Chinese Journal of Natural Medicines 2014, 12(10): 07380744

Chinese Journal of Natural Medicines

Gastroprotective effect of the iridoid fraction from Barleria prionitis leaves on experimentally-induced gastric ulceration Sunil K. Jaiswal1, Mukesh K. Dubey2, Sanjib Das2, Ch. V. Rao3* 1

Department of Pharmacy, Rameshwaram Institutes of Technology & Management, Lucknow 226020, Uttar Pradesh, India;

2

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004, Assam, India;

3

Pharmacognosy & Ethnopharmacology Div., National Botanical Research Institute (CSIR), Rana Pratap Marg, Lucknow 226001,

Uttar Pradesh, India Available online October 2014

[ABSTRACT] AIM: To study the gastroprotective effect and in vivo antioxidant potential of a standardized iridoid fraction from B. prionitis leaves (BPE) against different gastric ulcer models in rats. METHOD: The standardized iridoid fraction from BPE at 50, 100, and 200 mg/kg body weight was administered orally, twice daily for 5 days for prevention from aspirin, ethanol, cold-restraint stress (CRS), and pylorus ligation (PL)-induced ulcers. Estimation of the antioxidant enzyme activity was carried out in a CRS-induced ulcer model, and various gastric secretion parameters including volume of gastric juice, acid output, and pH value were estimated in the PL-induced ulcer model. RESULTS: BPE showed a dose-dependent ulcer protective effect in PL (18.67%–66.26% protection), aspirin (24.65%–63.25% protection), CRS (20.77%–59.42% protection), and EtOH (16.93%–77.04% protection)-induced ulcers. BPE treatment in PL-rats showed a decrease in acid-pepsin secretion, and enhanced mucin and mucosal glycoproteins. However, BPE reduced the ulcer index with significant decrease in LPO (P < 0.01–0.001), SOD (P < 0.01–0.001), and an increase in CAT (P < 0.01–0.001), activity in the CRS-induced model. CONCLUSION: The data shows that the iridoid fraction from BPE possesses anti-ulcerogenic and antioxidant potential. [KEY WORDS] Barleria prionitis; Iridoid; HPTLC; Anti-ulcer activity; Antioxidant enzymes

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2014)10-0738-07

Introduction Barleria prionitis L. (Family Acanthaceae; commonly known as Vajradanti), is an annual shrub, 1–3 feet high, widely distributed throughout Africa, India, Sri Lanka, and tropical Asia. In the indigenous system of medicine in India, the aerial parts are used in fever, toothache, inflammatory, and gastrointestinal disorders. The bark is used in whooping cough as an expectorant, and the whole plant and especially the roots are used as a tonic and diuretic [1-2]. The plant has also shown potent activity against respiratory syncytial virus [3], and [Received on] 17-May-2013 [*Corresponding author] Chandana V. Rao: Tel: 91-522-220583135Ext352, Fax: 91-522-2205836, E-mail: sunil_pharmachem90@ yahoo.co.in These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved

anti-arthritic, anti-inflammatory, and anti-fertility activities [4-5] . The iridoid-enriched fraction of the aerial parts (leaves and stems) was reported for hepatoprotective activity in various acute and chronic animal models [6]. The aerial parts were reported to contain a new phenylethanoid glycoside, barlerinoside, along with six known iridoid glycosides, namely, shanzhiside methyl ester, 6-O-trans-p-coumaroyl- 8-O-acetylshanzhiside methyl ester, barlerin, acetylbarlerin, 7-methoxydiderroside, and lupulinoside, barlerinoside, shanzhiside methyl ester, 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester, barlerin, acetylbarlerin, 7-methoxydiderroside, and lupulinoside [7] . This laboratory showed that B. prionitis possesses anti-inflammatory, anti-nociceptive, and antioxidant activity [8]. The pathophysiology of gastric ulcer has generally focused on an imbalance between aggressive and protective factors in the stomach, such as acid-pepsin secretion, mucosal barrier, mucus secretion, blood flow, cellular regeneration,

