, and antidyspeptic agent (Kaneda et al., 1991; Nshimo et al., 1993). Scientific evaluations on Muntingia calabura have revealed several pharmacological activities possessed by the plant. Muntingia calabura leaves have been reported to exhibit significant antiinflammatory, antipyretic (Zakaria et al., 2007c), antinociception (Zakaria et al., 2006b, 2007b, 2007c), antitumor (Kaneda et al., 1991; Su et al., 2003), antiproliferative, antioxidant (Zakaria et al., 2011) and antibacterial (Zakaria et al., 2006a) activities. Flavonoids, saponins, tannins, triterpenes and steroids have been detected in the leaves of Muntingia calabura (Zakaria et al., 2007c). Several types of flavonoids have been isolated and identified from the leaves, roots and stem barks of Muntingia calabura (Kaneda et al., 1991; Nshimo et al., 1993; Su et al., 2003; Chen et al., 2005; Sufian et al., 2013). Recently, we have reported the significant antiulcer activity of methanol extract of Muntingia calabura leaves and its modulation of endogenous nitric oxide and non-protein sulfhydryl compounds (Balan et al., 2013). Therefore, the present study was aimed to investigate the antiulcer mechanisms of methanol extract of Muntingia calabura leaf (MEMC) to provide a better understanding of the plant’s gastroprotective effect.

2. Materials and methods 2.1. Plant material 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 UPM IBS Laboratory of Natural Products Herbarium. 2.2. Preparation of methanol extract of Muntingia calabura The preparation of MEMC was done according to Zakaria et al. (2011). Matured leaves (500 g) were ground into fine powder after air-drying them at room temperature (27 72 1C) for 1–2 weeks. Methanol was used as the solvent. The powder was soaked in

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methanol at a ratio of 1:20 (w/v) for 72 h. Filter funnel, cotton and Whatman no. 1 filter paper were used to filter the mixture. The soaking and filtration were repeated on the residue for twice. The filtrate collected from each extraction was pooled and evaporated in a rotary evaporator at 40 1C under reduced pressure. 2.3. Phytochemical screening and HPLC profiling The phytochemical screening of MEMC was performed according to the conventional protocols as adopted by Zakaria et al. (2012). The HPLC profile of MEMC was done according to Zakaria et al. (2012) with slight modifications. Briefly, 10 mg of crude dried MEMC was dissolved in 1 ml methanol and filtered through a membrane filter with a pore size of 0.45 mm prior to analysis. A HPLC system (Waters Delta 600 with 600 Controller) with a photodiode array detector (waters 996) (Milford, MA, USA) was used to run the profiling. A Phenomenex Luna (5 mm) (Torrance, CA, USA) column was used (4.6 mm i.d.  250 mm). Two solvents denoted as A and B were used for elution of the constituents. A was 0.1% aqueous formic acid and B was acetonitrile. Initial conditions were 95% A and 5% B with a linear gradient reaching 25% B at t¼ 12 min. This was maintained for 10 min. At t ¼ 22 min, B was reduced to 15%, which was then maintained until t¼ 30 min. The programme returned to the initial solvent composition at t¼35 min. The flow rate used was 1.0 ml/min and the injection volume was 10 ml. The column oven was set at 27 1C and the eluent was monitored at 254, 300 and 366 nm. The retention time and UV spectra of major peaks were analyzed. MEMC was then spiked with a list of flavonoid compounds, which served as the standard, namely pinostrobin, hesperetin, flavanone, 4ʹ,5,7-trihydroxy flavanone, 2,4,4ʹ-trihydroxy chalcone, quercitrin, dihydroquercetin, fisetin, quercetin, rutin, quercitrin, naringenin, silibinin, and genistein using the same solvent system in order to determine the presence of these constituents in the extract. The HPLC analysis was carried out in the Laboratory of Phytomedicine, Medicinal Plants Division, Forest Research Institute of Malaysia (FRIM), Kepong, Malaysia. 2.4. Antioxidant activity of MEMC 2.4.1. Superoxide scavenging activity The method of Chang et al. (1996) with slight modification has been employed to determine superoxide scavenging activity of MEMC. Nitro-blue tetrazolium (NBT) solution (100 ml of 4.1 mM/l) was prepared by adding 3.15 g Tris-HCl, 0.1 g MgCl2, 15.0 mg 5-bromo-4-chloro-3-indolyl phosphate and 34.0 mg 4-nitro-blue tetrazolium chloride to 100 ml of distilled water. The reaction mixture (100 ml) was prepared by dissolving 0.53 g sodium carbonate (pH 10.2), 4.0 mg ethylene diamine tetraacetic acid (EDTA) and 500 mg xanthine in 0.025 mM NBT solution. The mixture was refrigerated at 4 1C. The reaction mixture (999 ml) was transferred into a microcuvette and placed in a 25 1C cell holder of spectrophotometer. Superoxide generation was initiated by adding 1.0 ml of xanthine oxidase (XOD) (20 U/ml). The optical density (OD) measurements were taken at 560 nm for 120 s using Lambda 2S spectrophotometer. MEMC was dissolved in the reaction mixture at the concentration of 200 mg/ml. The stock solution (200 ml) was added to 799 ml of the reaction mixture and placed in a cell holder to autozero. One microliter of XOD (20 U/ml) was then added, mixed thoroughly and similarly measured for the XOD curves. 2.4.2. DPPH radical scavenging activity Antioxidant reducing activity on DPPH radical was carried out according to the method of Blois (1958) with modification using a high-throughput microplate system. Fifty microliter of sample

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(1.0 mg/mL) was added to 50 μL of DPPH (FG: 394.32) (1 mM in ethanolic solution) and 150 μL of absolute ethanol in a 96-well microtiter plate in triplicates. The plate was shaken for 15 s at 500 rpm and left to stand at room temperature for 30 min. The absorbance of the resulting solution was measured spectrophotometrically at 520 nm.

