Scholarly article on topic 'Antioxidant and Anti-ulcer Effects of Ethyl Acetate Fraction of Merremia Tridentata (L.) Hallier F. Root'

Antioxidant and Anti-ulcer Effects of Ethyl Acetate Fraction of Merremia Tridentata (L.) Hallier F. Root Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Kandhasamy Sowndhararajan, Nyuk Ling Chin

Abstract The present study was aimed to determine the antioxidant and anti-ulcer effects of Merremia tridentata root (Arrow-leaf Morning Glory). Antioxidant potential of crude acetone extract and its fractions was evaluated by using various in vitro assays. In the anti-ulcer study, pre-treatment with ethyl acetate fraction (MEF) (50, 100 and 200mg/kg b.wt.) significantly protected the ethanol-induced gastric ulcer in rats. Simultaneously, the antioxidant enzyme levels of superoxide dismutase, catalase and glutathione in the stomach tissues were significantly increased while pre-treated with MEF. From the results, MEF has the therapeutical potential to prevent gastric ulceration induced by ethanol.

Academic research paper on topic "Antioxidant and Anti-ulcer Effects of Ethyl Acetate Fraction of Merremia Tridentata (L.) Hallier F. Root"

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Agriculture and Agricultural Science Procedia 2 (2014) 406 - 414

"ST26943", 2nd International Conference on Agricultural and Food Engineering, CAFEi2014"

Antioxidant and Anti-Ulcer Effects of Ethyl Acetate Fraction of Merremia tridentata (L.) Hallier F. Root

Kandhasamy Sowndhararajana,b*, Nyuk Ling Chinb

aDepartment of Biological Environment, Kangwon National University, Chuncheon 200 701, Republic of Korea bDepartment of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Abstract

The present study was aimed to determine the antioxidant and anti-ulcer effects of Merremia tridentata root (Arrow-leaf Morning Glory). Antioxidant potential of crude acetone extract and its fractions was evaluated by using various in vitro assays. In the antiulcer study, pre-treatment with ethyl acetate fraction (MEF) (50, 100 and 200 mg/kg b.wt.) significantly protected the ethanol-induced gastric ulcer in rats. Simultaneously, the antioxidant enzyme levels of superoxide dismutase, catalase and glutathione in the stomach tissues were significantly increased while pre-treated with MEF. From the results, MEF has the therapeutical potential to prevent gastric ulceration induced by ethanol.

© 2014 The Authors.PublishedbyElsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Peer-review under responsibility of the Scientific Committee of CAFEi2014

Keywords: Antioxidant; anti-ulcer; ethanol-induced ulcer; Merremia tridentata; root

1. Introduction

Gastric ulcer is a chronic disorder that affects large population of the world (Klein-Junior et al., 2010). It has been attributed to various causes such as Helicobacter pylori, pepsins, non-steroidal anti-inflammatory drugs, bile acids, and alcohol (Awaad et al., 2013). Consumption of excessive alcohol is one of the major factors in the formation of gastric ulcer (Franke et al., 2005). The current medicinal treatment for gastric ulcers includes antacids and histamine receptor antagonists-2 (H2-Ras) and proton pump inhibitors. But clinical evaluation of these drugs showed various side effects such as hypersensitivity, arrhythmia, impotence, gynecomastia and hematopoietic disorders in the long

* Corresponding author. Tel.: +82-33-250-6447; fax: +82-33-241-6440. E-mail address: sowndhar 19 82@gmail. com

2210-7843 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.Org/licenses/by-nc-nd/3.0/).

Peer-review under responsibility of the Scientific Committee of CAFEi2014

doi: 10.1016/j.aaspro.2014.11.057

run (Malfertheiner et al., 2009; Sheen and Triadafilopoulos, 2011). Therefore, there is a pressing need for alternative therapies to treat gastric ulcer especially from natural origin. In this scenario, plant extracts are the most important source of new drugs and relatively less expensive, safe to use even at higher doses.