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prostaglandins and epidermal growth factors [9]. The reactive oxygen species, especially hydroxyl radicals, play a major role in causing oxidative damage of mucosa in all types of ulcers [10]. Plant-based drugs are relatively less expensive, safe, and possess good tolerability, even in higher doses [11]. Although there are many products used for the treatment of gastric ulcers, most of these drugs produce several adverse reactions [12]. Plant extracts, however, are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of gastric ulcers [13]. In traditional medicine for example, several plants have been used to treat gastrointestinal disorders, including gastric ulcers [14-15]. Iridoids are a class of secondary metabolites found in a wide variety of plants and in some animals. They are monoterpenes biosynthesized from isoprene, and they are often intermediates in the biosynthesis of alkaloids. Chemically, the iridoids usually consist of a cyclopentane ring fused to a six-membered oxygen heterocycle. Cleavage of a bond in the cyclopentane ring gives rise to a subclass known as seco-iridoids, such as amarogentin. Iridoids are typically found in plants as glycosides, most often bound to glucose. They are found in many medicinal plants, and may be responsible for some of their pharmaceutical activities. Isolated and purified iridoids exhibit a wide range of bioactivities including cardiovascular, antihepatotoxic, choleretic, hypoglycemic, analgesic, anti-inflammatory, antimutagenic, antispasmodic, antitumor, antiviral, immunomodulator, and purgative activities [16-17]. Recently, it was shown that the methanol extract of the leaves of B. prionitis has gastroprotective activity [18]. The aim of the present investigation was to evaluate the gastroprotective and in vivo antioxidant potential of the iridoid fraction from B. prionitis leaves (BPE) on different experimental models of gastric lesions.

Material and Method Preparation and fractionation of extract The Barleria prionitis plant material was collected from Botanical Garden of the National Botanical Research Institute (NBRI), Lucknow, India in January. The plant material was identified and authenticated, and the voucher specimen number NBR-354 is deposited in the departmental herbarium (Pharmacognosy and Ethnopharmacology Division, NBRI, Lucknow) for future reference. The coarsely powdered leaves (5 kg) were exhaustively extracted with 50% ethanol by maceration for 2 days at room temperature with occasional shaking. The process of extraction was repeated four times, filtered, concentrated on rotavapour (Büchi, USA) and then freeze-dried (Freezone® 4.5, Labconco, USA) at high vacuum (133 × 10−3 mBar) and low temperature [(−40 ± 2) °C] to obtain at dry residue (yield 14.10%, w/w). The residue was dissolved in water and partitioned with n-hexane, chloroform, ethyl acetate, and butanol. All of the fractions were subjected to preliminary pharmacological study using aspirin-induced

ulcer model, and it was found that the butanol fraction showed the most promising activity, and was selected for detailed study. The butanol fraction was subjected to column chromatography (silica gel 0.5 kg) and eluted with 2.5 L each of 100% chloroform, 25% ethyl acetate in chloroform, 50% ethyl acetate in chloroform, 75% ethyl acetate in chloroform, 100% ethyl acetate, 2% methanol in ethyl acetate, 5% methanol in ethyl acetate, 10% methanol in ethyl acetate, 20% methanol in ethyl acetate, 30% methanol in ethyl acetate, 40% methanol in ethyl acetate, 50% methanol in ethyl acetate, and 100% methanol. The HPTLC (CAMAG, Germany) studies of the ethyl acetate:methanol fraction along with shanzhiside methyl ester (Aktin Chemicals, Inc., China) were carried out on pre-coated silica gel 60 F254 plate (E. Merck) of uniform thickness 0.2 mm as the stationary phase and ethyl acetate: toluene: methanol (8:1:1) as the mobile phase. The sample (2 µL) was applied with the help of Linomat 5 Applicato (CAMAG), and the eluted plate was then densitometrically scanned using a CAMAG scanner III at the respective wavelength or at multiple wavelengths under UV light using deuterium lamp with win CAT software. Test animals Male Sprague–Dawley rats (140–190 g) and female Swiss albino mice (25–30 g) were procured from CDRI, Lucknow and maintained in a 12 h light/dark cycle at a temperature 22 °C (± 3 °C). Rats were allowed to access standard rodent feed (Dayal, India) and water ad libitum. Food was withdrawn 18 h before the experiment and allocated to different experimental groups (six groups with six animals each). All of the studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. No. 222/ 2000/CPCSEA). Acute toxicity study A limit test for oral toxicity studies of the crude extract was performed as per OECD guidelines TG 425 with 2 000 mg·kg−1. Female Swiss albino mice were divided into six groups with six animals each. The crude extract was administered orally as a single dose to mice at different dose levels of 250, 500, 750, 1,250, 1,500, and 2,000 mg/kg b.w. The study revealed that the LD50 was more than 2,000 mg·kg−1 as none of the rats shown toxic symptoms after dosing of the extract for 14 days. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg·kg−1). Antiulcer activity−experimental procedure Animals were divided into five groups (six animals in each group). BPE in dose of 50, 100, and 200 mg·kg−1, the H2 receptor blocker ranitidine (RAN), and for gastric mucosal studies sucralfate (SFT), were used as standard drugs, in the dose of 50 mg·kg−1 and 250 mg·kg−1 were administered orally twice daily at 10:00 and 16:00, respectively for 5 days for acute ulcer protective studies. Animals of the control group received suspension of 1% carboxymethyl cellulose in distilled water (10 mL·kg−1) for the

