Gastro-protective effects of the phenolic acids of Macrotyloma uniflorum (horse gram) on experimental gastric ulcer models in rats

Food Bioscience 12 (2015) 34–46

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Gastro-protective effects of the phenolic acids of Macrotyloma uniflorum (horse gram) on experimental gastric ulcer models in rats Vandana Panda n, Swetha Suresh Department of Pharmacology & Toxicology, Prin. K.M. Kundnani College of Pharmacy, Jote Joy Building, Rambhau Salgaonkar Marg, Cuffe Parade, Colaba, Mumbai 400005, India

art ic l e i nf o

a b s t r a c t

Article history: Received 29 December 2014 Received in revised form 23 April 2015 Accepted 13 July 2015 Available online 14 July 2015

Macrotyloma uniflorum Lam. (Verdc.) known as horse gram is an underutilized and unexplored food legume distributed throughout Asia and Africa. Its seeds are rich in phenolic acids, p-coumaric acid being the most abundant phenolic acid. The present study evaluates the antiulcer and antioxidant activity of the hydroalcoholic extract of the seeds of M. uniflorum (MUSE) and p-coumaric acid against indomethacin (non-steroidal anti-inflammatory drug) and absolute ethanol (necrotizing agent) induced ulcers in rats. Pre-treatment with MUSE and p-coumaric acid showed a dose-dependent decrease in the ulcer index in both models. MUSE and p-coumaric acid elicited significant antioxidant activity by attenuating the ulcer elevated levels of malondialdehyde and restored the ulcer-depleted levels of reduced glutathione and the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. In conclusion, MUSE possesses potent antiulcer activity which may be attributed to an underlying antioxidant activity. & 2015 Published by Elsevier Ltd.

Keywords: Macrotyloma uniflorum seed extract p-Coumaric acid Absolute ethanol Indomethacin Antiulcer Antioxidant

1. Introduction Peptic ulcer or Peptic ulcer disease (PUD) is defined as a break in the mucosal lining of the gastrointestinal tract (Yuan, Padol, & Hunt, 2006). It occurs in that part of the gastrointestinal tract which is exposed to gastric acid and pepsin, i.e. the stomach and duodenum. The normal stomach mucosa maintains a balance between defensive and aggressive factors (Dimaline & Varro, 2007). Some of the main aggressive factors are gastric hydrochloric acid, abnormal motility, pepsin, bile salts, leukotrienes, free radicals, alcohol and nonsteroidal anti-inflammatory drugs (NSAID), as well microorganisms such as Helicobacter pylori. On the other hand, defensive factors such as the mucus-bicarbonate barrier, surface active phospholipids, prostaglandins, endogenous nitric oxide, endogenous antioxidants and normal tissue microcirculation protect against ulcer formation. Although the etiology of ulcer is still not fully understood, it is generally accepted that peptic ulcers develop when aggressive factors (endogenous, exogenous and/or infectious agents such as H. pylori) overcome mucosal defense mechanisms (Tulassay & Herszenyi, 2010). The incidence of PUD varies with age, gender and geographical location and is associated with severe complications including hemorrhage, perforation, gastrointestinal obstruction and malignancy (Brown & Wilson, n

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http://dx.doi.org/10.1016/j.fbio.2015.07.004 2212-4292/& 2015 Published by Elsevier Ltd.

1999). Thus, this clinical condition represents a serious health problem worldwide because of its high morbidity, mortality and economic loss. Modest approaches to control peptic ulcers include potentiation of the mucosal defense, reduction in acid secretion and its neutralization, enhancement of antioxidant levels in the stomach, stimulation of gastric mucin synthesis and inhibition of H. pylori growth. Several classes of pharmacological agents have proved to be effective in the management of acid peptic disorders. They include antacids (aluminum hydroxide and magnesium trisilicate), acid suppressive agents such as proton pump (H þ /K þ ATPase) inhibitors (e.g., omeprazole and lansoprazole), H2 receptor antagonists (cimetidine and ranitidine), anticholinergic agents (pirenzepine), cytoprotective agents (sucralfate and misoprostol) and antimicrobials for eradication of H. pylori (amoxicillin and clarithromycin) (Waller, Renwick, & Hillier, 2005). However, gastric ulcer therapy faces a major drawback because most of the currently available drugs in the market show limited efficacy and are often associated with severe side effects (Lehne, 1998). In this context, the use of medicinal plants is on a global rise for the prevention and treatment of different pathologies and natural products are regaining importance in the pharmaceutical industry as inspiring sources of new potentially bioactive molecules. Clinical research has confirmed the efficacy of several plants for the treatment of gastroduodenal diseases (Schmeda-Hirschmann & Yesilada, 2005). Most of these plant derived drugs augment the mucosal defensive factors which are thought to be important for