2.4.3. ORAC assay The ORAC assay was performed as described by Huang et al. (2002) with some modifications. 2,2ʹ-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) (0.65 g) was dissolved in 10 ml of 75 mM phosphate buffer (pH 7.4) to a final concentration of 240 mM (made fresh). A fluorescein stock solution (1 mM) was made in 75 mM phosphate buffer (pH 7.4) and stored, wrapped in foil at 5 1C. Immediately prior to use, the stock solution was diluted 1:100,000 with 75 mM phosphate buffer. The diluted sodium fluorescein was made fresh daily. The sodium fluorescein solution (150 ml) was added to the interior experimental wells. The blanks received 25 ml of Trolox dilution. The sample wells received 25 ml samples. The plate was then allowed to equilibrate by incubating for 10 min at 37 1C. BMG Omega Fluostar Fluorescent Spectrophotometer with injector was used with an excitation filter of 485 nm bandpass and emission filter of 528 nm bandpass. The plate reader was controlled by MARS data analysis software. Reactions were initiated by the addition of 25 ml of AAPH solution (240 mM) using the microplate reader’s injector for a final reaction volume of 200 ml. The addition of 25 ml of AAPH solution was followed by shaking at maximum intensity for 50 s. The fluorescence was then monitored kinetically with data taken every minute. The fluorescence of each well was measured by top reading every 60 s. ORAC values were calculated using MARS Data Analysis Reduction Software.

2.4.4. Total phenolic content Determination of total phenolic content (TPC) was performed using the Folin–Ciocalteu reagent according to the method of Singleton and Rossi (1965) with slight modifications. One milligram of MEMC was extracted for 2 h with 1.0 ml of 80% methanol containing 1.0% hydrochloric acid and 1.0% of distilled water at room temperature on the shaker set at 200 rpm. The mixture was centrifuged at 6000 rpm for 15 min, and the supernatant transferred into vials. TPC was determined using the supernatant. A 200 ml of supernatant extract was mixed with 400 ml of the Folin–Ciocalteu reagent (0.1 mL/0.9 mL) and incubated at room temperature for 5 min. This followed by the addition of 400 ml of sodium bicarbonate (60.0 mg/ml) and the mixture was incubated at room temperature for 90 min. Absorbance was measured at 725 nm. A calibration curve was generated by using the gallic acid standard OD. The TPC levels found in the samples were expressed as gallic acid equivalent (GAE)–TPC mg/100 g. 2.5. Animals All experiments were performed on male Sprague Dawley rats (180–200 g; 8–10 weeks old) obtained from the Animal Unit, Faculty of Medicine and Health Sciences, UPM, Malaysia. The animals were caged in polypropylene cages with wood shaving, fed with standard pellet and allowed free access to water. They were kept in room temperature (27 72 1C; 70–80% humidity; 12 h light/darkness cycle) in the Animal Holding Unit (UPM). The rats were fasted prior to all assays, standard drugs and MEMC were administered orally (p.o.) by gavage with 8% Tween 80 (10 ml/kg) as the vehicle. The use of animals in the following study was approved by the Animal Care and Used Committee (ACUC) of

Faculty of Medicine and Health Sciences, UPM (approval no. UPM/ FPSK/PADS/BR-UUH/00474). 2.6. Pharmacological assay 2.6.1. Pylorus ligation induced ulceration Pylorus ligation was carried out according to the method by Shay et al. (1945) with slight modifications. Thirty rats were divided into five groups. Group-I (control) was treated with vehicle (8% Tween 80), Group-II (positive control, ranitidine) was given at 100 mg/kg (p.o), Group-III, -IV and-V rats were treated with MEMC (100, 200 and 500 mg/kg, respectively). Pylorus ligation was performed 1 h after the administration of the test compounds on 48 h fasted rats. Under light anesthesia induced using ketamine HCl (100 mg/kg, intramuscular) and xylazine HCl (16 mg/kg, intramuscular), a 2 cm long incision was made in the abdomen just below the sternum. The stomach was exposed, and a thread was passed around the pyloric sphincter and tied in a tight knot. Care was taken while tying the knot to avoid involving blood vessels in the knot. The abdomen was sutured, and the skin was cleaned of any blood spots or bleeding. The animals were sacrificed 6 h after ligation by cervical dislocation. The stomachs were removed, and the contents were drained out, collected, and centrifuged. The stomach was opened along the greater curvature to determine the lesion damage as described by Balan et al. (2013). The percentage protection was calculated using the following formula: Protection ð%Þ ¼

