Gastroprotective Properties of Karanjin from Karanja (Pongamia pinnata) Seeds; Role as Antioxidant and H+, K+-ATPase Inhibitor

Plant extracts are the most attractive sources of newer drugs and have been shown to produce promising results for the treatment of gastric ulcers. Karanjin, a furano-flavonoid has been evaluated for anti-ulcerogenic property by employing adult male albino rats. Karanjin (>95% pure) was administered to these rats in two different concentrations, that is, 10 and 20 mg kg−1 b.w. Ulcers were induced in the experimental animals by swim and ethanol stress. Serum, stomach and liver-tissue homogenates were assessed for biochemical parameters. Karanjin inhibited 50 and 74% of ulcers induced by swim stress at 10 and 20 mg kg−1 b.w., respectively. Gastric mucin was protected up to 85% in case of swim stress, whereas only 47% mucin recovery was seen in ethanol stress induced ulcers. H+, K+-ATPase activity, which was increased 2-fold in ulcer conditions, was normalized by Karanjin in both swim/ethanol stress-induced ulcer models. Karanjin could inhibit oxidative stress as evidenced by the normalization of lipid peroxidation and antioxidant enzyme (i.e., catalase, peroxidase and superoxide dismutase) levels. Karanjin at concentrations of 20 mg kg−1 b.w., when administered orally for 14 days, did not indicate any lethal effects. There were no significant differences in total protein, serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase and alkaline phosphatase between normal and Karanjin-treated rats indicating no adverse effect on major organs. During treatment schedule, animals remained as healthy as control animals with normal food and water intake and body weight gain.


Introduction
Gastric hyperacidity and gastroduodenal ulcer are common global problems and are caused by a lack of equilibrium between the gastric aggressive and the mucosal defensive factors [1]. The etiology of gastroduodenal ulcers is influenced by various aggressive and defensive factors such as acid-pepsin secretion, parietal cell activation, reduction in mucous secretion, mucosal blood flow, cellular regeneration process and endogenous protective agents (prostaglandin and epidermal growth factors) [2]. Other factors that contribute to ulcers include improper dietary habits, excessive ingestion of non-steroidal anti-inflammatory agents, stress and infection by Helicobacter pylori [3].
Several pharmaceutical products have been employed consistently for the treatment of gastric ulcers aiming to reduce mortality and morbidity rates. However, adverse effects and limitations posed by them on the use of these drugs against only a set of population warranted alternative therapies. Despite the progress in ulcer therapy from vagotomy to anti-cholinergic drugs, histamine H2 antagonists, antacids, proton-pump inhibitors, and so forth, [4] in recent years growing interest has been toward the utilization of natural products, especially those derived from plant foods [5] and plant parts [6,7], which are often designated as complementary and alternative medicines (CAMs), particularly as nutraceutical [8] and herbal medicines [9], respectively.
Recent studies on complementary and alternative medicines (CAMs) in fact suggested that CAMs play a challenging role in inhibiting several steps of various diseases, including chronic diseases such as ulcers, cancers, diabetes, inflammation, and so forth, similar to those of allopathic 2 Evidence-Based Complementary and Alternative Medicine medicines [10,11], but with no or insignificant side effects [12]. The National Centre for Complementary and Alternative Medicine and the National Centre for Health Statistics, USA, indeed declared that ∼38% of Americans use CAMs [13] and this is evidenced by significant increase of demand in the world market for alternative drugs from plantsphytomedicines [14].
We have previously reported the gastroprotective properties from extracts of medicinal plants [15,16] and natural bioactive compounds isolated from microalgae [17]. In the current study, we explored the anti-ulcerative property of Karanjin, a furano-flavonoid isolated from karanja seeds. The study has been undertaken in the light of the previous observations [18], which have reported an anti-ulcer property in crude extracts of karanja, which potentially contain karanjin.
Pongamia pinnata (L.) Pierre (Leguminosae, Papilionaceae; synonym Pongamia glabra Vent.), is popularly known as "Karanj" or "Karanja" in Hindi [19]. It is one of the widely grown forest trees. The seed contains 33-36% oil, 20-28% protein and is characterized by the presence of minor constituents such as flavonoids. The seed oil is known as karanja oil and is recognized for medicinal properties [20]. Flavonoids, which occur naturally in plant foods, have been associated with reduced risk factor of cardiovascular diseases and are reported to possess antioxidant activity and antiulcerogenic and analgesic effects.
In the current study, we investigated the anti-ulcerative property of karanjin, isolated from karanja seeds. Proof of this bioactivity would envisage the dual activity of exploring karanjin isolation for medicinal purposes in addition to the extraction of karanja oil, currently being used for leather softening and in ayurvedic preparations because of its pharmacological values. This article highlights the anti-ulcer potential of karanjin in both in vitro and in vivo models.