V. Panda, S. Suresh / Food Bioscience 12 (2015) 34–46

protection of gastric mucosa (Tovey et al., 2011). The medicinal properties of plants are attributed mainly to the presence of flavonoids, but they may be also influenced by other organic and inorganic compounds such as coumarins, alkaloids, fatty acids, terpenoids, tannins, phenolic acids and micronutrients, such as copper, manganese, iron, zinc and selenium among others. Horse gram [Macrotyloma uniflorum Lam. (Verdc.)], previously [Dolichos biflorus] belonging to the family Fabaceae is a lesser known drought resistant legume grown throughout Asia, Africa and Australia and primarily utilized as a feed to animals and horses. In India it is known as the “poor man’s pulse” and used as a staple food. The horse gram seed is reported to be high in tannins and polyphenols when compared with other legumes (Kadam & Salunkhe, 1985). It is prescribed for persons suffering from jaundice or water retention and as part of a weight loss diet. It is useful in iron deficiencies and is considered helpful for maintaining body temperature in the winter season. The seeds of M. uniflorum are used in traditional medicine as bitter, thermogenic, astringent, anthelmintic, diaphoretic, diuretic, expectorant, ophthalmic and tonic. The seeds are also useful for hemorrhoids, tumors, bronchitis, splenomegaly and in asthma (Marimuthu & Krishnamoorthi, 2013). Reports of lipids obtained from horse gram in healing of peptic ulcers in rats successfully are available in literature (Jayaraj, Tovey, Lewin, & Clark, 2000). Seeds of M. uniflorum contain varying amounts of carbohydrates, proteins, amino acids, lipids, phenolic acids (3,4-dihydroxy benzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, chlorogenicacid, syringic acid and sinapic acid), flavonoids and tannins (quercetin, kaempferol and myricetin), fatty acids (hexanoic acid and hexadecanoic acid), phytosterols (stigmasterol and β-sitosterol), anthocyanidins (cyanidin, petudin, delphinidin and malvidin), saponins and minerals like iron, calcium and molybdenum (Kawsar, Huq, Nahar, & Ozeki, 2008). Phenolic acids obtained from M. uniflorum are considered to be potent antioxidants which act by scavenging free radicals and reactive oxygen species (Siddhuraju & Manian, 2007; Sreerama, Sashikala, & Pratape, 2010). Oxidative damage is considered to be a major mechanism in the pathogenesis of ulcer. Several phenolic acids such as caffeic, pcoumaric, ferulic and cinnamic acids have been documented to possess gastroprotective activity (Barros et al., 2008). Since, horse gram contains all these acids, the present study was undertaken to evaluate a polyphenol rich fraction of M. uniflorum seed extract for antiulcer activity. p-Coumaric acid being the most abundant phenolic acid, its antiulcer activity was also evaluated separately and compared with the extract.

2. Materials and methods 2.1. Plant material The seeds of M. uniflorum were collected from the Colaba market, Mumbai, India. The seeds were air dried under shade, powdered mechanically and stored in air tight containers. The powder was extracted using a mixture of ethanol: water (80:20) v/ v in a Soxhlet apparatus. This extract was dried and stored in a refrigerator for further use. The plant was authenticated at the Blatter Herbarium, St. Xavier’s College, Mumbai after matching with the existing specimen (specimen no. AD- 6). 2.2. Isolation of p-coumaric acid by preparative HPTLC After successful development of TLC, preparative High Performance Thin Layer Chromatography (HPTLC) of the hydroalcoholic extract of the seeds of M. uniflorum (MUSE) was carried out on the

35

CAMAG HPTLC System for isolation and identification of p-coumaric acid. The absorbance value of different bands in the crude extract after TLC separation was studied using the Desaga Scanner for the most possible wavelength absorption of p-coumaric acid. Prior to MUSE application, HPTLC plates (HPTLC Silica gel 60 F254, Merck) of 10  10 cm2 were activated at 110 °C for 30 min. MUSE (100 mg) was dissolved in 10 ml of methanol and 100 mL of this solution was applied as a single band of 180 mm length on the activated HPTLC plates using a Linomat V applicator (CAMAG, Switzerland). The plates were then developed with 10 ml of the solvent system comprising toluene: ethyl acetate: formic acid (6.8:2.3:0.9) (Medic-Saric, Jasprica, Smolcic-Bubalo, & Mornar, 2004) in a twin trough chromatographic chamber and examined at 315 nm for p-coumaric acid. After development, the constituent at Rf 0.55 was marked and scraped out from the plate. The scraped material was mixed with methanol and eluted from silica gel by centrifugation at 3000 rpm. The supernatant was evaporated on a water bath to get p-coumaric acid. Further confirmation of the isolated constituent was done by using UV–visible, IR and NMR spectroscopies for major functional groups. 2.3. Quantification of p-coumaric acid using HPLC 2.3.1. Chemicals and reagents HPLC grade methanol, acetonitrile and analytical grade formic acid were procured from Mercks India Ltd. (Mumbai). All the solvents and solutions were filtered through a membrane filter (Millipore filter paper, 0.45 μm pore size) and degassed before use. Standard p-coumaric acid was procured from Sigma-Aldrich Pvt. Ltd., MO, USA. 2.3.2. Instrumentation and materials Analysis was performed on Jasco HPLC system with Jasco PU2080 Plus Quaternary Gradient HPLC Pump and in-built Jasco MD2010 Plus PDA multi wavelength detector. Chromatographic software Chrompass was used for data collection and processing. The analytical column was LC–GC Qualisil BDS C18 (5 mm, 250 mm  4.6 mm). 2.3.3. Chromatographic conditions Chromatographic separation of p-coumaric acid was carried out using an isocratic mobile phase comprising water: acetonitrile (80:20) v/v at pH 3.5 (adjusted with formic acid) (Mas, Fonrodona, Tauler, & Barbosa, 2007). A well resolved and sharp peak for pcoumaric acid with a retention time (Rt) of 8 min was obtained. The flow rate was maintained at 1.0 mL/min and the detection carried out at 315 nm. 2.3.4. Preparation of standards A stock solution (1000 mg/ml) was prepared by dissolving 10 mg of p-coumaric acid in 10 ml of methanol. This solution was further diluted with the mobile phase to give a stock solution of 100 μg/mL. Further dilutions were made as required with the mobile phase. 2.3.5. Preparation of calibration curve The calibration curve was prepared by injecting various concentrations (10–50 mg/mL) of the standard p-coumaric acid solution. 2.3.6. Quantification of p-coumaric acid in MUSE MUSE (50 mg) was dissolved in 10 ml methanol to yield a solution of 5 mg/mL. This solution (20 mL) was injected and the elution was carried out using the mobile phase mentioned earlier. The amount of p-coumaric acid in MUSE was calculated from the calibration curve.

