Gastroprotective effect of swallow root (Decalepis hamiltonii) extract: Possible involvement of H+–K+–ATPase inhibition and antioxidative mechanism

Journal of Ethnopharmacology 112 (2007) 173–179

Gastroprotective effect of swallow root (Decalepis hamiltonii) extract: Possible involvement of H+–K+–ATPase inhibition and antioxidative mechanism Yogender Naik a , Smitha Jayaram a , M.A. Harish Nayaka a , Lakshman b , Shylaja M. Dharmesh a,∗ a

Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570020, Karnataka, India b Department of Pharmacology, Government College of Pharmacy, Subbaiah Circle, Bangalore, Karnataka, India Received 18 May 2006; received in revised form 12 January 2007; accepted 23 February 2007 Available online 27 February 2007

Abstract The present study reports the antiulcer potentials of aqueous extract of swallow root (Decalepis hamiltonii Wight & Arn, SRAE) belonging to the family Asclepiadaceae. Swim stress-induced ulcers with an ulcer index (UI) of 6.0 ± 0.01 was protected up to 43% and 72% at 100 and 200 mg/kg b.w. of SRAE, respectively, similar to protection offered by ranitidine (79%) at 30 mg/kg b.w. Depletion in antioxidant enzymes and increased Thiobarbituric Acid Reactive Substances (TBARS) were observed in ulcer-induced rats while SRAE fed rats showed normal levels. SRAE also normalized ∼3.1 and 2.4 folds of increased H+ –K+ –ATPase and gastric mucin, respectively, in ulcerous animals, similar to the levels found in healthy controls. SRAE also possessed reducing power, free radical scavenging ability with an IC50 of 0.17 ␮g/mL gallic acid equivalent (GAE), comparable to that of BHA (IC50 -0.08 ␮g/mL). DNA protection up to 80% at 0.2 ␮g was also observed. Toxicity studies indicated no lethal effects in rats fed up to 5 g/kg b.w. Antioxidant, proton pump inhibition as well as boosting of gastric mucin effects of SRAE have been implicated to be responsible for antiulcer property of SRAE. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Decalepis hamiltonii; Swallow root; Swallow root aqueous extract; Gastric ulcer; H+ –K+ –ATPase; Antioxidant

1. Introduction Hyper acidity is a common pathological condition caused due to uncontrolled hypersecretion of hydrochloric acid from parietal cells of gastric mucosa through the proton pump H+ –K+ –ATPase (Kishor et al., 2007). Apart from the damaging role of acid, reactive oxygen species especially the hydroxyl radical plays a major role in causing oxidative damage of mucosa (Bandyopadhyay and Chattopadhyay, 2006) in all types of ulcers including stress related gastric mucosal damage (Mitchell, 2004), non-steroidal anti-inflammatory drug-induced gastric lesions (Ivey, 1998) and Helicobacter pylori mediated gastroduodenal ulcers (Konturek and Konturek, 1994). The modern approach to control gastroduodenal ulceration is to

∗

Corresponding author. Tel.: +91 821 2514876; fax: +91 821 2517233. E-mail address: [email protected] (S.M. Dharmesh).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.02.021

scavenge reactive oxygen species, inhibiting H+ –K+ –ATPase pump to control increased acid secretion and eradication of H. pylori (Bandyopadhyay et al., 2002). A range of drugs like histamine blockers and proton pump inhibitors although has been used for efficient management of gastric hypersecretion, many of these drugs pose adverse effects like dizziness, drowsiness, gas accumulation, headache, nausea, vomiting, inflammation of the nose, etc. (Fort et al., 1995; Powers et al., 1995; Shimokawa et al., 1996; Martelli et al., 1998). Phytal sources have been popular, partly because of their low cost and minimal side effects. In the current study, we investigated Decalepis hamiltonii (Wight & Arn.) extract, as a potential alternatives to ulcer management. D. hamiltonii (swallow root - SR) commonly called as maredu kommulu or barre sugandhi or maradu gaddalu or makali beru is an endangered climbing shrub belonging to the family Asclepiadaceae and its roots have been used in Ayurveda, the ancient Indian traditional system of medicine to stimulate appetite, relieve flat-