ðUA control–UA pre  treated groupÞ  100% ðUA controlÞ

2.6.2. Determination of volume, pH, free and total acidity of gastric content The drained gastric content was centrifuged at 2500 rpm for 10 min. The volume and pH of the gastric juice were measured and were subjected to free and total acidity estimation. The method described by Srivastava et al. (2010) was employed in free and total acidity estimation. Free acidity was determined by titration with 0.01 N NaOH with methyl orange reagent until the color of the solution became yellowish. The volume of alkali added was noted. Then, two to three drops of phenolphthalein was added and the solution was titrated until a definite red tinge appear. The total volume of NaOH added was noted and this corresponds to total acidity. Acidity was calculated using the following formula: Acidity ¼

Volume of NaOH  normality of NaOH  100 meq=l 0:1

2.6.3. Estimation of gastric wall mucus content Gastric wall mucus content was determined by the method described by Corne et al. (1974) with slight modifications. The stomach was opened along the greater curvature, weighed, and immersed in 10 ml of 0.1% Alcian Blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8 for 2 h. The excessive dye was then removed by two successive rinses in 0.25 M sucrose solution (15 min each). The remaining dye complexed with the gastric mucus were extracted with 0.5 M MgCl2 for 2 h and shaken intermittently for 1 min in every 30 min interval. The blue extract was then shaken vigorously with an equal volume of diethyl ether and the resulting emulsion was centrifuged at 3600 rpm for 10 min. The OD of Alcian Blue in the aqueous layer was read at 580 nm using a spectrophotometer. The quantity of Alcian Blue extract per gram wet stomach was then calculated from a standard curve.

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2.7. In vitro effect of MEMC on nitric oxide 2.7.1. Cell culture and stimulation The murine monocytic macrophages’ cell line (RAW 264.7) was purchased from European Collection of Cell Cultures (Porton Down, UK) and maintained in DMEM supplemented with 10% FBS, 4.5 g/L glucose, sodium pyruvate (1 mM), L-glutamine (2 mM), streptomycin (50 μg/ml), and penicillin (50 U/ml) at 37 1C and 5% CO2. The cells (4  105 cells/well) were seeded into a 96-well plate and incubated in a CO2 incubator for 2 h at 37 1C to enable the attachment of the cells. The attached cells in the well were then triggered with stimuli (100 U/ml of IFN-g and 5 μg/ml of LPS) with or without the presence of MEMC tested at concentration ranging from 12.5 to 100 μg/ml. DMSO was used as a vehicle to dissolve the MEMC. The final concentration of DMSO was ensured to be 0.1% in all cultures. Cells were then incubated at 37 1C in a CO2 incubator for 17–20 h. The culture supernatant was subjected to Griess assay for nitrite determination and the cells remaining in the well were tested for cell viability assay. 2.7.2. Nitrite determination Griess assay was used to determine the concentration of nitrite (NO2  ), the stable metabolite of NO in culture medium, according to Lee et al. (2011). An equal volume of Griess reagent was mixed with culture supernatant. The color development was measured using at 550 nm. The amount of nitrite in the culture supernatant was calculated based on the standard curve of a sodium nitrite freshly prepared in deionized water, concentration ranging from 0 to 100 μM. Percentage of the NO inhibition was calculated by using the following formula: ½NO2  control –½NO2   sample  100% n ½NO2   control n

NO inhibitory ð%Þ ¼ n

control is the nitrite level of IFN-γ/LPS-induced group.

2.7.3. Cell viability The cytotoxicity of MEMC on the cultured cells was determined by assaying the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2 H-tetrazolium bromide (MTT) according to the method described by Lee et al. (2011). The MTT reagents (0.05 mg/ml) were dissolved in sterile PBS, pH 7.0. MTT reagents were added into each well after removing the supernatant. The remaining cells were incubated at 37 1C for 4 h. One hundred microliters of 100% DMSO was then added into the wells to dissolve the formazan salts formed. The absorbance was measured at 570 nm. The percentage of cell viability was calculated according to the following formula, where control is the cell viability of IFN-γ /LPS-induced group: Cell viability ð%Þ ¼

OD control  OD sample  100% OD control

2.8. In vitro anti-inflammatory effect of MEMC 2.8.1. Lipoxygenase assay Lipoxygenase inhibiting activity was measured using spectrophotometric method as described by Azhar-Ul-Haq et al. (2004). Sodium phosphate buffer 160 ml (0.1 M, pH 8.0), 10 ml of MEMC and 20 ml of soybean lipoxygenase solution were mixed and incubated for 10 min at 25 1C. An addition of 10 ml of the substrate in the form of sodium linoleic acid solution will then initiate the reaction. The enzymatic conversion of linoleic acid to form (9Z, 11E)-(13 S)13-hydroperoxyoctadeca-9,11-dienoate was followed by the change of absorbance measured at 234 nm over period of 6 min. MEMC and reference standards were dissolved in methanol. All reactions were performed in triplicates in a 96-well microplate.