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2.4. Drugs and chemicals Thiobarbituric acid (TBA), trichloroacetic acid (TCA), reduced glutathione, oxidized glutathione and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Himedia Laboratories, Mumbai, India. Epinephrine, histamine and 5,5′-dithiobis (2-nitrobenzoic acid)-(DTNB) were purchased from Sigma Chemical Co., St Louis, MO, USA. All other chemicals were obtained from local sources and were of analytical grade. 2.5. Animals Wistar albino rats (180–200 g) of either sex were used. They were housed in clean polypropylene cages under standard conditions of humidity (507 5%), temperature (25 72 °C) and light (12 h light/12 h dark cycle) and fed with a standard diet (Amrut laboratory animal feed, Pune, India) and water ad libitum. All animals were handled with humane care. Experimental protocols were reviewed and approved by the Institutional Animal Ethics Committee (Animal House Registration No. 25/1999/CPCSEA) and conform to the Indian National Science Academy Guidelines for the Use and Care of Experimental Animals in Research. 2.6. Preparation of test and reference drug solutions The hydroalcoholic extract of the seeds of M. uniflorum (MUSE) was dissolved in distilled water and the aqueous solution was used. The reference drug omeprazole was prepared as a suspension in 1% (w/v) aqueous carboxymethyl cellulose solution and used immediately. The toxicant drug indomethacin was prepared as a suspension in 1% (w/v) aqueous carboxymethyl cellulose solution and administered immediately. 2.7. Antiulcer activity The effects of MUSE were evaluated in ethanol and indomethacin induced ulcer models in rats. Omeprazole was used as a standard drug in both models for comparing the antiulcer potential of MUSE. 2.8. Indomethacin induced gastric ulceration All rats were fasted for 24 h but allowed free access to water. They were randomly divided into 6 groups of 6 rats each and treated in the following way:

The stomachs were then weighed, washed with ice cold saline and divided into two parts. One part was used to prepare a homogenate (10% w/v) in 1.15% (w/v) KCl. An aliquot of the homogenate was used for the estimation of lipid peroxidation (LPO). The homogenates were centrifuged at 7000  g measured at the bottom of the tube for 10 min at 4 °C and the supernatants were used for the assay of endogenous antioxidants like reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The remaining part of the stomach was fixed in 10% buffered formalin and used for histological studies. 2.9. Ethanol induced gastric ulceration All rats were fasted for 24 h but allowed free access to water. They were randomly divided into 6 groups of 6 rats each and treated in the following way: Group I (Normal Control): rats were administered distilled water (1 ml/kg, p.o.). Group II (Toxicant Control): rats received absolute ethanol (1 ml/ 200 g, p.o.). Group III (MUSE 250): rats received MUSE (250 mg/kg p.o.), 30 min prior to absolute ethanol (1 ml/ 200 g, p.o.) administration. Group IV (MUSE 500): rats received MUSE (500 mg/kg p.o.), 30 min prior to absolute ethanol (1 ml/ 200 g, p.o.) administration. Group V: rats received p-coumaric acid (250 mg/kg), 30 minutes prior to absolute ethanol (1 ml/ 200 g, p.o.) administration. Group VI (Standard): rats received omeprazole (20 mg/kg, p.o.), 30 min prior to absolute ethanol (1 ml/ 200 g, p.o.) administration. After 4 h all rats were sacrificed by an overdose of ketamine and the stomachs were removed and opened along the greater curvature. The stomachs were scored for ulcers, assayed for LPO, GSH, SOD, CAT, GPx and GR and studied for histo-architectural changes in a manner similar to the earlier model. 2.10. Ulcer assessment The stomachs were harvested, opened along the greater curvature and the mucosa was exposed for macroscopic evaluation. The ulcerated area was assessed and the ulcer index (UI) was calculated for each treatment by the method of Vogel and Vogel (1997). 2.11. Mean scoring

Group I (Normal Control): rats were administered distilled water (1 ml/kg, p.o.). Group II (Toxicant Control): rats received indomethacin (20 mg/kg, p.o.). Group III (MUSE 250): rats received MUSE (250 mg/kg, p.o.) and after 1 h were administered indomethacin (20 mg/kg, p.o.). Group IV (MUSE 500): rats received MUSE (500 mg/kg, p.o.) and after 1 h were administered indomethacin (20 mg/kg, p.o.). Group V: received p-coumaric acid (250 mg/kg, p.o) and after 1 h were administered indomethacin (20 mg/kg, p.o.). Group VI (Standard): received standard omeprazole (20 mg/kg, p.o.) and after 1 h were administered indomethacin (20 mg/kg, p.o.). After 4 h all rats were sacrificed by an overdose of ketamine and the stomachs were removed and opened along the greater curvature. Ulcers formed in the glandular portion of the stomach were observed under magnifying glass for scoring and ulcer index.

A score for the ulcer was made as follows: 0: Normal colouration 0.5: Red colouration 1: Spot ulcers 1.5: Hemorrhagic streaks 2: Ulcers 43 mm but o5 mm 3: Perforation

2.12. Ulcer index UI ¼ UNþUS þUP  10  1 UI ¼ Ulcer Index UN ¼Average of number of ulcers per animal US ¼ Average of severity score UP ¼ Percentage of animal with ulcers

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2.13. Lipid peroxidation

3. Results

The quantitative estimation of LPO was performed by determining the concentration of thiobarbituric acid reactive substances (TBARS) in the gastric tissue using the method of Buege and Aust (1978). The amount of malondialdehyde (MDA) formed was quantified by reaction with TBA and used as an index of lipid peroxidation. The results were expressed as nmol of MDA/mg protein using molar extinction coefficient of the chromophore (1.56  10  5/M/cm) and 1,1,3,3-tetraethoxypropane as standard.

3.1. Isolation, characterization and quantification of p-coumaric acid Fig 1a and b are HPLC chromatograms for standard p-coumaric acid and MUSE respectively. From the calibration curve (Fig. 2), the amount of p-coumaric acid present in MUSE was 1.375% w/w.

2.14. Glutathione estimation

3.1.1. UV spectrum (Fig. 3) The wavelength of maximum absorption (λmax) of the isolated constituent was observed to be at 288 nm which matches with that of standard p-coumaric acid (Fig. 3).

GSH was estimated in the stomach homogenate using DTNB by the method of Ellman (1959). The absorbance was read at 412 nm and the results were expressed as mmol of GSH/g of wet tissue.