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same administration period [19]. Pylorus ligated (PL) induced ulcers Drugs were administered for a period of 5 days and the rats were kept for 18 h fasting before pylorus-ligation, but water was allowed ad libitum. At the end of 24 h starvation, rats were anaesthetized with pentobarbitone sodium (35 mg·kg−1, i.p.), the abdomen was opened and pylorus ligation was done without causing any damage to its blood supply. The abdomen was then closed in two layers and rats were left in a cage with a false bottom of wide mesh wire gauze to prevent coprophagy. Water was withheld from 1 h before pylorus ligation and until the end of the 4 h period when the rats were sacrificed by overdosing with ether. Immediately afterwards, the abdomen was again opened, the cardiac end of the stomach was ligated, and the stomach removed. The stomach was then cut open along the greater curvature and the mucosa was washed under slow running tap water [20]. The ulcer index was calculated by adding the total number of ulcers per stomach and the total severity of the ulcers per stomach. The total severity of the ulcers was determined by recording the severity of each ulcer after histological confirmation as follows: 0, no ulcer; +, pin point ulcer and histological changes limited to superficial layers of mucosa and no congestion; ++, ulcer size less than 1 mm and half of the mucosal thickness showed necrotic changes; +++, ulcer size 1−2 mm with more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscular is remaining unaffected; ++++, ulcer either more than 2 mm in size or perforated with complete destruction of the mucosa with necrosis and hemorrhage, muscular is still remaining unaffected. The pooled group ulcer score was then calculated and the % protection calculated using the following formula [21]. Protection (%) = [(UIControl − UITreated ) / UIControl ] × 100 Aspirin (ASP)-induced ulcers Aspirin in a dose of 200 mg·kg−1 (20 mg·mL−1) was administered orally on the day of the experiment at about 10 am using an orogastric tube as an aqueous suspension, and animals were sacrificed after 5 h of administration [22]. The stomach was incised along the greater curvature and examined for ulcers as described earlier. Cold-restraint stress (CRS)-induced ulcers Rats of either sex weighing 150−175 g were immobilized for 2 h at 4 °C following the literature method [23]. Briefly, the animals were starved for 24 h with free access to water, and 60 min after receiving the corresponding treatment they were fully stretched and strapped to a wooden plank with adhesive tape after securing each limb to the plank individually. The animals were sacrificed after 2 h, and ulcers were scored as described above. Ethanol (EtOH)-induced ulcer The gastric ulcer was induced in rats by administering ethanol (EtOH, 100%, 1 mL/200 g, 1 h). EtOH was administered on the day of the experiment, the animals were scarified

