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Arbortristoside-A and 7-O-trans-cinnamoyl-6β-hydroxyloganin isolated from Nyctanthes arbortristis possess anti-ulcerogenic and ulcer-healing properties
Phytomedicine 20 (2013) 1055–1063
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Arbortristoside-A and 7-O-trans-cinnamoyl-6-hydroxyloganin isolated from Nyctanthes arbortristis possess anti-ulcerogenic and ulcer-healing properties Vaibhav Mishra b,c , Astha Shukla a , Sukanya Pandeti a , Manoj Kumar Barthwal b , Haushila Prasad Pandey c , Gautam Palit b,∗ , Tadigoppula Narender a,∗ a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, U.P., India Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226001, U.P., India c Department of Biochemistry (BHU), Varanasi 221005, U.P., India b
a r t i c l e
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Keywords: Nyctanthes arbortristis Arbortristoside-A 7-O-Trans-cinnamoyl6-hydroxyloganin Gastric ulcer Proton pump PGE2
a b s t r a c t Nyctanthes arbortristis Linn (Oleaceae) is widely distributed in sub-Himalayan regions and southwards to Godavari, India commonly known as Harsingar and Night Jasmine. In continuation of our drug discovery program on Indian medicinal plants, we isolated arbortristoside-A (AT) and 7-O-trans-cinnamoyl-6hydroxyloganin (6-HL) from the seeds of N. arbortristis. AT and 6-HL exhibited anti ulcer activity in experimentally induced ulcer models including cold restraint stress (CRU), alcohol (AL), pylorus ligationinduced gastric ulcer (PL) models and they also showed ulcer healing effect in chronic acetic acid-induced ulcer model (AC). © 2013 Elsevier GmbH. All rights reserved.
Introduction Nyctanthes arbortristis Linn (Oleaceae) is a Magnoliopsida class widely distributed in sub-Himalayan regions and southwards to Godavari (Kirtikar and Basu, 2000), commonly known as Harsingar and Night Jasmine. The indigenous people of Orissa state of India use N. arbortristis to cure various ailments. It has been also used extensively in Ayurveda, Sidha and Unani systems of medicines. The bioactive constituents such as polysaccharides, nyctanthoside, nyctanthic acid, -sitosterol, 6-hydroxyloganin, 7-O-Trans-cinnamoyl-6-hydroxyloganin, arbortristoside-A and arbortristoside-B (Purushothaman et al., 1985; Rathor et al., 1989; Stuppner et al., 1993) have been reported from N. arbortristis. Arbortristoside-A and arbortristoside-B have been reported to possess leishmanicidal (Tandon et al., 1991), antiplasmodial (Tuntiwahwuttikul et al., 2003), antispermatogenic (Gupta et al., 2006), antiallergic (Gupta et al., 1995), anti-inflammatory (Amrite et al., 2006; Patel et al., 1998; Saxena et al., 1984; Sanjita Das et al., 2008) antinociceptive (Sanjita Das et al., 2008) and analgesic activity (Saxena et al., 1987). It is evident from the literature and previous investigations that N. arbortristis also possesses significant anti-ulcer activity (Goyal et al., 2010). Peptic ulcer is one of the major gastrointestinal disorders which occur due to an imbalance between offensive and defensive factors.
∗ Corresponding authors. Tel.: +91 522 2612411; fax: +91 522 2623405. E-mail addresses: [email protected] (G. Palit), t [email protected], [email protected] (T. Narender). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.04.010
Major offensive factors are acid, pepsin, H. pylori and bile salts. Defensive factors mainly involve mucus-bicarbonate secretion and prostaglandins. Consequently reduction of gastric acid production as well as re-inforcement of gastric mucosal protection has been the major approaches for therapy of gastric ulcer disease (Hoogerwerf and Pasricha, 2006). Any potent anti-ulcer drug should possess both anti-ulcerogenic as well as ulcer-healing properties. Present study is done to establish the anti-ulcerogenic and ulcer-healing properties of the Arbortristoside-A (AT) (Fig. 1) and 7-O-trans-cinnamoyl-6hydroxyloganin (6-HL) (Fig. 1) in experimentally induced ulcer models including cold restraint stress (CRU), alcohol (AL) and pylorus ligation-induced gastric ulcer (PL). In addition, the healing effect of AT and 6-HL in chronic acetic acid-induced ulcer model (AC) was also examined to correlate the ethanopharmacological use of N. arbortristis. Materials and methods General chemistry IR spectra were recorded on Perkin-Elmer RX-1 spectrometer. Using either KBr pellets (or) in neat. 1 H NMR, 13 C NMR, DEPT-90 and DEPT-135 spectra were run on Bruker Advance DPX 300 MHz and 200 MHz in CDCl3 .Chemical shifts are reported as values in ppm relative to CHCl3 /DMSO with TMS as internal standard. ESI mass spectra were recorded on JEOL SX 102/DA-6000. Plates for thinlayer chromatography (TLC) were prepared from silica gel 60 GF254 (Merck) and activated by drying at 100 ◦ C for 2 h. Chromatography
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Fig. 1. Chemical structures of Arbortristoside-A (AT) and 7-O-trans-cinnamoyl-6-hydroxyloganin (6-HL) isolated from the Nyctanthes arbortristis.