3.1.2. IR spectrum (Fig. 4) The IR spectrum of the isolated constituent showed the following peaks and following were their assignments (Fig. 4):

2.15. Estimation of antioxidant enzymes

Wave number (cm  1)

Attributes

Superoxide dismutase (SOD) was assayed by the method of Sun and Zigman (1978) in which the activity of SOD was inversely proportional to the concentration of its oxidation product adrenochrome, which was measured spectrophotometrically at 320 nm. 1 unit of SOD activity is defined as the enzyme concentration required to inhibit the rate of auto-oxidation of epinephrine by 50% in 1 min at pH 10. Catalase (CAT) was estimated by the method of Clairborne (1985), which is a quantitative spectroscopic method developed for following the breakdown of H2O2 at 240 nm in unit time for routine studies of catalase kinetics. Glutathione peroxidase (GPx) estimation was carried out using the method of Rotruck, Pope, Ganther, Hofeman, and Hoekstra (1973), which makes use of the following reaction.

3356.25 2972 1600.97 1674.27 1626.05

OH stretching CH stretch of C ¼C 6 Membered aromatic stretching C ¼O stretching C ¼C stretching

H2O2 þ 2GSH-2H2O þGSSG (oxidized glutathione) GPx in the tissue homogenate oxidizes glutathione and simultaneously, H2O2 is reduced to water. This reaction is arrested at 10 min using trichloroacetic acid and the remaining glutathione is reacted with DTNB solution to result in a colored compound, which is measured spectrophotometrically at 420 nm. Glutathione reductase (GR) activity was determined by using the method of Mohandas, Marshal, Duggin, Horvath, and Tiller (1984), in which the following reaction is implicated. þ

þ

NADPH þH þGSSG-NADP þ2GSH In the presence of GR, oxidized glutathione undergoes reduction and simultaneously, NADPH is oxidized to NADP þ . Enzyme activity is quantified at room temperature by measuring spectrophotometrically at 340 nm, the disappearance of NADPH/min. 2.16. Histopathological studies The parts of the stomachs which were stored in 10% buffered formalin were embedded in paraffin, sections cut at 5 μm and stained with hematoxylin and eosin. These sections were then examined under a light microscope for histo-architectural changes. 2.17. Statistical analysis The results of antiulcer and antioxidant activities are expressed as mean 7SEM. Results were statistically analyzed using one-way ANOVA, followed by the Bonferroni test for individual comparisons. P o0.05 were considered to be significant. GraphPad In Stat version 4.00 of Graph Pad Software Inc., San Diego, USA was the software used for statistical analysis.

The above spectral findings matched with those of standard pcoumaric acid. 3.1.3. NMR spectrum (Fig. 5) The NMR spectrum of the isolated constituent showed the following chemical shifts (Fig. 5): Atom

Chemical shift (δ ppm)

Multiplicity

OH (1) CH (2,6) CH (3,5) CH (1′) CH (2′) OH (3′)

2.77 7.55 6.92 6.34 7.62 8.72

Broad singlet Doublet doublet Doublet doublet Doublet Doublet Broad singlet

The chemical shifts in the spectrum above matched with those of standard p-coumaric acid. 3.2. Indomethacin induced ulceration in rats Indomethacin administration caused marked ulcers along with mucosal defects that were circular and/or oval in shape and dispersed to all stomach surfaces as seen in the image of the indomethacin treated rats (Fig. 6b). There was remarkable hyperemia in the stomachs of indomethacin-administrated rats when compared with stomachs of untreated normal rats (Fig. 6a). However, there was a marked reduction in the damage in stomachs of omeprazole, MUSE (250 and 500 mg/kg) and p-coumaric acid treatment groups when compared with stomachs of the indomethacin control group of rats (Fig. 6c, d, e and f respectively). The effects of MUSE (250 mg/kg and 500 mg/kg) and p-coumaric acid treatment on mean ulcer score, ulcer index and percent ulcer inhibition in the indomethacin induced ulcer model are summarized in Table 1. Pretreatment of MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole to indomethacin administered rats caused significant reduction in the mean ulcer score and ulcer index in their stomachs when compared with those of the indomethacin control group.

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Fig. 1. (a) HPLC Chromatogram of standard p-coumaric acid at 315 nm. (b) HPLC chromatogram of MUSE at 315 nm.

Fig. 2. Calibration curve of standard ferulic acid by HPLC.

3.2.1. LPO, GSH and antioxidant enzymes The effect of MUSE and p-coumaric acid on endogenous antioxidants and LPO is summarized in Table 2. MDA, the lipid peroxidation marker was significantly elevated in the stomach homogenates of the indomethacin control group of rats when compared with the Normal group rats. Pretreatment of MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole to indomethacin administered rats attenuated significantly the elevated MDA levels. Significant depletion in GSH level was observed in the stomachs of the indomethacin treated group of rats when compared with the Normal group of rats. Treatment of MUSE 250, MUSE 500, p-coumaric acid and omeprazole to indomethacin intoxicated rats significantly restored the depleted GSH levels. SOD activity in the stomachs of the rats of the indomethacin

V. Panda, S. Suresh / Food Bioscience 12 (2015) 34–46

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Fig. 3. UV spectrum of isolated compound.

Fig. 4. IR spectrum of isolated compound.

treated group was examined to be strikingly lower than in the Normal group. MUSE 500, p-coumaric acid and omeprazole administration to indomethacin treated rats elevated significantly the indomethacin depleted SOD activity. CAT activity in the stomach homogenates of indomethacin treated rats was measured to be significantly lower than that in the Normal Control rats. MUSE, p-coumaric acid and omeprazole treatment to indomethacin intoxicated rats significantly restored the depleted CAT activity. Stomach GPx activity in the indomethacin treated group of rats showed a significant depletion when compared with the Normal group. MUSE, p-coumaric acid and omeprazole treatments to the toxicant treated rats significantly restored the GPx activity depleted by indomethacin. A significant decrease in GR activity in the stomachs of indomethacin treated animals was noted when compared with the Normal group. MUSE, p-coumaric acid and omeprazole treatment to the indomethacin treated rats restored significantly the GR levels depleted due to indomethacin.