by cervical dislocation, and the stomach was incised along the greater curvature and examined for ulcers. The ulcer index was scored, based on the product of length and width of the ulcer present in the glandular portion of the stomach (mm2/rat) [24]. Biochemical estimation The fundic part of the stomach of the animals from the CRS-induced ulcer group was homogenized (5%) in ice cold 0.9% saline with a Potter-Elvehjem glass homogenizer for 30 s. The homogenate was centrifuged at 800 r·min−1 for 10 min, followed by centrifugation of the supernatant at 1,200 r·min−1 for 15 min and the obtained mitochondrial fraction was used for the following estimations. Lipid peroxidation (LPO) product malondialdehyde (MDA) was estimated using 1, 1, 3, 3-tetraethoxypropane as the standard and expressed as μmol/mg protein [25]. Superoxide dismutase (SOD) activity was estimated by the inhibition of nicotinamide adenine dinucleotide (reduced)-phenazine methosulphate-nitrobluetetrazolium reaction system as adapted [26], and the results expressed as units (U) of SOD activity/mg protein. Decomposition of H2O2 in the presence of catalase (CAT) was followed at 240 nm [27], and the results expressed as mol of H2O2 consumed/min/mg protein. Gastric secretion studies The effective dose of 200 mg·kg−1 for 5 days was used for the secretion and mucosal studies. The gastric juice was collected 4 h after pylorus ligation and centrifuged for 5 min at 2 000 r·min−1 and the volume of the supernatant was expressed as mL/100g body weight. Total acid output was determined by titrating with 0.01 mol·L−1 NaOH, using phenolphthalein as indicator, and was expressed as μEq/mL concentration or μEq/4 h as output. Peptic activity was determined using hemoglobin as substrate, and was expressed as μmol of tyrosine/ml as concentration, or μmol of tyrosine/4 h as output. Dissolved mucosubstances were estimated in the 90% alcoholic precipitate of the gastric juice. The precipitate thus obtained was either dissolved in 0.1 mol·L−1 NaOH (1 mL) or 0.1 mol·L−1 H2SO4 (1 mL). The former was used for the estimation of protein [28], total hexoses, hexosamine, and fucose, while the latter was used for the estimation of sialic acid [29]. The results are expressed in µg·mL−1. The ratio of total carbohydrate (TC) (sum of total hexoses, hexosamine, fucose and sialic acid) to protein (P) was taken as the index of mucin activity [30]. Estimation of mucosal glycoproteins Samples of gastric mucosal scraping were homogenized in distilled water and treated with 90% ethanol. The carbohydrates and the proteins in the samples were estimated using the methods described above for gastric juice contents [31]. Statistical Evaluation All the data are presented as mean ± SEM and one-way analysis of variance (ANOVA) and Newman-Keuls Multiple Comparison Test were applied for determining the statistical significance between different groups.

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Results and Discussion Preliminary phytochemical studies on the 50% ethanolic extract of B. prionitis demonstrated the presence of alkaloids, flavonoids, glycosides, tannins, saponins, steroids, and triterpenoids. The extractive values of B. prionitis leaves were in n-hexane (7.5%), chloroform (15.35%), butanol (29.2%), and in the aqueous portion (55.10%). The ethyl acetate: methanol fraction was found to be a mixture of iridoid glucosides, which on TLC gave three spots of Rf 0.44, 0.64, and 0.69. One major compound (229 mg) with Rf 0.44 was further confirmed by co-TLC with a reference standard shanzhiside methyl ester (iridoid glucoside). In the acute toxicity study, no deaths or toxic symptoms were observed during the period of limit test. BPE showed significant gastric ulcer protective effect in doses of 50, 100,

and 200 mg·kg−1, twice a day for 5 days prevented the gastric ulcers induced by PL, ASP, CRS, and EtOH. The oral administration of BPE at 50–200 mg·kg−1 in pylorus ligature decreased the index of gastric lesion by (13.6 ± 1.9)–(5.6 ± 1.6), respectively (P < 0.05–P < 0.01) in comparison to control 16.6 ± 3.9. The oral administration of BPE in ASP-induced ulcers decreased the index of gastric lesion by (16.2 ± 1.6)–(7.9 ± 0.9) (P < 0.01–P < 0.001) in comparison to control (21.5 ± 3.4). At the same dose level, BPE also reduced CRS-induced ulcers, in which the total ulcer index (20.7 ± 1.4 in control group) was lessened by (16.4 ± 1.7)–(8.4 ± 1.2) (P < 0.01–P < 0.001) in those rats pre-treated with BPE at different doses. Administration of BPE 1 h before the induction of gastric lesions by ethanol showed significant activity, and decreased the total ulcer index of by (15.2 ± 1.2)–(4.2 ± 0.59), respectively (P < 0.001). Results for BPE are comparable to RAN at the dose of 50 mg·kg−1 (Fig. 1).

Fig. 1 Effect of the iridoid fraction from B. prionitis leaves (BPE twice daily for 5 days) on ethanol-, aspirin-, cold restraint stress (CRS)-, and pylorus ligation (PL)-induced gastric ulcers in rats (mean ± SEM, n = 6) # P < 0.001 vs their respective normal control group; *P

Gastroprotective effect of the iridoid fraction from Barleria prionitis leaves on experimentally-induced gastric ulceration.

To study the gastroprotective effect and in vivo antioxidant potential of a standardized iridoid fraction from B. prionitis leaves (BPE) against diffe...
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