Merremia tridentata (L.) Hallier f., belongs to the Convolvulaceae family, grows naturally as a perennial herb and is distributed from tropical Africa to tropical Asia, India and Sri Lanka, eastwards to China, and southwards throughout Malaysia, and northern Australia with local names of akar kelemak, karok relia or pungulang in Malaysia, irit-iritan, rangitan or jala ma tubu in Indonesia, thao tot maa in Thailand and maragta, talanuk or karadkad in Philippines. In traditional systems of medicine, the plant is used for various ailments such as piles, swellings, ulcers, rheumatic affections, stiffness of the joints, hemiplegia, urinary infections and general debility (Khare, 2007). Aschfalk et al. (2002) reported that M. tridentata was used as a supplementary feed to the grass Panicum maximum for young West African Dwarf Sheep. The aerial parts of the M. tridentata contain flavonoids, diosmetin, luteolin, and their 7-O-P-D-glucosides (Khare, 2007). Previous studies stated that M. tridentata have strong wound healing, anti-inflammatory and anti-arthritic activities (Hatapakki et al., 2004; Kamalutheen et al., 2009). The acetone extract of M. tridentata root registered higher antioxidant and free radical scavenging activities than the aerial parts (Sowndhararajan et al., 2010). Several reports are available about the gastroprotective effects are associated with plant extracts that are rich in antioxidants (Awaad et al., 2013). With this background, the present study was aimed to determine the antioxidant activity of acetone extract and its sub-fractions of M. tridentata root; to evaluate the protective effect of ethyl acetate fraction on ethanol-induced gastric ulcer in rat model; and to investigate the enzymatic antioxidant levels such as superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in the gastric tissues.

2. Materials and methods

2.1. Plant material and extract preparation

The fresh roots (4,000 g) of M. tridentata (Fig. 1A and B) were collected from Bharathiar University campus, Coimbatore, Tamil Nadu State, India in the month of November, 2010. The plant was authenticated by Prof. V.S Ramachandran and deposited in the Botany Herbarium, Bharathiar University with voucher number BUBH-2895. The sample of roots was cleaned thoroughly, shade dried at room temperature and powdered. The powdered sample (1,000 g) was macerated thrice with acetone at room temperature. The combined acetone extract (MCE - 54.7 g) was concentrated by low-pressure evaporation (<40 °C). The crude extract was dissolved in water and the aqueous solution was successively partitioned with petroleum ether (MPF - 3.9 g), ethyl acetate (MEF - 10.3 g) and n-butanol (MBF - 16.8 g). The obtained extracts, in addition to the aqueous solution (MWF - 22.6 g) remained after the extraction were filtered and dried under vacuum.

Fig. 1. Aerial parts (A) and roots (B) ofM. tridentata.

2.2. Determination of total phenolic content

The total phenolic content of crude acetone extract and its sub-fractions of M. tridentata root was determined by Folin-Ciocalteu method. The amount of total phenolics was calculated as the gallic acid equivalents (GAE) (Siddhuraju and Becker, 2003).

2.3. Ferric reducing antioxidant power (FRAP) assay

The ferric reducing capacity of the samples was estimated according to the method described previously by Pulido et al. (2000). The absorbance of the reaction mixture was measured at 593 nm. The values are expressed as mmol Fe (II)/g extract.

2.4. Phosphomolybdenum reduction assay

The total antioxidant activity of the samples was evaluated by the green phosphomolybdenum complex formation according to the previously described method of Prieto et al. (1999). The absorbance of the reaction mixture was measured at 695 nm. The results reported are mean values expressed as ascorbic acid equivalents (mg AAE/ g extract).

2.5. Free radical scavenging activity on DPPH

The DPPH (1,1-diphenyl-2-picrylhydrazil) radical scavenging activity of the samples was determined according to the method of Liyana-Pathirana and Shahidi (2005). IC50 values of the extract i.e., concentration of extract necessary to decrease the initial concentration of DPPH by 50% was calculated.

2.6. Antioxidant activity by the ABTS'+ assay

Radical scavenging activity of crude acetone extract and its sub-fractions of M. tridentata root was assessed spectrophotometrically by ABTS^+ cation decolorization assay (Re et al., 1999). IC50 values of the extract i.e., concentration of extract necessary to decrease the initial concentration of ABTS by 50% was calculated.