was executed with silica gel (60–120 mesh) using mixtures of chloroform, methanol and hexane as eluants. Visualization was obtained under UV light and spraying with 10% sulphuric acid in methanol. Background of plant N. arbortristis Linn (Oleaceae), commonly known as Harsingar and Night Jasmine. It is a shrub growing to 10 m tall, with flaky gray bark. The fruit is a flat brown heart-shaped to round capsule 2 cm diameter, with two sections each containing a single seed. Which have been claimed to possess multiple pharmacological activities like antibacterial, antifungal, anti-influenza, anti-inflammatory, analgesic, antipyretic, antihistaminic, anti ulcer, hypnotic, tranquilizing, hepatoprotective, antidiabetic, antianemic, immunobioactivities, antioxidant, antispermatogenic etc. Nyctanthes has been used as traditional medicine for the treatment of various diseases. Collection of medicinal plant N. arbortristis Linn (seeds) was purchased from the local market of Lucknow, Uttar Pradesh, India and the authentification was done by Botany Division of Central Drug Research Institute, Lucknow. Extraction Powdered N. arbortristis Linn (seeds) (4 kg) were placed in glass percolator with 95% ethanol (10.l) and allowed to stand for 24 h at room temperature. The percolate was collected and these processes were repeated for four times. The combined percolate was evaporated under reduced pressure at 50 ◦ C to afford ethanol extract. The weight of extract was found to be 300 g. Fractionation The ethanol extract was macerated with hexane. The hexane soluble fraction was separated and evaporated under reduced pressure to afford hexane fraction (F001, 80 g). Chloroform was added to hexane insoluble portion, and the resultant solution was evaporated under reduced pressure to afford chloroform fraction (F002, 120 g). n-Butanol was added to chloroform insoluble portion, the n-butanol soluble fraction was evaporated under reduced pressure at 60 ◦ C afford n-butanol fraction (F003, 80 g). Isolation and purification of iridoids n-Butanol fraction (80 g) was chromatographed on a column of silica gel (60–120 mesh) and eluted with chloroform and
methanol in increasing polarity. Fractions were collected and then combined on the basis of TLC pattern to get two subfractions (A and B). Fraction A was rechromatographed on silica gel, eluting with chloroform–methanol (96:4); recrystallization from methanol afforded AT (compound 1; 80 mg). Fraction B was rechromatographed on silicagel (60–120 mesh), eluting with chloroform - methanol (90:10); recrystallization from methanol afforded 6HL (compound 2; 200 mg). The compounds visualization was done under UV light, also shown brown spot by spraying with 10% sulphuric acid in methanol. Arbortristoside-A (1): IR (KBr) 3433, 2924, 2857, 2099, 1637, 1458, 1377, 1286, 1218, 1076, 1014, 768, 671 cm−1 ; 1 H NMR (Pyridine, 300 MHz) ␦ 7.98 (d, 1H), 7.73 (s, 1H), 7.52 (d, 2H) 7.00 (d, 2H), 6.69 (d, 1H), 5.79 (d, 1H), 5.40 (d, 1H), 3.68 (s, OMe), 3.55 (s, OMe), 2.49 (d, 1H), 2.43 (d, 1H), 1.27–1.22(q, 1H), 1.14(d, 3H); 13 C NMR (Pyridine, 75 MHz) 167.92, 166.84, 161.63, 153.04, 144.43, 130.01, 129.90, 127.36, 116.26, 114.55, 109.39, 100.70, 96.71, 79.51, 78.77, 77.38, 76.58, 74.51, 71.23, 62.41, 55.10, 50.92, 45.71, 38.98, 38.10, 36.19, 14.97; ESI-MS: 566 [M+H]+ . 7-O-trans-cinnamoyl-6ˇ-hydroxyloganin (2): IR (KBr) 3435, 2087, 1636, 1458, 1380, 1219, 1163, 1068, 772, 673 cm−1 ; 1 H NMR (CD3 OD, 300 MHz) ␦ 7.65 (d, 1H), 7.59 (s, 1H), 7.44 (d, 2H) 6.78 (d, 2H), 6.38 (d, 1H), 5.30 (d, 1H), 5.15 (d, 1H), 3.64 (s, OMe), 2.49 (d, 1H), 2.43 (d, 1H), 1.27–1.22(q, 1H), 1.14(d, 3H); 13 C NMR(CD3 OD, 75 MHz) 168.76, 167.67, 159.82, 152.84, 145.38, 129.83, 125.87, 115.48, 113.81, 108.99, 98.71, 96.31, 76.97, 76.55, 76.43, 76.22, 73.28, 70.18, 61.38, 48.93, 38.20, 36.00, 29.33, 13.64; ESI - MS: 552 [M+H]+ . Biological study Animals Experimental protocols were approved by the Institutional Ethical and Usage Committee of Central Drug Research Institute (CDRI), Lucknow. Adult female Sprague–Dawley rats, weighing 180–220 g were used in the study. Animals were housed in raised bottom mesh cages to prevent coprophagy and were kept in environmentally controlled rooms with temperature regulated at 22 ± 2 ◦ C, 12/12 h light and dark cycle. Three rats were housed per cage in a room Animals were fed with reference laboratory food pellets and water was provided ad libitum, except during the experimental period when food or water deprivation was applied. Drugs and reagents Sucralfate was obtained from Menarini pharmaceuticals, India, whereas omeprazole and other chemicals were purchased from Sigma (St. Louis, MD, USA). All other reagents used were of analytical grade.