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ulence and as a general tonic (Nayar et al., 1978). The plant is known to contain vanillin, salicylaldehyde, p-anisaldehyde, 2-hydroxy-4-methoxybenzaldehyde, bis-2,3,4,6-galloyl-␣/␤-dglucopyranoside, borneol, inositol, saponins, ketonic substances, sterols, amyrins and lupeols (Nagarajan et al., 2001; Thangadurai et al., 2002; Nagarajan and Rao, 2003; Harish et al., 2005; Srivastava et al., 2006a,b). Although the plant has been in use for centuries for its medicinal properties, the antiulcer property is yet to be elucidated. We demonstrate the antiulcer activity of swallow root aqueous extract (SRAE) in vivo animal models and provide a mechanism of action and biochemical basis for gastroprotectivity during ulceration. Phenolics with antioxidant, H+ –K+ –ATPase inhibitory and mucosal defense activity have been evaluated including the level of antioxidant enzymes in important organs (liver and brain) and gastric mucin levels in stomach. In addition, the effects of SRAE, various phenolic acids as well as omeprazole and lanzoprazole (known antiulcer drugs) on inhibition of H+ –K+ –ATPase activity were also investigated. Since very few studies (Murakami et al., 1991, 1992) have addressed the role of phenolics in blocking H+ –K+ –ATPase, the current study emphasizes the inhibition of H+ –K+ –ATPase by SRAE and phenolic acids in addition to their antioxidant potency and DNA protective ability. 2. Materials and methods 2.1. Chemicals

forest range located between 11–13◦ N and 77–78◦ E, SouthEast corner of Mysore district in May 2003 and identified by a taxonomist in the herbarium of Vivekananda Girijana Kalyana Kendra, B.R. Hills, Chamaraja Nagar, Karnataka, India, where a voucher specimen is deposited. 2.3. Preparation of aqueous extract of swallow root Swallow root was washed, dried and powdered to a particle size of 20 mesh. Swallow root powder (10 g) was defatted using hexane in a soxhlet apparatus. One gram of defatted powder was taken in 10 mL distilled water and boiled for 5 min, cooled and centrifuged at 1000 × g for 10 min. The clear supernatant was separated and referred as swallow root aqueous extract (SRAE). A total yield of 20 ± 3 g/100 g accounting to an average of 22% (w/w) was obtained with triplicate extractions. 2.4. Animals Animal experiments were carried out upon the clearance from the CFTRI Ethics committee, at the animal house facility of CFTRI, which has been registered with CPCSEA (Reg. No. 49, 1999), Government of India, New Delhi, India. Healthy Albino Wistar rats of both sex weighing 160–200 g were categorized into five groups, each containing six animals. The animals were allowed to acclimatize to the animal room conditions for 1 week and were fed with standard rat pellet feed and tap water ad libitum. 2.5. Ulcer induction and treatment

Nitroblue tetrazolium (NBT), 2-thiobarbituric acid (TBA), phenolic acids such as gallic, tannic, gentisic, caffeic, ferulic, vanillic, syringic, p-coumaric and proto catechuic acids, hydroxylamine hydrochloride, hydrogen peroxide, glutathione reductase, NADPH, reduced glutathione (GSH), 5,5 -dithionitrobenzoic acid (DTNB), butylated hydroxytoluene (BHT), 1,1,3,3-tetramethoxypropane (TMP), bovine serum albumin (BSA), ranitidine, adenosine triphosphate (ATP), ammonium molybdate, 4-(2-hydroxyethyl) piperazine1-ethanesulfonic acid (HEPES), monoclonal anti-human gastric mucin antibody (Mab-GM), Lanzoprazole, paranitrophenyl phosphate (PNPP), diethanolamine, Tris–HCl, Triton X-100, and Tween-20 were purchased from Sigma Chemical Co. (St. Louis, MO). Alkaline phosphatase conjugated-rabbit anti mouse IgG secondary antibody was procured from GENEI, Bangalore, India. Omeprazole, skimmed milk powder and other chemicals such as hexane, hydrochloric acid, trichloroacetic acid (TCA), phosphate buffer, carbonate buffer, magnesium chloride, potassium chloride, sucrose, ethylenediaminetetraacetic acid (EDTA) and solvents used were of the analytical grade purchased from local chemical company (Sisco Research Laboratories, Mumbai, India).