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2.8.2. Xanthine oxidase assay The spectrophotometric method from Noro et al. (1983) was employed to measure the xanthine oxidase inhibiting activity of MEMC. Potassium phosphate buffer 130 μl (0.05 M, pH 7.5), 10 μl of the test solution and 10 μl of xanthine oxidase solution were mixed and incubated for 10 min at 25 1C. The reaction was then initiated by the addition of 100 μl of the substrate in the form of xanthine solution. The enzymatic conversion of xanthine to form uric acid and hydrogen peroxides measured at absorbance of 295 nm. MEMC and reference standards were dissolved in DMSO. All reactions were performed in triplicates in a 96-well UV microplate. 2.9. Statistical analysis The results were expressed as mean 7SEM and analyzed using one way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. Results were considered significant when p o0.05.

3. Results 3.1. Phytochemical screening Phytochemical investigation on the crude extract revealed the presence of various compounds, such as flavonoids, tannins, polyphenols, saponins and steroids and the absence of triterpenes and alkaloids. On the other hand, the Muntingia calabura leaves powder was found to contain flavonoids, tannins, polyphenols, saponins, steroids and triterpenes, but not alkaloids (Table 1). 3.2. HPLC profiling The HPLC analysis of MEMC was measured at three different wavelengths, 254, 330, and 366 nm, and the best separation was obtained at two wavelengths, which were 254 and 366 nm (Fig. 1A). Ten major peaks were separated at these wavelengths, which were P1 (RT ¼2.846 min), P2 (RT ¼ 3.998 min), P3 (RT ¼ 14.584 min), P4 (RT ¼19.008 min), P5 (RT ¼ 19.456 min), P6 (RT ¼21.096 min), P7 (RT ¼20.349 min), P8 (RT ¼22.546 min), P9 (RT ¼23.234 min) and P10 (RT ¼27.805 min). Further analysis demonstrated that these 10 peaks showed λmax values in the region of 222.5–274.4, 221.3–272.0, 219.0–272.0, 219.0–278.0, 207.2–361.3, 254.3–367.2, 207.2–351.7, 207.2–343.4, 207.2–349.4 and 204.9–366.3 nm, respectively (Fig. 1B), suggesting, in part, the presence of flavonoid-based compounds. Comparison between chromatogram of the standard compounds with chromatogram of MEMC revealed the presence of rutin, fisetin, quercitrin and dihydroquercetin (Fig. 1C) as spiking of these compounds in MEMC increases the peak area. Table 1 Comparison on the phytochemical constituents of the leaves and methanol extract of Muntingia calabura (MEMC). Phytochemical constituent

Muntingia calabura powder

MEMC

Flavonoids Tannins and polyphenols Triterpenes Saponins Alkaloids Steroids

þþþ þþ þþ þ – þþþ

þ þþ – þþ – þ

For flavonoids, tannins, triterpenes, and steroids: þ , weak color; þ þ, mild color; þ þ þ , strong color. For saponins: þ , 1–2 cm froth; þ þ, 2–3 cm froth; þ þ þ, 43 cm froth. For alkaloids: þ , negligible amount of precipitate; þ þ , weak precipitate; þ þ þ, strong precipitate.

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Fig. 1. (A) The HPLC profile of MEMC at the wavelengths of 254 and 366 nm. (B) The UV spectra analysis of MEMC. The UV spoectra demonstrated the presence of 10 major peaks labeled as P1 (RT¼ 2.846 min), P2 (RT¼ 3.998 min), P3 (RT ¼14.584 min), P4 (RT¼ 19.008 min), P5 (RT¼ 19.456 min), P6 (RT¼ 21.096 min), P7 (RT ¼20.349 min), P8 (RT¼ 22.546 min), P9 (RT¼ 23.234 min) and P10 (RT¼ 27.805 min), which were observed at their respective λmax at the region of 222.5–274.4, 221.3–272.0, 219.0–272.0, 219.0–278.0, 207.2–361.3, 254.3–367.2, 207.2–351.7, 207.2–343.4, 207.2–349.4 and 204.9–366.3 nm, respectively, suggesting, in part the presence of flavonoid-based compounds. (C) Comparison between chromatogram of the standard compounds, namely rutin, quercitrin, fisetin and dihydroquercetin with chromatogram of MEMC at 366 nm showing the presence of rutin, quercitrin, fisetin and dihydroquercetin in MEMC.

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Table 2 The antioxidant activity and TPC value of MEMC determined using various assays. Samples

Superoxide scavenging (%)

DPPH (%)

ORAC (lM TE/100 g)

Total phenolic content (TPC) mg/100 g GAE

Sample concentration (lg/ml) 200 200 200 200 Standard Superoxide dismutase (SOD) (6  10–3 U/ml) Ascorbic acid (AA) 200 mg/ml Trolox standard curve Gallic acid (GAE) standard curve MEMC 99.32 7 0.18 (H) 98.687 0.38 (H) 24,955.007 1495.23 2751.26 7 10.51 (H) All values are expressed as mean 7 SEM. Superoxide scavenging and DPPH radical scavenging: H, high (70–100%); M, moderate (50–69%); L, low (0–49%). TPC 1. TPC value 4 1000 mg GAE/100 g is considered high total phenolic content. TPC 2. TPC expressed as milligram equivalent to gallic acid per 100 g of dry weight (mg GAE/100 g). ORAC value expressed as mM trolox equivalent (TE)/100 g is mean values from triplicate wells in duplicate experiments, with SEMo 20%.