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Experimental procedure After 1 week of acclimatization, the rats were randomly divided into groups, each consisting of 6 animals. Ulcer was induced in rats as described above and given same vehicle solutions as used for the dissolution of respective drugs and used for the purpose of comparison accordingly. Treatment groups for antiulcer studies: Rats were divided into three groups Group I (ulcer control): Rats were treated with vehicle 45 min before to the induction of ulcer. Group II (AT or 6-HL): Treament schedule was same as in group I rats were 45 min prior pretreated with AT or 6-HL (20 mg/kg, p.o.), before the induction of ulcer. Group III (reference drug): Rats were treated with reference drug omeprazole (10 mg/kg, p.o.) in CRU, Aspirin and Pyloric ligiation model. Sucralfate (500 mg/kg, p.o.) was used as reference drug in AL model, 45 min before to the induction of gastric ulcer. Treatment groups for ulcer healing: Treament schedule was same as above for ulcer studies Rats were divided into three groups. Group I (ulcer control): Rats were treated with vehicle after 3rd day of induction of ulcer. Group II (AT or 6-HL): Rats were treated with AT or 6-HL (20 mg/kg) for 10 days after 3 days of ulcer induction. Group III (reference drug): Rats were treated with reference drug omeprazole (OMZ) at a dose of 10 mg/kg body weight p.o. for 10 days after 3 days of ulcer induction. Anti-ulcer studies Cold restraint stress (CRU) induced gastric ulcer model in rats. The rats were subjected to cold restraint stress after 45 min of treatment of AT, 6-HL and reference drug omperazole (OMZ). Rats were immobilized in restraint cage and kept at 4 ◦ C in an environmental chamber (Levine, 1971). 2 h later the animals were killed and stomachs were observed and scored under Magnascope for ulcers. Alcohol induced gastric ulcers in rats (AL). The animals were divided into three groups according to the treatment. Absolute alcohol (1 ml/200 g, body weight of animals) was induced gastric ulcer in rats by administering orally (Robert, 1979). The AT, 6-HL and reference drug sucralfate (SUC) were administered 45 min before alcohol treatment. After 1 h of alcohol administration, the animals were sacrificed and stomach was cut open along the greater curvature to observe the gastric lesions which appear as hemorrhagic bands along the mucosal ridges of the stomach. The lengths of the lesions were measured using Biovis image analyzer software and summated to give a total lesion score. Aspirin induced gastric ulcer model in rats (AS). Aspirin at a dose of 150 mg/kg was administered to induce ulcer after 45 min of treatment of AT, 6-HL and reference drug omeprazole (OMZ). The animals were sacrificed 5 h after aspirin treatment (Djahanguiri, 1969) and the stomach was dissected out, incised along the lesser curvature and the lesion was scored. Pyloric ligation induced ulcer model in rats (PL) This was done by ligating the pyloric end of stomach of rats under chloral hydrate anesthesia (300 mg/kg, i.p.), after 45 min of pre treatment with AT, 6-HL and reference drug omeprazole (OMZ) ulcer was induced in rats by pyloric ligation. The abdomen was opened and the pyloric end of the stomach was ligated avoiding any injury to the adjacent blood vessels (Shay et al., 1945). Animals were allowed to recover and stabilize in individual cage and were deprived of water during postoperative period. After 4 h of surgery, rats were sacrificed and lesions scored and gastric fluid was collected and centrifuged at 2000 rpm for 10 min. The collected
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supernatant was used for the estimation of gastric secretion studies and mucin estimation and the gastric juice was collected for the estimation of free, total acids and mucin level. Gastric secretion analysis Free and total acidity was measured from the collected gastric juice by titrating against 0.01 N NaOH, using phenolphthalein as an indicator and expressed in terms of equiv./ml (Anoop and Jegadeesan, 2003). Mucin level in gastric juice was quantified by Crowther and Wetmore (1987). Evaluation of ulcer-healing activity Acetic acid-induced ulcer model in rats (AC). Acetic acid induced gastric ulcer on serosal surface of the stomach as described earlier by Okabe and Pfeiffer (1972) under chloralhydrate anesthesia. Stomach was exposed to 40% acetic acid for 90 s. Administartion with reference drug omeprazole and isolated compounds AT and 6-HL was started from the 3rd day of surgery, considering day 1 of treatment and was continued for another 3, 7 and 10 days. 10th day after the treatment, five rats of each group were sacrificed by cervical dislocation. 