Five groups—groups I–V of six animals each were categorized as healthy control, ulcer-induced, SRAE-treated at 100 and 200 mg/kg b.w. (dry weight) and ranitidine-treated groups, respectively. Animals were administered once daily for 14 dayssaline (groups I and II) or SRAE (groups III and IV). Group V received ranitidine (30 mg/kg b.w.) under similar experimental conditions. At the end of 14th day all rats were starved for 24 h and except the healthy control group all were subjected to swim stress (Kulkarni and Goel, 1996) by making them to swim in a jar of 30 cm height and 10 cm diameter containing water up to 15 cm height and maintained at 22 ◦ C ± 2 ◦ C for 3 h. Animals were sacrificed by cervical dislocation; stomach was examined for mucosal integrity and occurrence of ulcers. Mean ulcer scores of each experimental group was calculated and expressed as ulcer index (UI). After calculation of mean and standard deviation, Dunnets test was performed to obtain the significance between the treated and the control groups. A value of p < 0.05, p < 0.01 and p < 0.001 was considered as significant, very significant and highly significant, respectively, between the groups.

2.2. Plant

2.6. Collection and biochemical analysis of tissues

Decalepis hamiltonii Wight & Arn. (Asclepiadaceae) roots of Batch No. 4, 2003 procured from a local, Devaraja market, Mysore, India, had been originally collected from the Gumballi

The stomach, liver and brain tissues were collected, weighed and homogenized in chilled Tris-buffer (10 mM, pH 7.4) at a concentration of 5% (w/v). The homogenates were centrifuged at

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1000 × g at 4 ◦ C for 20 min using high speed cooling centrifuge (REMI C 24, Mumbai, India). The clear supernatant was used for the estimation of lipid peroxidation products as thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), gastric mucin (GM) and antioxidant enzymes-glutathione peroxidase (GPx), catalase (CAT) and, superoxide dismutase (SOD). 2.7. Toxicity studies Toxicity studies were carried out as described earlier (Bandyopadhyay et al., 2002) in Albino Wistar rats, kept at controlled environment and acclimatized to laboratory conditions for one week before study. Rats of both sex (150 ± 10 g) were orally fed once daily with SRAE (5 g/kg b.w.) for 14 days. The control group received the vehicle (distilled water) only. Twentyfour hours after the last dose, number of animals survived were noted and sacrificed by cervical dislocation, blood was collected and serum was used for estimation of TBARS, total protein and enzymes related to liver function tests [serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), and alkaline phosphatase (ALP)]. 2.8. Determination of gastric mucin (GM) by ELISA Gastric mucin was isolated from stomach tissue of the control, ulcer-induced and -treated groups and quantified by ELISA (Rajeshwari et al., 1998) employing a monoclonal antihuman gastric mucin antibody (Mab-GM). Briefly, wells in ELISA plate (Nunc) were incubated with 100 ␮L of GM in carbonate–bicarbonate buffer-pH 9.6 overnight at 4 ◦ C, wells were washed thrice with phosphate buffered saline (PBS) containing 0.05% Tween-20 and incubated with 5% skim milk powder in PBS for 1 h to avoid nonspecific adsorption of antibodies. Hundred microliters of Mab-GM and alkaline phosphatase conjugated rabbit anti-mouse IgG were employed as primary (1:1000; v/v) and secondary (1:10,000; v/v) antibodies, respectively. Hundred microliters of paranitrophenyl phosphate (1 mg/mL in diethanolamine buffer, pH 8.6) was added and optical density was measured at 405 nm in a microplate reader (Molecular Devices, Spectra max 340). 2.9. Antioxidant enzymes and antioxidant assays Antioxidant enzymes such as SOD (EC 1.15.1.1) CAT (EC 1.11.1.6) and GPx (EC 1.11.1.9) were assayed in the stomach and liver tissues as well as in the serum employing protocols described (Raja et al., 2007). Enzyme activity for SOD and CAT is expressed as units/mg protein, while the activity for GPx is expressed as n moles of NADPH oxidized/min/mg protein. Glutathione (GSH) content of homogenates was determined as described elsewhere (Raja et al., 2007). One milliliter aliquot of homogenate was mixed with equal volume of 10% TCA and protein precipitate was removed by centrifugation. The supernatant was added to equal volume of 0.5 M Tris–HCl, pH 9 containing 20 mM DTNB to yield yellow chromophore of thionitrobenzoic acid (TNB), which was measured at 412 nm. GSH was used as a reference standard.