3.2.2. Histopathological studies Histological observation of stomach slices of the Normal group of rats showed normal histo-architecture with an intact epithelium [Fig. 7a]. The stomach tissue of the indomethacin treated rats revealed several gastric lesions and moderate hemorrhage with desquamation of mucosal cells and inflammatory cell infiltration [Fig. 7b]. Omeprazole treated rats showed minimal infiltration and mild desquamation of mucosal cells [Fig. 7c]. Rats that received pretreatment with MUSE 250 showed milder inflammatory cell infiltration and mucosal desquamation in their stomachs when compared with the indomethacin control group stomachs [Fig. 7d]. MUSE 500 and p-coumaric acid treated rat stomachs showed the least inflammatory cell infiltration and mucosal desquamation when compared with the indomethacin treated rat stomachs, indicative of good ulcer healing [Fig. 7e and f]. 3.3. Ethanol-induced ulceration in rats Absolute ethanol administration caused a marked damage to the mucosa as seen in the image of the stomach of the ethanol

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Fig. 5. NMR spectrum of isolated compound.

Fig. 6. Observation of ulcers in indomethacin induced ulceration in rats. Note: (a) Normal (untreated) group; (b) Indomethacin (20 mg/kg) treatment group; (c) Omeprazole (20 mg/kg) treatment group; (d) MUSE (250 mg/kg) treatment group; (e) MUSE (500 mg/kg) treatment group; (f) p-coumaric acid (250 mg/kg) treatment group.

treated rat when compared with the stomach of the Normal group rat [Fig. 8a and b]. Ethanol produced characteristic lesions in the glandular portion of the stomach which appear as elongated thick bands of red and black. The stomach also showed prominent

ulcers. However, there was a marked reduction in the damage in stomachs of the omeprazole, MUSE (250 and 500 mg/kg) and pcoumaric acid treated groups when compared with the Ethanol Control group (Fig. 8c–f). The MUSE 500, p-coumaric acid and

14.78 7 0.43nnn 87.06 7 0.73nnn 9.838 7 0.166nnn 21.45 7 0.54nnn 2278.007 15.05nnn 13.98 7 0.22nnn 84.65 7 0.80nnn 9.4177 0.275nnn 19.53 7 0.50nnn 2019.007 36.12nnn

P value: o0.001 when Indomethacin (toxicant) Control compared with Normal Control. P value: o 0.05 when experimental groups compared with Indomethacin Control. nnn P value: o 0.001 when experimental groups compared with Indomethacin Control. n

a

3.3.2. Histopathological studies Histological observation of stomach slices of the Normal group

Values are Mean 7SEM; N ¼6 in each group. One-way ANOVA followed by Bonferroni’s test is applied for statistical analysis. 1 unit of CAT ¼mmol H2O2 consumed/min/mg protein. 1 unit of GPX ¼ mg GSH utilized/min/mg protein. 1 unit of GR ¼ nmol NADPH oxidized/min/mg protein. nn P value: o0.01 when experimental groups compared with Indomethacin Control.

3.3.1. LPO, GSH and antioxidant enzymes The effect of MUSE on gastric LPO, GSH and antioxidant enzymes (SOD, CAT, GPx and GR) is presented in Table 4. MDA, the lipid peroxidation marker was significantly elevated in the Ethanol Control group of rats when compared with the Normal group. Pretreatment of MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole to rats administered ethanol attenuated significantly the elevated MDA levels. Significant decrease in GSH levels was observed in the ethanol treated group of rats when compared with the Normal group of rats. Treatment of MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole to ethanol treated rats significantly restored the depleted GSH levels. SOD activity in the ethanol treated group of rats was examined to be strikingly lower than in the Normal group. MUSE (250 mg/kg & 500 mg/kg), p-coumaric acid and omeprazole treatment to ethanol administered rats elevated the SOD activities significantly. CAT activity in stomach homogenates of rats of the ethanol treated group was measured to be significantly lower than in the Normal group. Stomach CAT activities in MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole treatment groups were significantly higher than those in the absolute ethanol treated group. Stomach GPx activity in the ethanol treated group of rats showed a significant decline when compared with the Normal group of rats. MUSE 500, omeprazole and p-coumaric acid treatment to alcohol intoxicated rats elevated the GPx level significantly. GR activity was noted to be depleted significantly by absolute ethanol treatment. Stomach homogenates of rats pre-treated with MUSE (250 mg/kg and 500 mg/kg), p-coumaric acid and omeprazole followed by ethanol administration showed significant restoration of the ethanol depleted GR activity.

Table 2 Effect of MUSE and p-coumaric acid on stomach TBARS, GSH, SOD, CAT, GPx and GR in indomethacin induced ulcers in rats.

omeprazole treated stomachs showed only redness and no ulcers. However, MUSE 250 treatment group showed a little amount of ulceration. The effect of MUSE (250 and 500 mg/kg) and p-coumaric acid treatment on mean ulcer score, ulcer index and percent ulcer inhibition is summarized in Table 3. A marked reduction in the mean ulcer score and ulcer index was observed in the MUSE (250 and 500 mg/kg), p-coumaric acid and omeprazole treatment groups when compared with the ethanol treated group of rats.

8.963 7 0.471n 72.88 7 2.74 8.564 7 0.576n 9.299 7 0.638nnn 1678.007 13.76nnn

n

16.4 7 0.1285nnn 95.217 1.13nnn 10.25 7 0.04nnn 31.29 7 0.68nnn 2429.007 13.97nnn

P value: o 0.001. P value: o 0.01. P value: o 0.05.

nn

4.839 7 0.153a 70.90 7 1.34a 6.87 7 0.14a 5.1097 0.719a 773.30 7 25.97a

nnn

20.767 1.93 97.99 7 1.05 12.32 7 0.33 40.137 0.58 2632.007 39.74

Values are Mean 7 SEM; N¼ 6 in each group. One way ANOVA followed by Bonferroni’s test is applied for statistical analysis, treatment groups were compared with toxicant (Indomethacin) group.