2.7. Ethanol-induced gastric ulcer in rats

Male Wistar rats weighing 180-220 g were used for this anti-ulcer study and the animals were purchased from Orient Bio Inc. (Seoul, Republic of Korea). The animals were housed under standard conditions of temperature (23 ± 1°C), relative humidity (55 ± 1%), 12 h light: 12 h dark cycle, and allowed free access to food and water. All the studies conducted were approved by the Institutional Animal Ethical Committee, Kangwon National University, Chuncheon, South Korea. Animals described as fasted were deprived of food for 24 h, but had free access to water.

Based on the higher antioxidant potential, the ethyl acetate fraction was selected for the animal study. The rats were deprived of food but had ad libitum access to tap water for 24 h before ulcer induction. Gastric mucosal damage was induced in conscious rats by gavage of 5.0 mL/ kg b.wt. of absolute ethanol (99.5%). Group 1 (control) was treated with distilled water. Group 2 (Ethanol) was the disease control group. Group 3 was the standard drug control (omeprazole) and was given a dose of 20 mg/kg b.wt. Group 4, 5 and 6 were treated with MEF (50, 100 and 200 mg/kg b.wt., respectively). All the rats in groups (Groups 3 - 6) were pre-treated by gavage with MEF or omeprazole 60 min prior to the ethanol administration. The animals of Group 2 - 6 were subjected to ethanol administration (5.0 mL/ kg b.wt.) and the animals were sacrificed 60 min later. The stomach was excised, cut along the curvature and washed with 0.9% NaCl, followed by macroscopic determination of the gastric mucosal injury index.

Ulcer index (UI) was calculated in ethanol-induced rats. For the determination of ulcer index, the stomach was cut open along the greater curvature and the inner surface was examined for ulceration with the help of a simple dissecting microscope. The ulcer index was calculated according to the scoring method of Tan et al. (1996). Score 0

- no ulcer; Score 1 - vessel dilation and pointed ulcers; Score 2.5 - small ulcers < 4 mm long; Score 5 - large ulcers > 5 mm long. UI for each animal was calculated as mean ulcer score.

2.8. Histological procedure and assessment

Stomach samples were kept in 10% formaldehyde buffer, processed and embedded in paraffin wax. Five micrometer thick sections were prepared and stained with haematoxylin-eosin. The histopathological changes were examined and photographed.

2.9. Biochemical examinations of stomach tissues

A 10% stomach homogenate was prepared in phosphate buffer (100 mM, pH 7.4) and centrifuged at 10,000 g for 15 min at 4 °C. The resultant supernatant was used to evaluate the antioxidant enzyme activities such as SOD, CAT and GSH in rat stomach tissues using corresponding assay kits purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.10. Statistical analysis

The results were expressed as mean ± SEM. The statistical analysis was carried out by analysis of variance (ANOVA) followed by Dunnett's test. The data were evaluated with SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The significance level was set at P < 0.05.

3. Results and discussion

3.1. Antioxidant activities

Plant polyphenols, a diverse group of phenolic compounds (flavonoids, anthocyanins, phenolic acids, etc.) possess an ideal structural chemistry for free radical scavenging activity. Increasing experimental evidence has suggested that these compounds can affect a wide range of cell biological functions and has been effective in the prevention of various diseases such as cancer, ulcer, inflammation, diabetes and aging (Dixon et al., 2005; Rice-Evans, 2004). Therefore, it would be important to quantify the amount of total phenolics present in the plant extracts. Total phenolic content of acetone extract and its sub-fractions of M. tridentata root is given in Table 1. The amount of total phenolic content varied from 72.6 to 527.6 mg GAE/g extract. Among them, ethyl acetate fraction (MEF) was found to contain higher level of total phenolic content (527.6 mg GAE/g extract) followed by crude acetone extract (304.7 mg GAE/g extract).

Table 1. Total phenolic content and antioxidant activities of crude acetone extract and its sub-fractions of M. tridentata root.