Chemicals.
All the chemicals used were of highperformance liquid chromatography (HPLC) grade or analytical grade (E. Merck). Standard chemicals used were obtained from Sigma chemicals Co., USA.

Plant.
Mature karanja seeds were procured from M/s Suresh Forestry Network, Chickballapur, Karnataka, India.

Preparation of Karanjin.
Karanjin was prepared (>95% pure) in the laboratory. Extraction of oil from karanja seeds was carried out using petroleum ether (1 : 2 w/v). Oil was subjected to liquid-liquid extraction with methanol. Karanjin was obtained from methanolic extract by preparative HPLC. Karanjin thus obtained was characterized by 1 H and 13 C nuclear magnetic resonance (NMR) and mass spectral analysis.

2.2.2.
Preparation of H + , K + -ATPase. Gastric membrane containing H + , K + -ATPase was prepared [21] from mucosal stomach scrapings of sheep and was homogenized in 20 mM Tris-HCl buffer (pH 7.4). The homogenate was centrifuged for 20 min at 6000 g and the resulting supernatant was used to determine the H + , K + -ATPase activity and its inhibition, as standardized in our laboratory previously [22]. The protein content of the supernatant was determined by Bradford's method using bovine serum albumin as a standard [23].
The enzyme extract containing 300 µg protein was taken for testing the activity of H + , K + -ATPase. Reaction was carried out in 16 mM Tris buffer (pH 6.5). The reaction was initiated by adding substrate (2 mM ATP, 2 mM MgCl 2 and 10 mM KCl) and incubated for 30 min at 37 • C. The reaction was stopped by the addition of an assay mixture containing 4.5% ammonium molybdate and 60% perchloric acid. Phosphomolybdate formed was measured spectrophotometrically at 400 nm. Enzymatic activity was calculated as micromoles of inorganic phosphate (Pi) released per hour per milligram of protein.

Inhibition of H
The enzyme extract containing 300 µg of protein was taken for testing the activity of H + , K + -ATPase in the presence of different concentrations (8-56 µg mL −1 ) of karanjin. Lansoprazole, a known proton-pump blocker, was employed as a standard anti-ulcer drug for comparative studies. Karanjin was incubated with H + , K + -ATPase for 30 min. Subsequently, reaction was carried out as described above. Enzymatic activity was calculated as micromoles of Pi released per hour per milligram of protein at different concentrations of karanjin and results were expressed as percent inhibition of enzymatic activity at each concentration.

Animals.
Male Wistar albino rats, weighing ∼180-200 g and maintained under standard conditions of temperature, humidity and light, were provided with standard rat palette diet (Saidurga Feeds, Bangalore, India) and water ad libitum. The study was approved by the institutional ethical committee, which follows the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Reg. No. 49, 1999), Government of India, New Delhi, India.

Toxicity Studies.
Toxicity studies were carried out for 15 days in control and karanjin-treated (20 mg kg −1 b.w.) rats. Serum was used for the estimation of total protein and enzymes related to liver function tests, such as serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP).

Experimental Design.
All the animals were categorized into eight groups with six animals in each group, in two sets for studies on swim and alcohol stress-induced models. There were four control groups, such as healthy, karanjin (20 mg kg −1 b.w.), omeprazole as positive control (20 mg kg −1 b.w.) and vehicle control groups. Karanjin was administered at two concentrations, that is, 10 and 20 mg kg −1 b.w. Omeprazole was given to one group at 20 mg kg −1 b.w. concentration. Ulcer was induced in one Evidence-Based Complementary and Alternative Medicine 3 group of animals without any pre-treatments. Karanjin and omeprazole were administered orally once daily, for 14 days. At the end of the 14th day, animals were fasted for 18 h and on the 15th day they were subjected to ulcer-inducing treatment.

Induction of Ulcer and Determination of Ulcer Index.
Ulcers were induced in the first set of rats by administering 100% ethanol (5 mL kg −1 b.w.) and animals were sacrificed after 1 h of ethanol treatment [24]. In other set, ulcers were induced by forced swim stress as per a published protocol [25], in which rats were briefly subjected to forced swim stress by making them swim in a jar 30-cm high and of 10 cm diameter which contained water up to 15 cm height for 3 h. Animals were sacrificed under deep ether anesthesia and the inner layer of the stomach was examined for the occurrence of ulcers. Low-to-high grading was assigned to milder to severe symptoms, respectively. The following are descriptions of ulcers groups; 0.5-red coloration, 1.0spot ulcers, 1.5-hemorrhagic streaks, 2.0-ulcers >3 mm and <5 mm, 3.0-ulcers >5 mm. Mean ulcer scores for each experimental groups were calculated and expressed as the ulcer index (UI) [26]. Stomach and liver-tissue homogenate and serum were collected from all animals and analyzed for various biochemical parameters.