Ulcer area (mm2)

15

***

10

5

*** ***

***

***

0 50

0 25

0 10

R an iti di ne

Ve

hi cl e

0

MEMC (mg/kg) Fig. 2. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg) or MEMC (100, 250, and 500 mg/kg) on pylorus ligation-induced ulcer. The ulcerated area (mm2) was expressed as mean7SEM for six animals. One way ANOVA was followed by Dunnett’s post hoc test, ***po0.001 vs. vehicle.

3.3. Antioxidant assays The antioxidant activities of the Muntingia calabura were investigated by superoxide scavenging, DPPH radical scavenging, and ORAC assays. At the concentration of 200 μg/ml, MEMC produced high superoxide and DPPH scavenging activity (Table 2). ORAC assay was used to evaluate the total antioxidant capacity of Muntingia calabura. The ORAC value was based on the net area under the curve (AUC) of fluorescent decay curve for various concentrations of MEMC and the values have been expressed as relative Trolox equivalents (TE) (Table 2). On the other hand, evaluation of the total phenolic content showed a high value indicating the high level of phenolic present in the MEMC (Table 2). 3.4. Gastroprotective study 3.4.1. Pylorus ligation-induced gastric lesion Gastric lesion measurements of pylorus-ligated rats showed that MEMC significantly decreased the ulcer area at the doses of 100, 250, and 500 mg/kg. Ranitidine (100 mg/kg), the standard drug used as the positive control in the study, also showed significant reduction of gastric lesion. The ulcer inhibition was found to be 77.6, 79.3, and 77.6%, respectively, while ranitidine showed 87.9% protection in comparison to the control group (Fig. 2). 3.4.2. Evaluation of gastric juice parameters The effects of MEMC upon baseline acid secretion acid collected after 6 h of pylorus ligature in rats are shown in Table 3. MEMC, at

all the three doses (100, 250, and 500 mg/kg) significantly decreased the volume of gastric secretion by 62% (p o0.01), 72% (p o0.001) and 71% (p o0.001), respectively. As for the pH of the gastric juice, all the doses of MEMC showed an increment in the pH value but the values were not significantly different as compared to the negative control. Meanwhile, the free acidity of MEMC was also found to be not significantly different. However, the total acidity was significantly reduced in the doses of 100 and 500 mg/kg of MEMC by about 25% (p o0.05) and 36% (p o0.001), respectively, while 250 mg/kg of MEMC failed to significantly decrease the total acidity of the gastric secretion. Ranitidine (100 mg/kg) caused a reduction in the volume of secretion by 83% (po0.001), increased the pH by about 2.2 folds (po0.001) and decreased the total acidity of the gastric juice by about 41% (po0.001) as compared to the control group.

3.4.3. Determination of mucus in the gastric mucosa As observed in Fig. 3, pre-treatment with MEMC caused a significant increment in gastric wall mucus content in all the doses administered (100, 250, and 500 mg/kg) whereby the amount of mucus recorded was significantly higher (p o0.01) when compared with the control animals pretreated with vehicle alone. The rats that received ranitidine also increased the mucus content significantly (p o0.001).

3.5. In vitro effect of NO The RAW 264.7 cells were induced into an inflammatory state by treatment with LPS/IFN-γ causing synthesis and secretion of excessive NO. Nitrite ions (NO2  ), which is the stable metabolite of NO, was detected in the induced culture medium at a mean concentration of 49.65 μM. Trace amount of NO was found in the cells that were not induced. MEMC at the highest concentration tested (100 mg/ml) produced significant (po 0.05) inhibitory activity (Fig. 4), upon IFN-g/LPS treated macrophages. At the lower concentration of MEMC (50, 25, and 12.5 mg/ml), NO was not significantly inhibited (Fig. 4). L-NAME, a standard NOS inhibitor, used as a positive control in the assay, caused a significant inhibition of NO (p o0.05).

3.6. Cytotoxicity of MEMC MTT assay was conducted to determine the cytotoxicity of the tested extract on the viability of RAW 264.7 cells. In this study, the viability of RAW 264.7 cells upon treatment of MEMC was high for the concentration of 100, 50, 25, and 12.5 mg/ml (Fig. 4). The result showed that the extract did not cause toxicity to the cells and the NO suppressive action of the extract at the highest concentration was not due to the cytotoxicity effect.

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Table 3 Effect of MEMC on several gastric content parameters in pylorus-ligated rat model. Treatment

Volume of gastric juice (ml)

pH

Free acidity (mEq/l)

Total acidity (mEq/l)

Control (8% Tween 80) Ranitidine

6.177 1.20 1.03 7 0.22***

1.25 7 0.04 2.777 0.31***

70.00 77.86 54.83 74.93

131.83 7 9.01 77.677 4.85***

MEMC 100 mg/kg 250 mg/kg 500 mg/kg

2.357 0.62** 1.687 0.49*** 1.60 7 0.50***

1.617 0.17 1.82 7 0.19 2.03 7 0.32

60.33710.18 60.83 79.92 45.83 77.32

99.50 7 5.61* 121.007 7.80 84.83 7 8.45***

Values are expressed as mean7 SEM for six animals in each group. One way ANOVA was followed by Dunnett’s post hoc test. n

po 0.05 as compared to the control group within the respective column. p o0.01 as compared to the control group within the respective column. nnn p o0.001 as compared to the control group within the respective column.