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2.10. Measurement of lipid peroxidation Lipid peroxidation products (TBARS) in serum, fundic stomach, liver and brain homogenates were estimated as TBARS (Possamai et al., 2007). One milliliter of homogenates were mixed with 2 mL of TCA–TBA–HCl reagent (15% TCA, 0.375% TBA in 0.25N HCl) containing 0.05% BHT and heated for 15 min in boiling water bath. The solution was cooled to room temperature. The precipitate was removed by centrifugation at 1000 × g for 10 min and the absorbance of the supernatant was measured at 532 nm using 1,1,3,3-tetramethoxypropane as standard. 2.11. Assay of H+ –K+ –ATPase activity Proton potassium ATPase was prepared (Cheon et al., 2001) from mucosal scrapings of stomach of sheep obtained from slaughterhouse and experimental rats were homogenized in 20 mM Tris–HCl, pH 7.4, centrifuged for 10 min at 5000 × g and the resulting supernatant was subsequently centrifuged at 5000 × g for 20 min. The protein concentration in the supernatant was determined using the method of Bradford with bovine serum albumin as standard (Bradford, 1976) and thus prepared parietal cell extract was employed to determine the H+ –K+ –ATPase inhibition. The H+ –K+ –ATPase activity was assayed (Ricardo et al., 2006) in presence and absence of different doses of SRAE and omeprazole. The reaction mixture (1 mL) contained enzyme in 20 mM Tris–HCl, pH 7.4, 2 mM MgCl2 and 2 mM KCl. Reaction was started with the addition of 2 mM ATP and incubated for 30 min at 37 ◦ C. The reaction was terminated by the addition of ammonium molybdate and trichloroacetic acid mixture followed by centrifugation at 2000 × g. The amount of inorganic phosphate released from ATP was determined spectrophotometrically at 400 nm. 2.12. Antioxidant activity in SRAE Antioxidant activity of SRAE was determined as reducing power, DPPH radical scavenging and DNA protective ability as described earlier (Suresh Kumar et al., 2006). Total phenolic content of SRAE was determined (Singleton and Rossi, 1965) using gallic acid as standard and expressed as gallic acid equivalents (GAE) in milligrams per gram sample. The antioxidant activity was expressed for GAE in terms of total absorbance at 700 nm, IC50 (concentration required to scavenge 50% of DPPH radical) and percent for reducing power, DPPH radical scavenging activity and DNA protection ability, respectively. 2.13. Statistical analysis of data The statistical analysis of all the results was carried out using one-way ANOVA followed by Dunnets multiple comparisons using Graphpad Prism Statistical software and all the results obtained were compared with the stress-induced group. Values are expressed as mean ± S.D. (n = 6). A value of p < 0.05,

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Table 1 Effect of SRAE on mucin content, reduced glutathione, lipid peroxidation products and H+ –K+ –ATPase in stomach of healthy, ulcer-induced and -treated rats Group

Mucin content (ELISA units/g)

Control (healthy) Stress-induced SRAE-treated Ranitidine-treated

6.2 3.2 7.82 7.38

± ± ± ±

0.02*** 0.01 0.02*** 0.01***

Glutathione (n moles/g) 236.8 99 272 241

± ± ± ±

16*** 12 19*** 26***

H+ –K+ –ATPase (U/mg protein)

TBARS (n moles/g) 0.72 1.54 0.69 1.13

± ± ± ±

0.06** 0.19 0.04** 0.3*

0.826 2.606 1.006 1.48

± ± ± ±

0.09*** 0.19 0.08*** 0.16**

Each value is expressed as mean ± standard deviation (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, Dunnets test as compared to stress-induced.

by SRAE and ranitidine at 200 mg/kg b.w. and 30 mg/kg b.w., respectively. Mean ulcer index of 6.0 ± 0.01 was reduced to 3.4 ± 0.1 and 1.63 ± 0.438 at 100 and 200 mg/kg b.w. of SRAE, respectively. An ulcer index of 1.250 ± 0.403 was observed in case of ranitidine treatment while control (healthy) showed no signs of ulcers; hence an ulcer index of zero. Significant antiulcer effect (p < 0.01) with approximately 4.4- and 4.8-fold reduction in ulcer index in rats treated with SRAE and ranitidine were observed, respectively.

p < 0.01, p < 0.001 and was considered significant, very significant, and highly significant, respectively. 3. Results 3.1. Toxicity studies with SRAE Comparative analysis between healthy controls (HC) and SRAE-treated (ST) rats indicated no significant differences in the serum total protein (HC/ST—275.8 ± 19.65/266.3 ± 22.10 mg/dL), SGOT (HC/ST—10.48 ± 0.395/ 10.29 ± 3.984 U/mg protein), SGPT (HC/ST—12.03 ± 1.77/ 12.33 ± 1.009 U/mg protein), ALP (HC/ST—21.81 ± 3.879/ 27.55 ± 6.725 U/mg protein) and TBARS (HC/ST—0.1023 ± 0.04/nmol/0.071 ± 0.01 mg protein) levels. Data thus suggest no adverse effects on the major organs. Animals after the experiment schedule remained healthy as that of control animals with normal food and water intake, body weight gain and behavior.