3.6217 0.170nnn

25.92561 55.83263 55.0634

4.3727 0.124nnn

8.763 70.076nnn 5.225 70.072nnn 5.316 70.093nnn

71.25951

7.439 7 0.039nnn

1.583 70.583n 0.9167 70.3745nn 1.167 70.460nn

3.4100 70.0218

– nnn

3.229 7 0.361nnn

11.83 70.251 nnn

10.3517 0.455a

0.3333 70.2108

% Ulcer inhibition

2.756 7 0.335

4.083 70.676

Ulcer index

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TBARS (nmol MDA/mg protein) GSH (mmol/g wet tissue) SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) GR (U/mg protein)

Indomethacin (20 mg/ kg) Omeprazole (20 mg/ kg) MUSE (250 mg/kg) MUSE (500 mg/kg) p-Coumaric acid (250 mg/kg)

Mean ulcer score

Group I Normal Control Group II Indomethacin (20 mg/ kg)

Treatment group and dose

Biochemical parameters

Table 1 Effect of MUSE and p-coumaric acid on ulcer parameters in indomethacin induced ulcers in rats.

Group III Omeprazole (20 mg/kg) Group IV MUSE (250 mg/kg) Group V MUSE (500 mg/kg) Group VI p-coumaric acid (250 mg/ kg)

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Fig. 7. H&E staining of stomach slices in indomethacin induced ulcers in rats. Note: (a) H&E staining of stomach of normal rat 10  10  ¼100  . (b) H&E staining of stomach of Indomethacin (20 mg/kg) treated rat 10  10  ¼ 100  . (c) H&E staining of stomach of omeprazole (20 mg/kg) and Indomethacin (20 mg/kg) treated rat 10  10  ¼ 100  . (d) H&E staining of stomach of MUSE (250 mg/kg) and Indomethacin (20 mg/kg) treated rat 10  10  ¼ 100  . (e) H&E staining of stomach of MUSE (500 mg/kg) and Indomethacin (20 mg/kg) treated rat 10  10  ¼ 100  . (f) H&E staining of stomach of p-coumaric acid (250 mg/kg) and Indomethacin (20 mg/kg) treated rat 10  10  ¼ 100  .

of rats showed normal histo-architecture with an intact epithelium [Fig. 9a]. The stomach tissue of the ethanol treated rats showed several gastric lesions and severe hemorrhage with inflammatory cell infiltration [Fig. 9b]. Omeprazole treated rats exhibited only a mild inflammatory cell infiltration [Fig. 9c]. Rats that received pretreatment with MUSE 250 showed lesser inflammatory cell infiltration and milder hemorrhage in the stomach when compared with the ethanol treated rats [Fig. 9d]. MUSE 500 and p-coumaric acid treated rat stomachs showed minimal inflammatory cell infiltration when compared with the ethanol treated group, indicative of good ulcer healing [Fig. 9e and f].

4. Discussion Phenolic acids are naturally occurring compounds found in the plant kingdom with unique structural similarities and a presence of carboxylic groups, as in caffeic, gallic, p-coumaric, vanillic, ferulic, and protocatechuic acids. Phenolic acids from grape seed (Saito, Hosoyama, Ariga, Katapka, & Yamaji, 1998), cacao liquor (Osakabe, Sanbongi, Yamagishi, Takizawa, & Osawa, 1998), Brazilian green propolis (Barros et al., 2008) and prickly pear cactus (Galati et al., 2003) have been shown to possess good antiulcer activity. The antioxidant activity of phenolic acids may be an important contributing factor for their antiulcer effect, since free radicals/ reactive oxygen species (ROS) are related to the occurrence of ulcers (Das, Bandyopadhyay, Bhattacharjee, & Banerjee, 1997). Studies have shown that phenolic acids such as p-coumaric acid, ferulic acid, caffeic acid and cinnamic acids display antisecretory effects and anti-histaminic effects. They have been shown to enhance mucosal defensive factors and increase the

prostaglandin content and mucus formation in the gastric mucosa, showing a cytoprotective effect (Barros et al., 2008). Phenolic acids play a major role in down regulating parietal cell H þ , K þ -ATPase; cinnamic acid and p-coumaric acid exhibiting the maximum inhibitory activity (Nanjundaiah, Nayaka, & Shylaja, 2011). They also inhibit the ulcerogen H. pylori (Nanjundaiah et al., 2011). The M. uniflorum seed has been found to be rich in phenolic acids and around eight phenolic acids have been identified in it. The phenolic acids obtained from M. uniflorum are considered to be potent antioxidants which act by scavenging free radicals and ROS (Siddhuraju & Manian, 2007). The present study has evaluated the antiulcer and antioxidant activities of MUSE and tried to investigate if antioxidant activity is an underlying mechanism for its antiulcer activity. Chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin is often associated with the development of adverse gastrointestinal disorders such as gastric erosion, gastric or duodenal ulceration and other complications such as gastrointestinal hemorrhage or perforation that often limits their wide spread clinical use (Sostres, Gargallo, Arroyo, & Lanas, 2010). Indomethacin is known to induce gastric ulceration by inhibiting the biosynthesis of gastric mucosal PGE2 which is cytoprotective to the gastric mucosa (Sostres et al., 2010). This effect occurs via COX 1 isoform inhibition, creating a gastric environment that is more susceptible to topical attack by endogenous and exogenous aggressive factors (Vane & Botting, 1995). Moreover, the inhibition of the COX 1 blocks platelet production of thromboxane, which increases bleeding when an active gastrointestinal bleeding site is present (Sostres et al., 2010). The COX 1 expression in stomach produces prostaglandins (PG) that play a protective role by