Preparation of Tissue Homogenate for Biochemical
Analysis. The stomach and the liver tissues were collected, weighed and homogenized in chilled phosphate buffer (20 mM, pH 7.4). The homogenates were centrifuged at 1000 g at 4 • C for 20 min using a high-speed cooling centrifuge (Remi C 24, Mumbai, India). The clear supernatants were analyzed for various biochemical parameters.

Estimation of Gastric Mucin.
Gastric mucin was estimated by Alcian blue-binding method [27]. A sample of 100 mg of stomach tissues from animals of each group was taken and incubated for 2 h in acetate buffer (pH 5.8, 0.05 M) containing 0.16 M sucrose and 1.0% Alcian blue dye. Absorbance of the supernatant was read at 498 nm.

Estimation of H
Equal weight of gastric tissue from animals of each group was homogenized in Tris-HCl buffer (16 mM, pH 6.5). The homogenates were centrifuged at 6000 g for 20 min at 4 • C. The activity of H + , K + -ATPase in the supernatant was assessed as described previously.

Estimation of Oxidant/Antioxidant and Antioxidant
Enzymes. Lipid peroxidation products of serum, stomach and liver homogenates were determined as thiobarbituricacid reactive substances (TBARS). The malondialdehyde (MDA) formed was quantified using the molar extinction coefficient of the MDA molecule [28]. Glutathione (GSH) content was determined [29]. The activity of superoxide dismutase (SOD) was determined by measuring the reduction in the auto-oxidation of epinephrine in the presence of SOD [30]. Catalase (CAT) was assayed by decomposition of H 2 O 2 in the presence of CAT at 240 nm [31]. Glutathione peroxidase (GPx) was estimated based on the degradation of H 2 O 2 in the presence of GSH and the decrease in absorbance was read at 340 nm [32].
SOD activity was expressed as units per milligram of protein per minute (1 unit = milligram of protein required to inhibit 50% of epinephrine auto-oxidation) and CAT activity was expressed as micromoles of H 2 O 2 utilized by milligram of protein per minute. The activity of GPx was expressed as nanomoles of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized per minute per milligram of protein.
The protein content of the homogenate was determined by Bradford's method.

Histopathological Studies.
Gastric tissue samples were fixed in 10% buffered formalin for 24 h. The processed tissues were embedded in paraffin blocks and sections made were stained with hematoxylin and eosin dye [33]. The sections were analyzed by observing under light microscope at ×10 magnification.

Statistical Analysis.
All the values are expressed as mean ± SD. Significant difference between healthy, treated and ulcer-induced groups was tested (P < .05) by Duncan multiple-range test using the Statistical Package for Social Sciences (SPSS; SPSS Inc., version 10.0.5) software.  (Figure 1). (Table 1) with karanjin, carried out in rats for safety evaluation, indicated no lethal effect up to 20 mg kg −1 b.w. when orally fed for 14 days. Parametric values, however, showed slight variation as indicated by P-values; nevertheless, the values are within the reference range as per the range of values provided by the National Institute of Nutrition Manual [34] indicting no adverse effect on major organs at the ingested concentrations. After the treatment schedule, animals remained as healthy as control animals with normal food and water intake and body weight gain.

Effect of Karanjin on Swim/Ethanol Stress-Induced
Gastric Ulcer. Healthy rats did not show ulcer lesions in their stomachs (Figure 2(a)), while rats treated with forced swim stress and ethanol stress showed damage in the gastric wall with a hemorrhagic form of lesions and intraluminal bleeding (Figures 2(d) and 2(g)). Rats treated with only karanjin (Figure 2(c)) showed no lesions, which is similar to those of controls indicating no toxicity. Omeprazole, at 20 mg kg −1 b.w., showed protective effect in case of both swim and ethanol stress-induced ulcers (Figures 2(j) and 2(k)). In case of swim stress, karanjin at 10 and 20 mg kg −1 b.w. reduced ulcers up to 50% in a dose-dependent manner (Figures 2(e) and 2(f)). However, karanjin showed marginal protection in case of ethanol stress-induced ulcers (Figures 2(h) and 2(i)). Quantitative reduction in UI percentage in treated rats, compared with ulcer-induced rats is depicted in Figure 3.