5 ml of 10% formalin was used to fixed ulcers. After 5 min, stomach was cut open along the greater curvature. The longitudinal and abscissal length of ulcer base was deliberated with micrometer and area (mm2 ) was calculated. Stomach was again dipped in formalin for 24 h. Paraffin blocks were prepared by embedding tissue for 4 h in an automatic block making machine. A section of 5 m was made with the help of automated microtome. Hematoxylin and eosin staining (HE) was done and stained slides were visualized with stereomicroscope under 40× magnification and photos were taken for respective slides. Measurement of ulcer lesion. Ulcers formed in stomach of rats in CRU, pyloric ligated, aspirin and acetic acid induced gastric ulcer models were scored according to the arbitrary scoring system and graded as following: (i) shedding of epithelium = 10; (ii) petechial and frank hemorrhages = 20; (iii) one or two ulcers = 30; (iv) more than two ulcers = 40; and (v) Perforated ulcers = 50 (Srivastava et al., 1991). In AL model after sacrificed the rats take images of stomach by Olympus trinocular zoom microscope and then length of the lesions were measured using Biovis image analyzer software (Expert Vision Lab Private Ltd., Mumbai, India) and summated to give a total lesion score. In vitro assay of H+ K+ -ATPase activity. Gastric microsomes isolated from normal fasted rat stomach (Berglindh, 1990). H+ K+ -ATPase activity was assayed in gastric microsomes incubated with or without different concentrations of AT and 6-HL as well as reference drug omeprazole (OMZ) for 10 min at 37 ◦ C, were added to an assay buffer containing (in mM) 150 KCl, 10 PIPES, 1 MgSO4 , 5 Mg ATP, 1 EGTA and 0.1 ouabain, at pH 7.2 and 10 g/ml valinomycin, 2.5 g/ml oligomycin. The reaction was carried out at 37 ◦ C for 20 min and was stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation (2000 g for 1 min), inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength (Sanui, 1974) and expressed as M/hr/mg protein. PGE2 estimation. Prostaglandin was determined in mucosal tissue samples obtained from control, treatment and reference drug groups. Briefly, mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH-7.4) containing 1 mM EDTA and 10 M indomethacin. The homogenate was centrifuged (10,000 rpm, 10 min, 4 ◦ C), and the supernatant was processed for PGE2 estimation using the Biotrak enzyme immunosorbent assay
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Fig. 2. Effect of graded dose of AT (10, 20 and 40 mg/kg, p.o.) and reference drugs omeprazole (OMZ) (10 mg/kg p.o.) on percentage protection of ulcer against cold restraint induced gastric ulcer models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
kit (Caymen EIA kit), following the manufacturer’s instructions. Results were expressed as pg PGE2 /mg protein. Estimation of alteration in gene expression by RT-PCR. Total RNA was extracted from gastric samples using TRIZOL Reagent (Sigma, USA). CDNA was generated from 5 g of total RNA using RETRO script kit (fermentos, USA) following manufacturer’s instructions. Genes for TNF-a, IL-1 and -Actin were amplified with specific primer sets. cDNA samples were annealed at 94 ◦ C (5 min) and amplified for 35 cycles with the following cycling conditions: 94 ◦ C for 1 min; respective annealing temperature for COX-1 70 ◦ C, COX-2 63 ◦ C, TNF-a 70 ◦ C, IL-1 65 ◦ C, and -Actin 55 ◦ C for 1 min; 72 ◦ C for 1 min followed by a final extension at 72 ◦ C for 10 min and was run on Bioer XP Cycler. PCR products were electrophoresed on a 1.0% agarose gel using 100-bp ladder (Amersham Biosciences, Buckinghamshire, UK) and intensity was measured using Biovis gel documentation software and expressed as relative intensity of PCR-product/-actin ratio. Statistical analysis. All values shown in the figures and tables represent the means ± S.E.M. IC50 values with 95% confidence limits were estimated using Maximum Likelihood Iterative Procedure (Finney, 1952). Statistical analysis was performed with Prism version 3.0 software using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05 was considered to be statistically significant. Results and discussion Effect of AT and 6-HL against cold restraint ulcer in rats In our pilot study, we have screened AT and 6-HL at graded doses (10, 20 and 40 mg/kg, p.o.) in CRU model exhibited (29.17%, 50.0%, 58.5%) and (37.5%, 62.5%, 66.67%) respectively compared with reference drug omeprazole (OMZ) (77.74%). From our preliminary study we found that 20 mg/kg dose as effective and selected for further studies (Figs. 2 and 3). Gastric ulcer caused by CRU in which more acid secretion and peripheral sympathetic activation carry