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Fig. 8. Observations of ulcers in ethanol induced ulceration in rats. Note: (a) Normal (untreated) group; (b) ethanol treatment group; (c) Omeprazole (20 mg/kg) treatment group; (d) MUSE (250 mg/kg) treatment group; (e) MUSE (500 mg/kg) treatment group; (f) p-coumaric acid (250 mg/kg) treatment group. Table 3 Effect of MUSE and p-coumaric acid on ulcer parameters in ethanol induced ulcers in rats. Treatment groups ans dose Mean Ulcer score Ethanol (1 ml/200 gm) Omeprazole (20 mg/kg) MUSE (250 mg/kg) MUSE (500 mg/kg) p-Coumaric acid (250 mg/ kg)

3.583 70.554 0.8333 70.4014nnn 1.511 70.365nn 0.751 70.281nnn 0.8333 70.3073nnn

Ulcer index

% Inhibition

10.81 7 0.12 5.1587 0.055nnn 8.7167 0.056nnn 6.9417 0.087nnn 6.8667 0.054nnn

– 52.28492 19.37095 35.79093 36.48474

Values are mean7 SEM; N ¼6 in each group One-way ANOVA followed by Bonferroni’s test is applied for statistical analysis, Treatment groups were compared with Ethanol (toxicant) group. nnn nn

P value: o 0.001. P value: o 0.01.

stimulating the synthesis and secretion of mucus and bicarbonate, by increasing mucosal blood flow and by regulating mucosal cell turnover and repair (Aly, 1987). So, the COX-1-mediated PG synthesis is mainly responsible for maintaining gastric mucosal integrity at baseline. The observed antiulcer property of MUSE may be due to an increased synthesis of mucus and/or prostaglandins. Neutrophil- and oxygen radical-dependent microvascular injuries are important events that lead to gastric mucosal injury induced by indomethacin (Naito & Yoshikawa, 2006). Reactive oxygen species (ROS) produced by activated neutrophils after indomethacin treatment cause gastric mucosal injury via ROSmediated oxidation of important biomolecules such as lipid, protein, and DNA. Indomethacin seems to initiate peroxidative degradation of membrane lipids and endoplasmic reticulum rich in PUFA, leading to formation of lipid peroxides which in turn give

products like MDA that cause loss of integrity of cell membrane and damage to the gastric mucosa (Basivireddy, Vasudevan, Jacob, & Balasubramanian, 2002). In rats treated with indomethacin alone, the increase in MDA levels indicates enhanced peroxidation leading to a failure of the antioxidant defense mechanism to prevent formation of excess free radicals. Pretreatment with MUSE (250 mg/kg and 500 mg/kg) and p-coumaric acid prevented significantly the increased formation of MDA. Reduced glutathione is one of the most abundant non-enzymatic biological antioxidants present in the stomach. Together with GPX, GR and CAT–SOD couple, it efficiently scavenges ROS produced by indomethacin (Townsend, Tew, & Tapiero, 2003). As a substrate for antioxidant enzymes GPX and glutathione S-transferase (GST), it protects cellular constituents from the damaging effects of peroxides formed during metabolism and other ROS (Mannervik, 1987). Stomach injury has been observed when GSH stores are markedly depleted. Decreased GSH levels in indomethacin administered rats may be due to its increased utilization for enhancing the activities of GSH related enzymes GPX and GR. The reduced glutathione levels were significantly elevated with MUSE (250 mg/kg and 500 mg/kg) and p-coumaric acid treatment. It may be understood that the effect of MUSE and pcoumaric acid may be due to an initial reduction in peroxidative activities followed by inhibition of the activities of GSH related enzymes, thereby leading to restoration of the GSH content. It is known that SOD, CAT and GPx constitute a mutually supportive enzyme system of the first line cellular defence against oxidative injury, decomposing O2 and H2O2 before their interaction to form the more harmful hydroxyl (OH  ) radical (Ji, Stratman, & Lardy, 1988). In the present study, SOD activity decreased significantly in the indomethacin treated animals due to an excessive formation of superoxide anions. These excessive superoxide anions might inactivate SOD and decrease its activity. In the

0.73517 0.0263nnn 121.30 7 2.34nnn 34.767 0.21nnn 23.737 0.69nnn 577.90 7 7.34nnn

P value: o0.001 when Ethanol (toxicant) Control compared with Normal Control. P value: o 0.01 when experimental groups compared with Ethanol Control. P value: o 0.001 when experimental groups compared with Ethanol Control. nnn

nn

a

Values are Mean 7SEM; N ¼ 6 in each group. One-way ANOVA followed by Bonferroni’s test is applied for statistical analysis. 1 unit of CAT ¼mmol H2O2 consumed / min / mg protein. 1 unit of GPX ¼ mg GSHutilized / min / mg protein. 1 unit of GR ¼ nmol NADPHoxidized / min / mg protein. n P value: o 0.05 when experimental groups compared with ethanol Control.

0.4067 70.0250nnn 103.11 72.00nnn 28.22 70.74nn 7.361 70.355 316.50 719.05nn 0.7984 70.0271nnn 114.80 71.87nnn 35.72 70.50nnn 27.83 70.88nnn 587.30 76.05nnn 0.238 7 0.018a 79.36 7 2.13a 24.43 7 0.65a 4.436 7 0.431a 145.60 7 12.95a 1.0717 0.022 123.617 2.21 37.917 0.46 43.277 0.72 636.20 7 8.01

0.6448 7 0.0185nnn 115.007 1.72nnn 33.65 7 0.28nnn 22.737 1.04nnn 567.707 5.91nnn

6.3617 0.329nnn 7.4767 0.442nnn 11.521 70.436nnn 6.237 70.215nnn 21.681 7 0.816a 5.683 7 0.132

TBARS (nmol MDA/mg protein) GSH (mmol/g wet tissue) SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) GR (U/mg protein)

Biochemical parameters

Group I Normal Control Group II ethanol (1 ml/ 200 gm)

Group III Omeprazole 20 mg/kg) Group IV MUSE (250 mg/kg) Group V MUSE (500 mg/kg) Group VI p-Coumaric acid (250 mg/ kg)

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Table 4 Effect of MUSE and p-coumaric acid on stomach TBARS, GSH, SOD, CAT, GPx and GR in ethanol induced ulcers in rats.