1.5

***

*** ***

1.0

**

0

25

50

0

0 10

R an iti di ne

0.0

MEMC (mg/kg) Fig. 3. Effect of oral administration of vehicle (Tween 80, 8%), ranitidine (100 mg/kg) or MEMC (100, 250 and 500 mg/kg) on gastric wall mucus produced in the stomach. The gastric wall mucus content (mg Alcian Blue/g wet tissue) was expressed as mean7SEM for six animals. One way ANOVA was followed by Dunnett’s post hoc test, ***po0.001, **po0.01 vs. vehicle.

150

Inhibition (%)

assays. The final concentration of the sample was 100 mg/ml. In the lipoxygenase assay, the result exhibited a high inhibition of enzyme activity. Meanwhile, when tested using the xanthine oxidase assay, the extract showed moderate inhibition (Table 4).

4. Discussion

0.5

Ve hi cl e

Gastric wall mucus content (µg Alcian Blue/g wet tissue)

nn

L-NAME (250 µM) MEMC 100 µg/ml MEMC 50 µg/ml MEMC 25 µg/ml MEMC 12.5 µg/ml

100

* * 50

0

Nitric oxide

Cell viability

Fig. 4. Effect of MEMC tested against nitric oxide (NO) production and RAW 264.7 cell viability. The assays were done in triplicate. One way ANOVA was followed by Dunnett’s post hoc test, *p o 0.05 shows significant difference as compared to the IFN-γ/LPS-treated group (inflammation induced group).

Table 4 Effect of MEMC on the anti-inflammatory mediators using the in vitro lipoxygenase and xantine oxidase assays. Sample

MEMC

Concentration (mg/ml) Lipoxygenase (%) Xantine oxidase (%)

100 87.65 7 4.21 (H) 51.89 7 4.58 (M)

All values are expressed as mean7 SEM. Note: H, high (71–100%); M, moderate (41–70%); L, low (0–40%); NA, not active.

3.7. Effects of plant extract on anti-inflammatory mediators MEMC exhibited high anti-inflammatory properties in the two assays tested, namely the lipoxygenase and xanthine oxidase

The present study aimed to investigate the antiulcer mechanism(s) of MEMC. In our previous study, pharmacological evaluation of MEMC revealed that this extract was effective in inhibiting ulcers caused by ethanol and indomethacin (Balan et al., 2013). Meanwhile, in the current study, MEMC administration showed a significant protection against ulcers induced by pylorus ligation. This is one of the most widely used models to study the effect of drugs on gastric acid and mucus secretion. Ulcers developed by ligating the pyloric end of the stomach are caused by an increase in gastric hydrochloric acid (HCl) secretion and/or stasis of acid, leading to auto digestion of the gastric mucosa and breakdown of the gastric mucosal barrier (Kumar et al., 2011). Parietal or oxyntic cells are the principle cell in gastric glands, which secrete gastric acid (HCl) to promote proteolytic digestion of foodstuffs, iron absorption, and killing pathogens (Rang et al., 2012). The three most important mediators, namely acetylcholine, gastrin, and histamine, interact with specific receptors located at the basolateral membrane of the parietal cells that stimulate gastric acid secretion (Oiry et al., 1999). The regulation of gastric acid secretion by the parietal cells is an important factor in the pathogenesis of peptic ulcer. Therefore, the inhibition of gastric acid secretion is a key therapeutic target for the ulcer diseases (Jain et al., 2007). Our findings in this study clearly demonstrated that MEMC inhibited the aggressive factor by significantly reducing the volume of gastric secretion and total acidity in the rats. This may possibly be due to the anti-secretory property of MEMC. Gastric mucus plays an important role in the gastric ulcer defense mechanism, whereby it forms a continuous mucus gel-like protective barrier coating the entire gastric mucosa that maintains the mucosal surface at a pH of 6–7 in the acidic environment (pH 1–2). In gastric ulcers, in spite of low acid secretion, weakening of mucosal defenses can also lead to severe injury (Jain et al., 2007). The important criteria that determine the status of mucosal defense barrier against the unpleasant attack of acid and pepsin are the quality and quantity of gastric mucus secretion (Rachchh and Jain, 2008). According to Venables (1986), rise in amount of mucus secreted by the gastric mucosal cells prevent ulcer formation by acting as an effective barrier to the back diffusion of hydrogen ions, improving the buffering of gastric acid juice and reducing stomach wall friction during peristalsis. The mucus comprises of mucin-type glycoproteins that can be detected by amounts of Alcian Blue binding (Corne et al., 1974). Our present study revealed that administration of MEMC was able to increase