3.3. Biochemical changes in stomach, liver, brain and serum of ulcer-induced rats upon SRAE and ranitidine treatment 3.3.1. Gastric mucin, H+ K+ ATPase, GSH and TBARS A 2/2.7-fold reduction in mucin/GSH levels observed during ulcer condition (3.2 ± 0.01 U/g/99 ± 12 nmol/g) compared to that of healthy control (3.2 ± 0.01 U/g/99 ± 12 nmol/g) was normalized by SRAE (7.82 ± 0.02 U/g/272 ± 19 nmol/g) and ranitidine (7.38 ± 0.01 U/g/241 ± 26 nmol) treatments (Tables 1 and 2). Similarly, the gastroprotective effect of SRAE was evident by observing the normalization of 2.1- and 4.2fold raised TBARS levels in the stomach (Table 1) as well as in liver/serum (Table 2), respectively. Ranitidine being an antiulcer agent via blocking histamine receptor did not have effective control over TBARS levels (Tables 1 and 2).

3.2. Antiulcer effect of SRAE in swim stress-induced ulcer animal model High inflammatory and blood clotting areas were observed in swim stress-induced ulcerous animals, while the group treated with either SRAE or ranitidine indicated normalization as that of control. Ulcer index suggests 77% and 79% protection offered

Table 2 Effect of SRAE on biochemical parameters in ulcer-induced rats; comparison with healthy controls and ranitidine-treated groups Parameter

Control (healthy)

Stress-induced

Serum Total protein (mg/dL) SOD (U/mg protein) Catalase (U/mg protein Peroxidase (U/mg protein) Reduced GSH (U/mg protein) TBARS (n moles/mg protein)

7.533 47.58 3.78 1.075 7.447 0.412

± ± ± ± ± ±

0.389NS 3.424NS 0.290*** 0.059*** 1.310* 0.017**

7.883 48.15 1.66 0.240 1.363 1.744

± ± ± ± ± ±

0.673 4.721 0.30 (2.2 fold↓) 0.04 (4.4 fold↓) 0.064 (5.7 fold↓) 0.284 (4.2 fold↓)

6.783 54.02 4.12 0.941 11.92 0.432

± ± ± ± ± ±

0.311* 2.408* 0.36*** 0.199** 1.67** 0.018**

7.633 48.263 1.91 0.226 6.887 1.408

± ± ± ± ± ±

0.417NS 3.97NS 0.312NS 0.072NS 1.304* 0.175NS

Liver Total protein (mg/g) SOD (U/mg protein) Catalase (U/mg protein Peroxidase (U/mg protein) TBARS (n moles/mg protein)

32.25 160.3 64.35 123.3 1.908

± ± ± ± ±

2.136* 14.38*** 5.481*** 13.86*** 0.291**

46.5 60.67 30.60 80.03 3.679

± ± ± ± ±

5.694 16.97 (2.6 fold↓) 5.391 (2.1 fold↓) 8.00 (1.5 fold↓) 0.552 (1.9 fold↓)

37.32 123.7 50.47 105.3 1.463

± ± ± ± ±

0.479NS 6.960** 2.831** 8.110*** 0.372**

31.12 101.6 61.28 96.4 2.8

± ± ± ± ±

1.86* 5.210* 4.814*** 7.21** 0.382*

Brain Total protein (mg/g) TBARS (n moles/mg protein)

6.150 ± 0.302*** 0.277 ± 0.03***

7.600 ± 0.349 0.738 ± 0.013 (2.7 fold↓)

SRAE-treated

6.200 ± 0.491** 0.2628 ± 0.02***

Ranitidine-treated

7.133 ± 0.4944* 0.635 ± 0.029**

Each value is expressed as mean ± standard deviation (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, NS -Not significantly different than stress-induced, Dunnets test as compared to stress-induced.