Fig. 3. Effect of graded dose of 6-HL (10, 20 and 40 mg/kg, p.o.) and reference drug omeprazole (OMZ) (10 mg/kg, p.o.) on percentage protection of ulcer against CRU models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
main function (Djahanguiri et al., 1973). AT and 6-HL imparted significant protection in this model. This interesting finding in CRU model intrigued us to additional explores its effect on other gastric ulcer models in rats. Effect of AT and 6-HL against alcohol induced gastric ulcer in rats AT and 6-HL showed significant anti-ulcer activity against ethanol induced ulcer having 72.41% (p < 0.01) and 80.67% (p < 0.001) protection whereas the reference drug, sucralfate (SUC), showed 65.67% protection (p < 0.01) as depicted in Fig. 4. AT and 6-HL exerted a protective effect against ethanol in contrast to reference drug. The necrotizing agents produce ulceration in gastric mucosa by depleting gastric mucus and breaking the mucosal barrier (Devenport, 1967). Alcohol is necrotizing agent causes abruption of gastric mucosa and inhibits the release of mucosal prostaglandins (Miller and Henagan, 1984). Hence a cytoprotective agent, who increases mucus secretion, will be effective in this model. In our studies we have observed that AT and 6-HL has significantly reduced the ulcer in AL model. 6-HL is more significantly protects the gastric mucosa as comparison with AT. Effect of AT and 6-HL against aspirin induced gastric ulcer in rats To confirm the cytoprotective effect of AT and 6-HL we further checked AT and 6-HL in aspirin induced ulcer model. AT and 6-HL exhibited 48.45% and 52.5% protection respectively (Fig. 4). Reference drug omeprazole (OMZ) showed 58.50% protection in comparison to control. AT and 6-HL reduced ulcer incidence, which further supports cytoprotective effect, which may be mediated by prostaglandins. Effect of AT and 6-HL against pyloric ligation induced gastric ulcer models in rats Anti-ulcer effect of AT and 6-HL were also studied against pyloric ligation induced ulcer model in rats where they showed protection of 50.0% and 62.50% (p < 0.01) respectively whereas
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Fig. 5. Effect of AT, 6-HL and reference drug omeprazole (OMZ) on H+ K+ -ATPase activity in the rat gastric microsomes. Dots and lines are mean ± S.E.M. of experiments performed in triplicates (n = 3). *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
of PGE2 . The PGE2 generation in the ulcer control group was 2551 ± 222.9 pg/mg tissue protein. The PGE2 value of AT, 6-HL and reference drug omeprazole (OMZ) treated group was found to be 3857 ± 141.2 (Pb < 0.05), 4724 ± 441.8 (Pb < 0.001), 4168 ± 320.8 (Pb < 0.01) respectively (Fig. 6). Fig. 4. Effect of AT and 6-HL (20 mg/kg, p.o.) and reference drugs (SUC and OMZ) on percentage protection of ulcer against alcohol, aspirin and pyloric ligation induced gastric ulcer models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
reference drug omeprazole (OMZ) showed 69.42% (p < 0.01) protection (Fig. 4). In this model, auto-digestion of mucosa by gastric acid results in the development of ulcers (Goel and Bhattacharya, 1991). AT and 6-HL significantly reduced free and total acidity in this model, which suggests its anti-secretory potency. Effect of AT and 6-HL on gastric secretion The effect of AT and 6-HL on offensive factors like free and total acidity, and defensive factors like mucin secretion, which play a crucial role in the pathogenesis of gastric ulcers, was studied by collecting gastric juice from stomachs in PL model. As shown in Table 1, treatment with AT and 6-HL at a dose of 20 mg/kg body weight has significantly reduced the free 40.28 and 47.56%, respectively and total acidity by 18.61 and 28.83%, respectively (Table 1). On the other side, AT and 6-HL at a dose of 20 mg/kg body weight has increased the mucin secretion by 54.90 and 64.06%, respectively in comparison to control (Table 1).