Sample Total phenolics (mg GAE/g extract) FRAP (mmol Fe(II)/g extract) Phosphomolybdenum (mg AAE/g extract) IC50 of DPPH (^g/mL) IC50 of ABTS (^g/mL)

MCE 304.7 ± 12b 2656.7 ± 20.5b 567.8 ± 8.5b 26.6 ± 1.1b 12.8 ± 0.5b

MPF 72.6 ± 03e 714.9 ± 12.1e 96.5 ± 11.2e 176.3 ± 3.2e 106.3 ± 4.7e

MEF 527.6 ± 38a 3317.4 ± 63.5a 659.4 ± 41.5a 17.5 ± 0.7a 7.3 ± 0.3a

MBF 237.9 ± 18c 1865.6 ± 33.9c 329.5 ± 32.4c 29.1 ± 0.8c 16.2 ± 0.7c

MWF 165.2 ± 11d 1213.1 ± 15.2d 219.7 ± 19.8d 48.7 ± 1.2d 22.9 ± 2.3d

Values are means of three replicate determinations (n = 3) ± standard deviation. MCE, M. tridentata crude acetone extract; MPF, M. tridentata petroleum ether fraction; MEF, M. tridentata ethyl acetate fraction; MBF, M. tridentata butanol fraction; MWF, M. tridentata water fraction. Mean values followed by different superscript in a column are significantly different (P < 0.05).

In the previous study, acetone extracts of M. tridentata root (37.5 g TAE/100 g extract) and aerial parts (35.1 g TAE/100 g extract) had the highest amount of total phenolic content than other solvent extracts (Sowndhararajan et

al., 2010). Previously, many authors have reported that the positive correlation between phenolic content and antioxidant potential of extracts from various plants (Socha et al., 2009; Zhang and Wang, 2009). Polyphenols act as reducing agents, hydrogen donors, free radical quenchers, metal chelators, decomposers of peroxides and regulation of various enzyme activities (Itagaki et al., 2009; Chang et al., 2011).

In the present study, antioxidant activity of crude acetone extract and its sub-fractions was evaluated using ferric reducing, phosphomolybdenum reduction, DPPH, and ABTS radical scavenging methods. The results of ferric and phosphomolybdenum reducing potential of crude acetone extract and its sub-fractions are presented in Table 1. MEF resulted in the highest FRAP activity (3,317.4 mmol Fe (II) / g extract) followed by crude acetone extract (2,656.7 mmol Fe (II)/ g extract). Antioxidant compounds present in the extracts that act as reducing agents exert their effect by donating a hydrogen atom to the ferric complex and thus breaking the radical chain reaction (Shakirin et al., 2010). The phosphomolybdenum reduction activity is expressed as mg ascorbic acid equivalent/g extract and higher activity found in the order of MEF (659.4) > MCE (567.8) > ABF (329.5) > AWF (219.7) > APF (96.5). The result suggests that the MEF and MCE registered higher phosphomolybdenum reduction activity than the five Phyllanthus species (245-376 mg ascorbic acid/g extract) reported by Kumaran and Karunakaran (2007).

Several methods are used to evaluate the antioxidant potential of plant extracts. Among them, DPPH and ABTS radical scavenging methods are the oldest and most usually used in vitro methods based on the transfer of hydrogen between the free radicals and the antioxidants (Stratil et al., 2006). DPPH and ABTS radical scavenging activities of acetone extract and its sub-fractions of M. tridentata root are presented in Table 1. All the extracts exhibited appreciable levels of DPPH (IC50 17.5 - IC50 176.3 ¡¡g/mL) and ABTS (IC50 7.3 - IC50 106.3 ¡¡g/mL) radical scavenging activities with the exception of petroleum ether fraction (MPF). Among the various fractions, MEF registered higher DPPH and ABTS radical scavenging activities with the IC50 values of 17.5 and 7.3 ¡g/mL, respectively. The results of the in vitro antioxidant studies revealed that the DPPH and ABTS radical scavenging activities were closely related to the higher phenolic contents detected in the crude acetone extract and its subfractions.