Analysis of Gastric Mucin.
It is well established that gastric wall mucus is damaged during ulcer development and becomes the first target for stress-induced reactive oxygen species (ROS); this is followed by mucin oxidation or degradation and the mucus subsequently loses the protective effect [35]. Since Alcian blue binds to carboxylated mucopolysaccharides as well as sulfated and carboxylated glycoproteins, any disruption in structure results in the reduction in dye binding, which can be quantified. The gastric mucin was decreased to 15 mg g −1 tissue in swim stress-induced ulcerous rats, when compared to that of controls (54 mg g −1 tissue). Rats treated with 10 and 20 mg kg −1 b.w. karanjin showed ∼3-fold increase in mucin level. Similar results were obtained in rats that were treated with omeprazole. In ethanol stress-induced ulcerous rats however, there was no improvement in the mucin regeneration despite karanjin treatment ( Table 2).

H + , K + -ATPase
Activity. H + , K + -ATPase enzyme was increased 2-fold in ulcerous animals over healthy controls. Karanjin could normalize the levels in vivo in both swim/ethanol stress-induced models. The extent of inhibition was comparable to that of the known anti-ulcer drug omeprazole (Table 2).

Oxidant/Antioxidant/Antioxidant Enzymes and Lipid
Peroxidation Levels. Approximately 3-fold increase in TBARS levels (0.44 nmol mg −1 of protein) shown in the  Results are expressed as mean ± SD (n = 6). Range was provided by Duncan multiple-range test at P < .05. Different letters a-d in the column represent values that are significantly different when ulcer-induced group was compared with healthy control and sample-treated groups. a less or not significant; b moderately significant; c less significant and d very significant.
stomach homogenate in ulcer condition was normalized (0.17 nmol mg −1 protein) by karanjin at 20 mg kg −1 b.w., similar to the extent of protection offered by omeprazole (0.14 nmol mg −1 of protein; Tables 3 and 4). Similarly, 2-fold-depleted antioxidant enzymes-SOD, GPx and CAT-during ulcer conditions were normalized with the treatment of rats with karanjin at 10 and 20 mg kg −1 b.w. (Tables 3 and 4).

Histopathological Analysis.
Healthy controls showed intact mucosal epithelium (Figure 4(a)). Deep erosions with discontinuous mucosal layer were observed in ulcer-induced rats (Figures 4(d) and 4(g)). In case of swim stress, almost complete recovery of the mucosal layer was observed (