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absence of adequate SOD activity, superoxide anions are not dismuted into H2O2, which is the substrate for the H2O2 scavenging enzymes CAT and GPX. As a result, there is inactivation of the H2O2 scavenging enzymes CAT and GPX, leading to a decline in their activities. Pretreatment with MUSE and p-coumaric acid significantly prevented the decrease in SOD, CAT and GPX activities, which may be attributed to the scavenging of radicals by MUSE and p-coumaric acid resulting in protection of these enzymes. GR is a cytosolic enzyme involved in the reduction of GSSG (an end product of GPX reaction) to GSH. With indomethacin treatment, there was a marked reduction in GPX activity, leading to reduced availability of substrate for GR, thereby decreasing the activity of GR. MUSE (at both doses) and p-coumaric acid treatment restored significantly the GR activity depleted due to indomethacin, thus accelerating the conversion of GSSG to GSH. Alcohol absorption into the bloodstream occurs throughout the gastrointestinal tract and its direct contact with the mucosa can induce numerous metabolic and functional changes. These alterations may lead to marked mucosal damage, which can result in a broad spectrum of acute and chronic diseases, such as gastrointestinal bleeding and ulcers (Bode & Bode, 1997). Oral treatment with ethanol causes focal hyperemia, edema, necrosis and submucosal hemorrhage, as well as circulatory disturbances (Bode & Bode, 1997). Ethanol is metabolized to the cytotoxic acetaldehyde by the microbes present in the normal human gut (Tuma & Casey, 2003). Acetaldehyde in turn is oxidized to acetate by aldehyde oxidase or xanthine oxidase, giving rise to ROS via CYP 2E1. These ROS may be involved in acute and chronic ulceration in the gastric mucosa by producing oxidative stress. The intracellular oxidative stress produces mitochondrial permeability transition and mitochondrial depolarization, which precede cell death in gastric mucosal cells (Hirokawa et al., 1998). Acetaldehyde is a toxic and reactive compound and could be a pathogenetic factor in H. pyloriassociated gastric injury (Roine, Salmela, & Salaspuro, 1995). Studies have shown that acetaldehyde inhibits gastric mucosal regeneration and form stable adducts with mucosal proteins (Tuma & Casey, 2003). Both of these mechanisms could cause gastric injury. These data suggest that antioxidant compounds could be active in this experimental model, producing antiulcerogenic effects. This effect is known as cytoprotection. Oral treatment with ethanol clearly resulted in the expected characteristic zone of necrotizing mucosal lesions in the stomachs of the Ethanol Control group of rats. Treatment with MUSE and p-coumaric acid significantly decreased the ulcer score and the ulcer index. These results indicate that MUSE and p-coumaric acid display an antiulcerogenic effect relating to a cytoprotective activity, since they significantly reduced ethanol-induced ulcers. Ethanol-induced generation of free radicals triggers cell damage through two mechanisms viz., covalent bonding to cellular macromolecules and peroxidative degradation of membrane lipids and endoplasmic reticulum rich in PUFA (Bode & Bode, 1997). This leads to the formation of lipid peroxides, which in turn yield products like MDA, which cause loss of integrity of cell membranes and damage to stomach tissue. Elevation in the levels of end products of lipid peroxidation was observed in the stomachs of ethanol treated rats. The increase in MDA levels in the stomach suggests enhanced peroxidation leading to tissue damage and failure of the antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with MUSE and p-coumaric acid significantly reversed these changes. Hence, it is likely that the mechanism of gastroprotection of MUSE and p-coumaric acid is due to inhibition of LPO and a free radical scavenging activity. The ethanol treated group of animals showed a decrease in GSH levels and SOD, CAT, GPx and GR activities, which was reversed by MUSE and p-coumaric acid treatment. This was probably by a

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Fig. 9. H&E staining of stomach slices in ethanol induced ulcer in rats. Note: (a) H&E staining of stomach of normal rat 10  10  ¼ 100  . (b) H&E staining of stomach of ethanol treated rat 10  10x ¼ 100  . (c) H&E staining of stomach of omeprazole (20 mg/kg) and ethanol treated rat. 10  10  ¼100  . (d) H&E staining of stomach of MUSE (250 mg/kg) and ethanol treated rat. 10  10  ¼100  . (e) H&E staining of stomach of MUSE (500 mg/kg) and ethanol treated rat 10  10  ¼100  . (f) H&E staining of stomach of p-coumaric acid (500 mg/kg) and ethanol treated rat 10  10  ¼100  .

mechanism similar to that explained in the indomethacin induced ulceration model. The histo-architecture of the stomach slices of rats treated with MUSE and p-coumaric acid showed protection of gastric mucosa and lesser infiltration of inflammatory cells in both the ulcer models. The histological studies, thus, provided additional substantial evidence of the gastroprotective effect of MUSE and pcoumaric acid and supported the biochemical findings. The results obtained from the p-coumaric acid treatment group were similar to those of the MUSE 500 mg/kg treated group, suggesting that MUSE’s antiulcer activity may be attributed, at least in part, to p-coumaric acid present in it. 5. Conclusion In conclusion, MUSE and p-coumaric acid afforded significant antiulcer activity by enhancing the antioxidant potential of the gastric mucosa, thereby reducing mucosal damage. The present study aims to bring about value addition to the health benefits of M. uniflorum seeds, establish it as a potent “functional food” and promote its use in people’s diets. Besides, it is cheap, readily available to all strata of society, with medicinal properties attributed to it. Further research on horse gram is warranted to isolate the active constituents and study their exact mechanism of action in cytoprotection.

Conflict of interst statement We, the authors state that there are no conflicts of interest for this MS.

Acknowledgment We acknowledge the All India Council for Technical Education (AICTE) (New Delhi, India) for providing grant-in-aid under its research promotion scheme for this study. We are extremely grateful to Glenmark Pharmaceuticals Ltd., India for providing animals for this study.

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