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3.3.2. Total protein and antioxidant enzymes Further, there were no significant differences in the total protein among control, ulcer-induced, SRAE and ranitidine-treated groups in serum, brain, or liver tissues (Table 2). However, the antioxidant enzyme profile indicated some changes in both serum and liver. In serum, the SOD level was not affected while ∼2 to 3-fold reduction in SOD and catalase levels were observed in the liver homogenate of ulcerous rats. The SOD/CAT enzymes were recovered near to normal by SRAE (77/63%) and ranitidine (78/95%) treatments. Significantly depleted (4.4 folds) peroxidase in the serum of ulcerous animals was normalized up to 88% by SRAE, while ranitidine did not show any recovery. Data clearly suggested that antioxidants and antioxidant enzymes are highly applicable to control ulcer disease via management of oxidative stress while ranitidine may offer protection by blocking the histamine receptor. Obviously SRAE and ranitidine both are effective via two different mechanisms.

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than other dietary/vegetable sources studied in our laboratory (data not shown). The reducing power of SRAE at a dose of 0.1 mg was 0.178 Units (Fig. 2a) with a total reducing power of 52 U/mg GAE. These results clearly indicate that the significant antioxidant activity of SRAE may be related to their reducing power. Besides, Fig. 2b and c illustrates the scavenging effect of SRAE on DPPH radical and DNA protective ability, respectively. Fifty percent scavenging ability was observed at a concentration of 0.17 ␮g/mL GAE of SRAE, as opposed to BHA (IC50 -0.08 ␮g/mL). As indicated in Fig. 2c, the damaged DNA migrated fast, while protected DNA moved slowly as that of normal, untreated DNA. Image analysis indicated recovery of DNA to >90%.

3.4. Mechanism of antiulcer/gastroprotective effect of SRAE 3.4.1. SRAE inhibits H+ –K+ –ATPase Since H+ –K+ –ATPase is a prime enzyme in inducing acidity, we studied the ability of SRAE in inhibiting H+ –K+ –ATPase isolated from sheep stomach (Fig. 1) as well as from the experimental rats (Table 1). SRAE inhibited H+ –K+ –ATPase activity in a concentration-dependent manner with an IC50 of 36 ␮g/mL when compared to known anti-ulcer drug-omeprazole (IC50 -27 ␮g/mL) under similar experimental conditions. 3.4.2. SRAE multipotent antioxidant activity The swallow root aqueous extract possessed phenolics as antioxidant source with 34 mg GAE/g, which is 40-fold higher

Fig. 1. Effect of SRAE and omeprazole on H+ –K+ –ATPase activity: H+ –K+ –ATPase activity was measured without (control) and with 10–50 ␮g/mL of SRAE and omeprazole.

Fig. 2. Antioxidant activity in SRAE: The reducing power (a), free radical scavenging (b) and DNA protective ability (c) of SRAE was measured at indicated doses. Values are mean ± S.D. (n = 3).

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4. Discussion Ulcer is a recurrent disease affecting large populations in all geographical regions and reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide variety of clinical disorders and gastric damage. Although prolonged anxiety, H. Pylori infection, emotional stress, hemorrhagic surgical shock, burns and trauma are known to cause severe gastric irritation, the mechanism is still poorly understood (Saggioro and Chiozzini, 1994). It is generally accepted that ulcer results from an imbalance between aggressive factors and the maintenance of mucosal integrity through the endogenous defense mechanisms. To regain the balance, different therapeutics including spice and plant extracts have been used (Goel and Sairam, 2002; Jainu and Devi, 2006). The present study was undertaken to evaluate the antiulcer property of SRAE that had been used traditionally for gastric disturbances. SRAE at 200 mg/kg b.w. protected swim stress-induced ulcer lesions up to 77% similar to that of ranitidine (79%), a known antiulcer drug at 30 mg/kg b.w. Bloody streaks, inflammations, oozing of blood into the lumen of the stomach, etc., observed in ulcerous animals were not found in SRAE ingested animals, similar to those of healthy rats indicating the gastroprotective effect of SRAE. Further, we followed the protective effect investigating the biochemical parameters such as alterations in the gastric mucin, oxidants, GSH, H+ –K+ –ATPase and antioxidant enzymes level including catalase, superoxide dismutase, peroxidase, etc., in the ulcerated organ-stomach as well in the metabolizing organ-liver and reflux controlling organ-brain in all groups of rats-healthy, ulcerated and SRAE/ranitidine-treated. Preventive antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) are the first line of defense against reactive oxygen species (ROS). Administration of SRAE resulted in a significant increase in the SOD, catalase and reduced GSH levels (Table 2) similar to those of control animals, suggesting the efficacy of SRAE in preventing free radicalinduced damage during ulceration. In our experimental model ∼2 fold reduction in gastric mucin and reduced glutathione as well as increased oxidative product-TBARS in the stomach were normalized by SRAE (Table 1) treatment. Gastric ulcers are often a chronic disease and it may persist for 10–20 years characterized by repeated episodes of healing and re-exacerbations (Bakumov et al., 2003). Stress-induced ulcer better resembles clinical ulcers in chronicity, severity and practicality of experiencing stress due to varietal patterns of lifestyle in day to day life and serves the most reliable model to study ulcer healing process (Thomas et al., 1980; Jones, 2006). The incidence of swim stress-induced ulcer is predominant in the glandular part of the stomach leading to gastric mucosal/mucin damage. SRAE significantly prevented ulcers both by reducing the oxidative stress as well as boosting the mucosal defense. Further, during our study, we evaluated the possible mechanism of protection to gastric ulcer apart from upregulation of antioxidant and antioxidant enzyme levels. Gastric H+ –K+ –ATPase located in the apical membrane of parietal cells, pumps protons into the gastric lumen, using energy derived from the hydrolysis of ATP, and is thus involved in gastric acid