Discussion
Pongamia pinnata is a medium-sized glabrous tree, found throughout India and further distributed eastwards, mainly in the littoral regions of Southeast Asia and Australia [19,36]. The seed and seed oil are in use for the treatment of various inflammatory and infectious diseases, such as leucoderma, leprosy, lumbago and muscular and articular rheumatism [37]. Although only a few efforts have been made to rationalize the conventional uses of karanja, some pharmacological properties have been established, which  Figure 3: Effect of karanjin on gastric lesions in swim/ethanol stress-induced ulcer models; Ulcers were scored as described under the methods and expressed as ulcer index. The letters "a" to "e" represents level of significant differences among healthy, ulcerinduced and treated groups, where, a, less or not significant; b, less significant; c, moderately significant; d, very significant and e, most significant.
indicate that sequential extraction with different solvents exhibits differential bioactivity including ulcer healing. However, the active constituents responsible for the activity are not clearly understood. Ulcers result from an imbalance between aggressive factors and maintenance of mucosal integrity through the endogenous defense mechanisms. To regain the balance, different therapeutics, including spice and plant extracts, are used. In the previous papers we had shown that free and bound phenolics of several food sources, including ginger [38], swallow root [15] and mango ginger [39], possessed potential ulcer-preventive activity in vitro, including inhibition of H + , K + -ATPase and H. pylori growth. However, in view of addressing a question whether karanjin, a furanoflavonoid being a major component in karanja extract, can be attributed to observed gastroprotective properties of karanja extract [18], we evaluated in vitro and in vivo ulcer-preventive properties of isolated and purified karanjin.
Results of the study indicated the presence of significant (95%) levels of karanjin and structural studies including HPLC, liquid chromatography-mass spectroscopy (LC-MS) and NMR confirmed that the specifically extracted karanjin indeed is homogenous and pure. Karanjin could inhibit swim stress-induced ulcers by 50 and 74% at 10 and 20 mg kg −1 b.w.; however, only marginal protection was observed in ethanol stress-induced ulcerous rats at similar concentrations of karanjin. Karanjin controls and vehicletreated animals showed neither the toxicity of karanjin as evaluated by analysis of liver marker enzymes (Table 1) nor protection by vehicle alone suggesting that the differential results observed in two different models are specific changes brought about by karanjin. Furthermore, data also may highlight that karanjin might protect significantly from the acid-induced mucosal damage and ulcerations by blocking H + , K + -ATPase activity in swim stress model while the ethanol stress-induced mucosal damage that is induced by ethanol via a mechanism initiated by inadequate microcirculation may not be inhibited. Figure 5 depicts the induction of ulcers via different mechanisms by swim/ethanol stress models. ROS, however, accumulate in both the models and cause activation of H + , K + -ATPase leading to gastric acidity, mucosal layer damage and gastric ulcerations in addition to the inadequate microcirculation encountered during ethanol stress. Multi-step inhibitory effect has been highlighted in the scheme. Lacunae in the effective protection ability of karanjin in ethanol stress model while complete protection in swim stress model suggests the inability of protective effect of karanjin against inadequate microcirculation while potent H + , K + -ATPase inhibitory and antioxidant properties that can offer effective protection against swim stress-induced ulcers.
Etiology of induction of ulcers in different models needs to be considered to understand the differential role of karanjin. Ethanol has been known to damage the plasma membrane and leads to intracellular accumulation of sodium and water by increasing the membrane permeability. These changes ultimately cause cell death and gastric mucosal exfoliation [40]. Obviously, inadequate microcirculation in mucosal cell results in mucosal injury. Recovery of these damages requires processes such as active release of prostaglandin E 2 that enhances the proliferation of mucosal cells to produce mucin and rejuvenate the damaged layer, as revealed by our previous study [41]. Furthermore, ethanol is known also to release the endogenous ulcerogenic mediators, which could rapidly induce mucosal injury either by causing vascular changes such as mucosal edema and increased mucosal permeability [42] or by non-vascular effects such as mucus depletion and enzyme release in the stomach [43]. Swim stress, on the other hand, induces activation of parietal-cell membrane H + , K + -ATPase enzyme which enhances the influx of H + into the lumen of the stomach leading to acidity and acid-induced mucosal injury at later stages [15]. Association between severe physiological stress and gastrointestinal (GI) ulceration is well established. The pathology of stress-related mucosal damage has not been described completely, but there is a strong evidence that hypo-perfusion of the upper GI tract is a major cause. Aggressive management of the underlying disease is the most important factor in the prevention of stress ulcerations. Consideration of the mechanism of injury certainly may help in understanding the differential role of karanjin in two different models.
The current result of karanjin that was effective in regenerating mucin which is important for mucosal protection up to 76% and 86% at 10 and 20 mg kg −1 b.w. in the swim stress model. However, <47% mucin recovery in ethanol stress-induced models together with effective blocking of H + , K + -ATPase activity in both the models may suggest that, in swim stress, initiation of ulcer pathogenicity may be due to activation of H + , K + -ATPase activity; conversely, in ethanol stress-induced models, it is due to exfoliation and aberrant microcirculation followed by increase in H + , K + -ATPase activity leading to acidity and ulcerations. Thus, karanjin may be believed to protect swim stress-induced ulcers by virtue of inhibition of H + , K + -ATPase activity. This indication was substantiated by the poor improvement of UI in ethanol stress-induced ulcerated rats. Karanjin may be ineffective in preventing the ethanol stress-induced mucosal cell damage, although it could inhibit injury-mediated activation of H + , K + -ATPase. Further, studies suggest that proton-pump blockers may be effective against stress-induced ulcers. Omeprazole, a known, potent H + , K + -ATPase blocker, worked effectively in both the models suggesting the multi-step action of omeprazole [44]. It is also interesting to observe that the levels of antioxidant enzymes were brought to normal levels in both swim and ethanol stress-induced ulcerated rats upon treatment with karanjin. Observed marginal (47%) levels of gastric protection in ethanol stress-induced ulcer model could be by virtue of the antioxidant property of karanjin, although to a lesser extent. Data could thus imply that karanjin can be an effective anti-ulcer agent. Further, being non-toxic, it may also be used in combination with other nutraceuticals for effective management of oxidative stress-induced disease conditions.   (8) induction is via accumulation of reactive oxygen species (ROS) (4), activation of H + , K + -ATPase (5), increase in acidity (6) and damage of mucosal layer (7) while, in ethanol stress, it is more direct and via damage of mucosal layer due to lack of microcirculation. Karanjin protects multi-steps which includes inhibition of ROS (A), inhibition of H + , K + -ATPase (B) and mucosal protection (C). It is also possible that karanjin, like most of the phenolics, may just regulate proton pump via dehydrogenase coupling.