After acetic acid induced ulcer, healing effect of AT and 6-HL AT and 6-HL (20 mg/kg, p.o.) showed ulcer healing effect (55.94% and 59.97%) respectively after 10 days of treatment (Fig. 7). Histopathology after chronic treatment of AT and 6-HL Control rats showed sharply defined mucosal ulcer in the stomach at the site of exposure to 40% acetic acid (Fig. 8). After intraluminal application of acetic acid injured mucosal cells, glands, inflammatory exudates, proliferated fibroblasts, mixed leukocytic infiltrate and cellular debris were found in the ulcerated wall of the stomach. The control rats showed mucosal ulcer in the stomach at the site of acetic acid exposure. After 10 days control rats groups exhibited decreased inflammatory exudates along with mucosal regeneration, glandular organization and reduced size of the ulcer. AT and 6-HL treated rats at this time point showed much clearer evidences of restoration of mucosal epithelium (i.e., re-epitheliazation), continued clearance of inflammatory exudates and better secretory activity of normally arranged glands (Fig. 9) reference drug omeprazole (OMZ) also showed restoration of mucosal epithelium (Fig. 10).
Effect of AT and 6-HL on H+ K+ -ATPase activity The gastroprotective activity of the compounds AT and 6-HL is not well established. Thus, we investigated the effect of AT and 6-HL on H+ K+ -ATPase inhibitory activity in isolated gastric microsomes from rat stomach. AT and 6-HL inhibited the proton pump activity with an IC50 103.86 g/ml and 65.01 g/ml respectively comparable to reference drug omeprazole with an IC50 30.24 g/ml. it was signifying the anti-secretory activity of the AT and 6-HL (Fig. 5). Effect of AT, 6-HL and omeprazole on PGE2 level AT and 6-HL significantly reduced ulcer, which further supports cytoprotective effect of AT and 6-HL, which may be mediated by prostaglandins. Especially PGE2 , have been paid much attention as major defensive factors. AT and 6-HL been identified as a major defensive factor by increasing the level
Fig. 6. Effect of AT, 6-HL and reference drug omeprazole (OMZ) on gastric PGE2 level in comparison to ulcer control group. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
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Table 1 Effect of AT, 6-HL reference drug omeprazole (OMZ) on free acidity, total acidity and mucin contents in pyloric ligation model (n = 6 in each group). Treatment
Free acid (equiv./ml)
Total acid (equiv./ml)
Mucin (g/ml)
Control AT(20 mg/kg) 6-HL(20 mg/kg) Omz(10 mg/kg)
74.19±4.234 44.3±8.167* 38.9±5.408** 36.24±8.197**
127.43±6.310 103.71±9.215* 90.68±2.890** 90.75±3.247**
2245.174±205.2 4979.127±341.8* 6248.248±419.6** 4643.294±192.4*
* **
Statistically significant at p < 0.05. p < 0.01, in comparison to control n = 6 in each group.
Fig. 7. Images of ulcerated stomach obtained from rats of control groups and treated with AT, 6-HL and reference drug omeprazole (OMZ) in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group).
Effect of on AT and 6-HL cyclo-oxygenase 1 and 2 We investigated AT and 6-HL effect on the gene expression of COX-1 and COX-2. After 10 day of treatment the mRNA
expression levels of COX-2 was significantly increased in the ulcer control group in comparison to sham. COX-1 was constitive gene and an expression level is normal in all groups (Figs. 11 and 12).
Fig. 8. Sections of ulcerated stomach obtained from rats of control groups and sham in acetic acid-induced ulcer model in rats (n = 6 in each group). HE 40×.
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Fig. 9. Sections of ulcerated stomach obtained from rats of treated with AT and 6-HL in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group). HE 40×.
Effect of on AT and 6-HL pro-inflammatory cytokines (TNF-˛ & IL-1ˇ)
Fig. 10. Sections of ulcerated stomach obtained from rats of treated with OMZ in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group). HE 40×.
Fig. 11. Values are expressed as mean ± SEM. **p < 0.01 in respect to control.