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the amount of mucus secretion, supporting the point that one of the potential mechanisms of the gastroprotective effect elicited by MEMC is a result from enhancement of the gastric mucosal defense action. Overall, treatment of MEMC appears to significantly weaken the gastric aggressive factors by decreasing the amount of gastric secretion and total acidity while enhancing the cytoprotective factor by strengthening the gastric mucosal barrier. This explains the antiulcer effect of MEMC in the pylorus ligation induced ulcer model, whereby MEMC was able to exert significant protection as compared to the control group. The positive control used was ranitidine, which also revealed significant gastroprotective effect. On the other hand, our findings also showed that there was an increase in the pH and free acidity of the gastric secretion, but not significantly higher as compared to the control group. Other than the imbalance between the aggressive and defensive factors, increase in free radicals and oxidative processes also strongly contributes to the ulcer disease. For this reason, it is hypothesized that the ability of an extract/compound to scavenge free radicals and elicit antioxidant properties could also be one of the pathways in which the extract exerts its protective effect. Pathogenesis of gastric ulcer involves oxidative stress with antioxidants plays a very important role in order to protect gastric mucosa (Trivedi and Rawal, 2001) and repairing gastric damage. In our in vitro experiment, MEMC inhibited superoxide and DPPH radical activity effectively. Thus, it can be postulated that MEMC exhibited high antioxidant activity in both the pathways. On the other hand, ORAC is a classic tool for measuring the antioxidant capacity of natural products (Prior et al., 2007). This assay was used to measure the peroxyl radical absorbing capacity of the extract. The ORAC method is unique in its analysis as it takes into account the inhibition time and degree of inhibition into a single quantity by measuring the area under the curve (Mukherjee et al., 2010). Our findings demonstrated that MEMC exhibited high ORAC value indicating a potent antioxidant activity of the extract. In addition, the chemical analysis revealed that the MEMC has high total phenolic content, which explains the high antioxidant activity of MEMC exhibited in various antioxidant pathways. The inflammatory processes are thought to be responsible for producing various mediators, which are involved in the production of reactive oxygen species (ROS) and NO that contribute to the pathogenesis of ulcer disease. Nitric oxide is a ubiquitous mediator and plays an important role as an endogenous modulator of numerous physiological functions. In the gastrointestinal tract (GIT), NO participates in the modulation of the smooth musculature tone, such as the regulation of intestinal peristaltism, gastric emptying, and antral motor activity (Martín et al., 2001). It also helps in maintaining the gastric mucosal blood flow, barrier function, alkaline production, and regulates gastric mucus and acid secretion (Calatayud et al., 2001). In physiological conditions, NO modulates both the integrity and repairing of the tissues (Martín et al., 2001). However, overproduction of NO by the inducible isoforms associated with the tissue injury in the gut during inflammatory reactions including peptic ulcer, chronic gastritis, gastrointestinal cancer, bacterial gastroenteritis, celiac or chronic inflammatory bowel diseases (Barrachina et al., 2001). This shows the double-edged role played by NO in gastrointestinal ulcerative and inflammatory diseases, whereby Barrachina et al. (2001) have reviewed in detail the dual role of nitric oxide in modulating gastrointestinal mucosal defense and injury. Our present study demonstrated significant inhibition of NO by MEMC upon inflammation induced macrophages at its highest concentration, 100 mg/ml, as compared to the positive control group. In contrary, our previous study reported the participation of NO in the gastroprotection exhibited by MEMC (Balan et al., 2013). Therefore, it can be postulated that MEMC exhibited its gastroprotective activity via modulation of NO.

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Besides that, MEMC was also tested for its ability to inhibit xanthine oxidase (XO). XO is an enzyme that generates ROS from the chemical reaction it catalyzes. ROS react with cellular lipids, causing the formation of lipid peroxides, which are metabolized to malondialdehyde, a major product of lipid peroxidation (Abdelwahab et al., 2013). ROS is an important pathogenic method for gastric mucosal injury associated with ethanol consumption (Smith et al., 1996). Huh et al. (1996) also reported that alcoholinduced gastric mucosal damage may be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme, leading to oxidative stress. An endogenous antioxidant defense mechanism may constantly remove the continuous production of ROS during normal metabolism (Lemos et al., 2012). Thus, the moderate inhibition of xanthine oxidase exerted by MEMC in our finding may contribute to its antiulcerative effect via its antioxidant, especially the radical scavenging property as reported earlier in this study. Besides xanthine oxidase, MEMC exhibited high inhibition of lipoxygenase (LOX) enzyme, in vitro. LOX are involved in the metabolism of leukotrienes (LTs), which make it a likely target for biochemical manipulation (Rask-Madsen et al., 1992). The LTs possess potent inflammatory actions that could contribute in the pathogenesis of gastric damage and disturbs the gastric mucosal integrity. According to Boughton-Smith (1989), acute gastric damage caused by ethanol showed a marked increase in the mucosal formation of LTC4 and LTB4, whereby LTC4 causes vasoconstriction in the gastric mucosa. LOX inhibitors are used in the treatment of inflammatory bowel disease (IBD) as there is an increased generation of LTs in the inflamed mucosa (RaskMadsen et al., 1992). Therefore, it can be postulated that LOX partially mediate the production of gastric mucosal lesions induced by damaging agents. The high LOX inhibition exerted by MEMC could partially contribute to the gastroprotective activity of the plant extract. Phytochemical screening of MEMC revealed the presence of flavonoids, saponins, steroids, condensed tannins and polyphenols. Su et al. (2003) and Chen et al. (2005) showed that flavonoids, such as pinocembrin, pinobanksin, pinostrobin, chrysin, isoliquiritigenin and gnaphaliin, are present in the leaves of Muntingia calabura. Flavonoids have been reported to act in the gastrointestinal tract, exhibiting antispasmodic (Lima et al., 2005), anti-secretory, antidiarrhoeal (Di Carlo et al., 1993) and antiulcer properties (La Casa et al., 2000). Flavonoids were also reported to exhibit antioxidant (Tapas et al., 2008; Ferreira et al., 2010) and anti-inflammatory (Sandhar et al., 2011) activities. Flavonoids are able to activate the mucosal defense system through stimulation of gastric mucus secretion and scavenge for the ROS and free radicals produced by ethanol (Abdelwahab et al., 2011). In addition, flavonoids able to decrease ulcerogenic lesions by promoting the formation of gastric mucosa inhibit the production of pepsinogen and diminish acid mucosal secretion (La Casa et al., 2000). Mota et al. (2009) have summarized the literature on 95 flavonoids with varying degrees of antiulcerogenic activity, confirming that flavonoids have a therapeutic potential for the more effective treatment of peptic ulcers. On the other hand, tannins are known to “tan” the outer most layer of the mucosa and to render it less permeable and more resistant to chemical and mechanical injury or irritation (Asuzu and Onu, 1990). Tannins form a protective pellicle by promoting precipitation of protein on the ulcer in order to prevent ulcer development. This pellicle helps in preventing toxic substance absorption and combat the attack of proteolytic enzymes (John and Onabanjo, 1990; Nwafor et al., 1996). Furthermore, the saponins may exert its protective activities in ulceration by the activation of mucous membrane protective factors (Choudhary et al., 2013). Considering all these reports on different substances found in MEMC, it is plausible to suggest that the gastroprotective activity