secretion. Accordingly, the activity of gastric H+ –K+ –ATPase was measured in the stomach homogenate, which showed 3fold upregulation of the enzyme in ulcer condition and was normalized by treatment with SRAE (Table 1). Results were further substantiated by sheep H+ –K+ –ATPase inhibition by SRAE (Fig. 1) with an IC50 of 36 ␮g/mL on par with omeprazole (IC50 -27 ␮g/mL), indicating the potential multi-targeted effect of SRAE in preventing swim stress-induced ulcers in experimental rats. SRAE may find itself more useful as a H+ –K+ –ATPase (proton pump) inhibitor than the existing pump inhibitors, since they have adverse effects as reported particularly under conditions of pregnancy/lactation and alcohol or any other drug consumption. Least toxicity of SRAE may also find SRAE as useful alternative source for ulcer healing therapeutics. This is the first report to show that SRAE is highly potent in inhibiting gastric ulceration in experimental stress-induced ulcer in animal models. SRAE blocks acid secretion by inhibiting the activity of H+ –K+ –ATPase as does omeprazole/lansoprazole. Therefore SRAE has potentials for therapeutic application as a preventive measure for hyperacidity in any form of drug therapy. Besides, SRAE also exhibited reducing power and prevented free radical-induced lipid and DNA peroxidation. This antioxidative property also contributes significantly to reduce ulcer condition and justifies the ethno medical claims. 5. Conclusions The present study clearly demonstrated that aqueous extract of swallow root was able to protect the gastric mucosa from stress-induced mucosal lesions and inhibits gastric acid secretion probably by blocking H+ –K+ –ATPase action and offering antioxidant protection against oxidative stress-induced gastric damage. The results confirm the popular use of D. hamiltonii for its medicinal properties in Ayurveda and folklore medicines. These results further suggest the use of swallow root for gastric disorders that needs to be considered as possibilities for new therapeutic approaches. Acknowledgements The authors thank Dr. V. Prakash, Director, CFTRI, for his keen interest in the work and encouragement. Authors are also thankful to Dr. S.G. Bhat, Head, Department of Biochemistry and Nutrition, for his valuable suggestions. Dr. SMD thanks CSIR for financial assistance. References Bakumov, P.A., Shchepotkin, I.V., Dugina, Y.L., Sergeeva, S.A., Epstein, O.I., 2003. Clinical efficiency of epigam in patients with exacerbation of peptic ulcer disease of the stomach and duodenum. Bulletin of Experimental Biology and Medicine 135, 159–162. Bandyopadhyay, D., Chattopadhyay, A., 2006. Reactive oxygen species-induced gastric ulceration: protection by melatonin. Current Medicinal Chemistry 13, 1187–1202. Bandyopadhyay, U., Biswas, K., Chatterjee, R., Bandyopadhyay, D., Chattopadhyay, I., Ganguly, C.K., Chakraborty, T., Bhattacharya, K., Banerjee, R.K., 2002. Gastroprotective effect of Neem (Azardichta indica) bark extract: pos-

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