3.2. Ethanol-induced anti-ulcer activity

Based on the results of in vitro antioxidant assays, MEF was chosen as the most potent extract and subsequently used to evaluate its anti-ulcer potential against ethanol-induced ulcer model. Oral administration of ethanol interrupts the mucosal defense system, thereby aggravating mucosal damage that might bring about necrosis as well as apoptosis of gastric mucosal cells (Liu and Cho, 2000; Ko et al., 2004). The severity of gastric ulcer could be evaluated by ulcer lesions and histological changes. Goel and Sairam (2002) reported that plant extracts specifically inhibit the gastric acid secretion, or to stimulate the mucosal defense mechanism by increasing the mucus production, protecting the surface epithelial cells. Therefore, the present study was carried out to evaluate the anti-ulcerogenic effect of MEF against ethanol-induced ulcer model in rats.

The anti-ulcer effects of MEF against ethanol induced ulcer model in rats are shown in Figures 2 and 3. The ulcer control group that received ethanol (5 ml/kg b.wt.) produced severe gastric lesions including hemorrhagic bands and spots in the surface mucous cells (Fig. 2B) with an ulcer index score of 29.7 ± 0.73. The rats received MEF (200 mg/kg b.wt.) showed a significant reduction in ulcer index (4.2 ± 0.17) and nearly equal to that of the standard drug omeprazole (3.7 ± 0.13). The results of the present study demonstrated that animals pretreated with MEF (Fig. 2D -F), presented a dose dependent protection of mucosal layer damage. Consumption of excessive alcohol usually elevates the risk of gastric mucosal damage by generating oxygen-derived free radicals such as superoxide anions, hydroxyl radicals and lipid peroxides (Li et al., 2008; Pan et al., 2008). In the present study, administration of MEF exhibited remarkable protection against ethanol-induced depletion of gastric mucosa. The anti-ulcer activity of MEF was compared with the standard anti-ulcer drug omeprazole. Omeprazole is a proton pump inhibitor, exhibits an anti-secretory and protective effect. It is effective in treating peptic ulcer disease and gastroesophageal reflux in short- and long-term use (Schneeweiss et al., 2006).

Fig. 2. Effect of ethyl acetate fraction of M. tridentata root on ethanol-induced gastric ulcer in rats. A, Control group; B, Ethanol group 5 mL/kg b. wt.; C, Omeprazole 20 mg/kg b.wt.; D, Ethyl acetate fraction 50 mg/kg b. wt.; E, Ethyl acetate fraction 100 mg/kg b.wt.; F, Ethyl acetate fraction 200 mg/kg b.wt.

Control Ethanol Omeprazole MEF - 50 MEF-100 MEF - 200 Treatment group

Fig. 3. Effect of ethyl acetate fraction of M. tridentata root at different doses on ulcer index in ethanol-induced gastric tissues. Control (vehicle), distilled water p.o.; Ethanol, 5 mL/kg p.o.; Omeprazole, 20 mg/kg p.o.; MEF - 50, Ethyl acetate fraction 50 mg/kg p.o. MEF - 100, Ethyl acetate fraction 100 mg/kg p.o.; MEF - 200, Ethyl acetate fraction 200 mg/kg p.o. Each value represents the mean ± SEM of six rats in each group. #P < 0.001 compared to the control group; **P < 0.01, ***P < 0.05 compared to the ethanol group.

Histopathological changes of rat stomach tissue due to the intragastric administration of ethanol are depicted in Figure 4 (A-F). In microscopical observation, the stomach tissues of ulcer control group showed that the ruptured and distorted epithelial cells (Fig. 4B). However, the animals pre-treated with MEF (200 mg/kg b.wt.) or omeprazole promoted a healing of gastric ulcer with the restoration of the pathological injuries (Fig. 4C-F). Previous studies

reported that the oral administration of ethanol provoked significant histological changes in the gastric mucosal tissue such as disruption and exfoliation of the superficial gastric epithelium, disappearance of the mucous cells, vacuolization, necrosis, and karyopyknosis in the superficial gastric epithelium cells (Arun and Asha 2008; Liu et al. 2012).