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of MEMC involved, partly, synergistic action of flavonoids, condensed tannins, and saponins. Our HPLC profiling of MEMC also showed the presence of flavonoid-based compounds. According to Tsimogiannis et al. (2007) flavonoids are divided into five major subgroups, namely flavones, flavonols, flavanonols, flavanones and dihydroflavonols. The UV–vis spectra of flavonoids falls within two absorbance bands called Band A and Band B. Band A, which represents flavones or flavonols, lies in the range of 310–350 nm or 350– 385, respectively, while Band B falls between 250 and 290 nm, resembling much the same in all the aforementioned flavonoid subgroups. Thus, the presence of flavonoids detected in the extract is in consistent with the finding obtained from the HPLC profiling. Furthermore, MEMC, when spiked together with a list of flavonoids, the chromatogram analysis revealed the possible presence of rutin, dihydroquercetin, fisetin and quercitrin. Rutin and quercetin had been reported to prevent gastric mucosal ulceration in several animal models (Barnaulov et al., 1982, 1983, 1985; Martin et al., 1993; Alarcón de la Lastra et al., 1994; Izzo et al., 1994; Martin et al., 1998; La Casa et al., 2000; Kahraman et al., 2003; Rao et al., 2003). Izzo et al. (1994) reported that the protective effect of rutin was mediated by endogenous platelet-activating factor (PAF) whereas La Casa et al. (2000) said that the compound’s antioxidant properties may be another possible mechanism underlying the antiulcer effect of rutin. A latest study revealed that rutin exerts antiulcer effect by inhibiting the gastric proton pump (Dubey et al., 2013). On the other hand, quercetin’s mechanism of action involves an increase in mucus production (Alarcón de la Lastra et al., 1994), endogenous PAF (Izzo et al., 1994), antihistaminic properties, inhibition of Helicobacter pylori (Beil et al., 1995), and its antioxidant properties (Martin et al., 1998; Kahraman et al., 2003). Quercetin was also reported to promote gastric healing (Motilva et al., 1992). Barnaulov et al. (1982) demonstrated the antiulcer effect of quercitrin in reserpin-induced ulceration whereas Kandhare et al. (2011) reported the therapeutic value of fisetin in the prevention of experimental gastric ulcer by virtue of its antioxidant mechanism. In conclusion, our findings from the present study showed that MEMC exerts its gastroprotective effects via various mechanism(s). The ability of MEMC to reduce gastric secretion and total acidity while increase mucus production suggests the balanced protection of MEMC against the aggressive and defensive factors of gastric ulcer. Furthermore, the modulation of NO and inhibition of lipoxygenase, xanthine oxidase and high radical scavenging activity show the mechanisms underlying the gastroprotective effect of MEMC. High phenolic content and presence of phytochemicals such as flavonoids, tannins and saponins explain the effectiveness of the extract in protecting against gastric damages. Further studies are in progress to identify the bioactive compound (s) that may be present, which could be responsible for the antiulcer properties of Muntingia calabura.

Acknowledgment This study was supported by the Science Fund Research Grant (Reference no. 04-01-04-SF1127) awarded by the Ministry of Science Technology and Innovation (MOSTI), Malaysia, and the Research University Grant Scheme (Reference no. 04-02-111395RU) from the UPM, Malaysia. The authors thanked the Faculty of Medicine and Health Sciences, UPM, Malaysia, for providing the facilities to carry out this study. References Abdelwahab, S.I., Mohan, S., Abdulla, M.A., Sukari, M.A., Abdul, A.B., Taha, M.M., Syam, S., Ahmad, S., Lee, K.H., 2011. The methanolic extract of Boesenbergia

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