Fig. 4. Effect of ethyl acetate fraction of M. tridentata root on histological findings of gastric damage induced by ethanol in rats. A, Control group; B, Ethanol group 5 ml/kg b.wt.; C, Omeprazole 20 mg/kg b.wt.; D, Ethyl acetate fraction 50 mg/kg b. wt.; E, Ethyl acetate fraction 100 mg/kg b.wt.; F, Ethyl acetate fraction 200 mg/kg b.wt. Red arrows indicate loss of continuity in epithelial cells. Black arrows indicate continuity in epithelial cells similar to control group.

In a number of pathophysiological conditions associated with oxidative stress, reactive oxygen species (ROS) have been proposed to mediate cell damage through various mechanisms including inactivation of antioxidant enzymes. They also played an important role in the gastric mucosal injury associated with ethanol consumption (Ajaikumar et al., 2005; Halici et al., 2005; Cadirci et al., 2007). The primary role of CAT is to decompose hydrogen peroxide. Increased SOD activity can protect cells against threat of reactive oxygen species, especially superoxide anion radicals. GSH is regarded as a crucial enzyme which catalyses the reduction of hydrogen peroxide. A decrease in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects including loss of integrity and function of cell membranes. Similar to previous studies, the activities of the antioxidant enzymes SOD, CAT and GSH were significantly decreased subsequent to intragastric administration of ethanol (Alimi et al., 2011; Liu et al., 2012). As shown in Table 2, there was a significant increase of CAT, SOD, and GSH activities in the pre-treatment groups (MEF or omeprazole) than the ethanol control group. The data suggests that MEF demonstrated anti-ulcer activity, which may be related to the reduction of ROS and associated with the increase in the activities of enzymatic antioxidants (SOD, CAT and GSH). The gastroprotective effect of MEF is possibly due to the presence of higher level of total phenolics, which known to have potent antioxidant properties.

Table 2. Effect of ethyl acetate fraction of M. tridentata root (MEF) on SOD, GSH and CAT activities in rat stomach tissues.

Group SOD CAT GSH

(U/mg protein) (^mol/min/mg protein) (nmol/mg protein)

Control group 221.3 ± 14.1 5.21 ± 0.16 1.35 ± 0.03

Ethanol group 93.3 ± 7.3*** 1.93 ± 0.04*** 0.43 ± 0.02***

Omeprazole - 20 mg/kg 207.3 ± 9.2** 4.90 ± 0.21** 1.21 ± 0.06**

MEF - 50 mg/kg 112.3 ± 8.5* 2.82 ± 0.08* 0.58 ± 0.02*

MEF - 100 mg/kg 172.7 ± 9.5** 3.73 ± 0.12** 0.82 ± 0.04**

MEF - 200 mg/kg 210.3 ± 11.9** 4.85 ± 0.15** 1.28 ± 0.08**

Values are means of three replicate determinations (n = 3) ± standard deviation. Mean values followed by different superscript in a column are significantly different (P < 0.05). SOD - U/mg protein; GSH - nmol GSH oxidized/min/mg protein; CAT - jimol H2O2 consumed/mg protein. P < 0.05 as compared to ethanol group; P < 0.01 as compared to ethanol group; P < 0.001 as compared to control group.

4. Conclusions

The present investigation suggested that the ethyl acetate fraction of M. tridentata root (MEF) exhibited higher antioxidant and free radical scavenging potentials. The results also demonstrated that ethyl acetate fraction significantly protected the gastric damage against ethanol induced ulcer by increasing the activities of enzymatic antioxidants (SOD, CAT and GSH). It can be concluded that M. tridentata root could serve as therapeutic agents in protecting ethanol mediated gastric ulcer. Further studies in relation to isolation and characterization of effective anti-ulcer compounds from the ethyl acetate fraction of M. tridentata root are under progress.

Acknowledgements

This study was supported by Agriculture and Life Sciences Research Institute, Kangwon National University, Chuncheon, Republic of Korea.

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Accepted for poster presentation in CAFEi2014 (December 1-3, 2014 - Kuala Lumpur, Malaysia) as paper 232.