To verify whether AT and 6-HL is imparting any effect in controlling the inflammatory events initiated by ulcer we investigated its effect on the levels of gene expression of proinflammatory cytokines like TNF-␣, IL-1. The mRNA expression levels of TNF␣, IL-1 were significantly increased in the ulcer control group in comparison to sham. As evident from Fig. 13, induction of ulcer caused a significant increase in the levels of TNF-␣ (p < 0.001), IL-1 (p < 0.01) signifying the proinflammatory nature of ulcer. Administration of AT and 6-HL at a dose of 20 mg/kg, p.o. suppressed the levels of expression of these cytokines significantly after 10 day of treatment (Figs. 13 and 14). In the gastric ulcer healing, the proinflammatory response, which results in an increase in the level of TNF-a and IL-1B. Thus after AT and 6-HL treatment it reduces the expression levels of TNF-a and IL-1B (10 days treatment after the 3rd day of formation of ulcer formation of a gastric ulcer) the necrotic cell death associated with gastric ulcer formation to activate proinflammatory, resulting in the stimulation of gastric ulcer. This notion is consistent with the idea that after AT and 6-HL orally adminstration it accelerated the healing process.
Fig. 12. Values are expressed as mean ± SEM. ***p < 0.001 in comparison to sham.
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Acknowledgments Authors are thankful to Dr. T.K. Chakraborty, Director, CSIR-CDRI for constant encouragement for the program on natural products of biological importance. Vaibhav Mishra gratefully acknowledge CSIR, New Delhi, for providing financial support. We also thank the ICMR, New Delhi for providing research grant and fellowship. SAIF, CDRI for spectral data. This is CDRI communication No. 8448.
References
Fig. 13. Values are expressed as mean ± SEM. **p < 0.01 in comparison to control.
Fig. 14. Values are expressed as mean ± SEM. ***p < 0.001 in comparison to control.
Conclusions For the treatment of gastric ulcers it is important not only to prevent further ulcer formation, but also to enhance ulcer healing. In our current studies AT and 6-HL isolated from N. arbortristis exhibited both properties. They not only prevented the formation of irritant-induced gastric ulcers but also enhanced gastric ulcer healing. Conflict of interest The authors have no conflict of interest. Disclosure statement The authors have nothing to disclose.
Amrite, O., Thurakal, J., Gadgoli, C., 2006. Evaluation of anti-inflammatory activity of Nyctanthes arbortristis and Onosma echioides. Pharmacognosy Magazine 2, 258–260. Anoop, A., Jegadeesan, M., 2003. Biochemical studies on the anti-ulcerogenic potential of Hemidesmus indicus R. Br. var. indicus. Jounal of Ethnopharmacology 84, 149–156. Berglindh, T., 1990. Gastric glands and cells: preparation and in vitro methods. Methods in Enzymology 192, 93–107. Crowther, R.S., Wetmore, R.F., 1987. Fluorometric assay of O-linked glycoproteins by reaction with 2 cyanoacetamide. Analytical Biochemistry 163, 170–174. Devenport, H.W., 1967. Salicylate damage to the gastric mucosal barrier. New England Journal of Medicine 276, 1307–1312. Djahanguiri, B., Taubin, H.L., Landsburg, L., 1973. Increased sympathetic activity in the pathogenesis of restraint ulcer in rats. Journal of Pharmacology and Experimental Therapeutics 184, 163–168. Djahanguiri, B., 1969. The production of acute gastric ulceration by indomeathacin in the rat. Scanadian Journal of Gastroenterology 4, 265–267. Finney, D.J., 1952. A statistical treatment of the sigmoidal response curve, 2nd ed. Cambridge Univ. Press, New York London, pp. 318. Goel, R.K., Bhattacharya, S.K., 1991. Gastroduodenal mucosal defence and mucosal protective agents. Indian Journal of Experimental Biology 29, 701–714. Goyal, S., Daniel, K., Daniel, V., Silawat, N., Trivedi, R., Porwal, P., 2010. Evaluation of anti-ulcer activity of ethanolic extract of leaves of Nyctanthes arbortritis Linn. by different model in rats. Inventi Journal, 1. Gupta, P.P., Srimal, R.C., Srivastava, M., Singh, K.L., Tandon, J.S., 1995. Anti allergic activity of arbortristosides from Nyctanthes arbortristis. International Journal of Pharmacognosy 33, 70–72. Gupta, R.S., Kachhawa, J.B.S., Sharma, R., 2006. Antispermatogenic effect of Nyctanthes arbortristis in male albino rats. Pharmacology 2, 261–273. Hoogerwerf, W.A., Pasricha, P.J., 2006. Pharmacotherapy of gastric acidity, peptic ulcers, and gastroesophageal reflux disease. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), “Goodman and Gilman’s” The Pharmacological Basis of Therapeutics. , 11th ed. Mc Graw Hill, New York, pp. 967–981. Kirtikar, K.R., Basu, B.D., 2000. Indian Medicinal Plants. Sri Satguru Publications, Delhi. Levine, R.J., 1971. In: Pfeiffer, C.J. (Ed.), A method for rapid production of stress ulcers in rats. Peptic Ulcer Munksgaard, Copenhagen, pp. 92–97. Miller, T.A., Henagan, J.M., 1984. Indomethacin decreases resistance of gastric barrier to disruption by alcohol. Digestive Diseases and Sciences 29, 141–149. Okabe, S., Pfeiffer, C.J., 1972. Chronicity of acetic acid ulcer in the stomach. Digestive Diseases and Sciences 7, 612–629. Patel, U.M., Patel, K.M., Patel, M.P., Bhavasar, G.C., 1998. Anti-inflammatory activity of Nyctanthes arbortristis. Indian Journal of Natural Products 14, 9–11. Purushothaman, K.K., Venkatanarasimhan, M., Sarada, A., 1985. Arbortristoside A and B, two iridoid glucosides from Nyctanthes arbortristis. Phytochemistry 24, 773–776. Rathor, A., Juneja, K.K., Tandon, J.S., 1989. An iridoid glucoside from Nyctanthes arbortristis. Phytochemistry 28, 1913–1917. Robert, A., 1979. Cytoprotection by prostaglandins. Gastroenterology 77, 761–767. Sanjita Das, Dinakar, Sasmal, Saumya Priya, Basu, 2008. Anti-inflammatory and antinociceptive activity of arbortristoside-A. Journal of Ethnopharmacology 116, 198–203. Sanui, H., 1974. Measurement of inorganic orthophosphate in biological materials: extraction properties of butyl acetate. Analytical Biochemistry 60, 489–504. Saxena, R.C., Gupta, B., Saxena, K.K., Srivastava, V.K., Prasad, D.N., 1987. Analgesic analgesic antipyretic and ulcerogenic activity of Nyctanthes arbortristis leaf extracts. Journal of Ethnopharmacology 19, 193–200. Saxena, R.S., Gupta, B., Saxena, K.K., Singh, R.C., Prasad, D.N., 1984. Study of anti-inflammatory activity in the leaves of Nyctanthes arbortristis Linn – an Indian medicinal plant. Journal of Ethnopharmacology 11, 319–330. Shay, M., Kamarov, S.A., Fels, D., Meraaze, D., Gruenstein, H., Siplet, H., 1945. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 5, 43–61.
V. Mishra et al. / Phytomedicine 20 (2013) 1055–1063 Srivastava, S.K., Nath, C., Gupta, M.B., Vrat, S., Sinha, N.J., Dhawan, N.K., Gupta, G.P., 1991. Protection against gastric ulcer by verapamil. Pharmacological Research 23, 81–86. Stuppner, H., Müller, E.P., Mathuram, V., Kundu, A.B., 1993. Iridoid glycosides from Nyctanthes arbor-tristis. Phytochemistry 32, 375–378.
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Tandon, J.S., Srivastava, V., Guru, P.Y., 1991. Iridoids: a new class of leishmanicidal agents from Nyctanthes arbortristis. Journal of Natural Products 54, 1102–1104. Tuntiwahwuttikul, P., Rayanil, K., Taylon, W.C., 2003. Chemical constituents from the flowers of Nyctanthes arbortristis. Science Asia 29, 21–30.
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Phytomedicine journal homepage: www.elsevier.de/phymed
Arbortristoside-A and 7-O-trans-cinnamoyl-6-hydroxyloganin isolated from Nyctanthes arbortristis possess anti-ulcerogenic and ulcer-healing properties Vaibhav Mishra b,c , Astha Shukla a , Sukanya Pandeti a , Manoj Kumar Barthwal b , Haushila Prasad Pandey c , Gautam Palit b,∗ , Tadigoppula Narender a,∗ a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, U.P., India Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226001, U.P., India c Department of Biochemistry (BHU), Varanasi 221005, U.P., India b
a r t i c l e
i n f o
Keywords: Nyctanthes arbortristis Arbortristoside-A 7-O-Trans-cinnamoyl6-hydroxyloganin Gastric ulcer Proton pump PGE2
a b s t r a c t Nyctanthes arbortristis Linn (Oleaceae) is widely distributed in sub-Himalayan regions and southwards to Godavari, India commonly known as Harsingar and Night Jasmine. In continuation of our drug discovery program on Indian medicinal plants, we isolated arbortristoside-A (AT) and 7-O-trans-cinnamoyl-6hydroxyloganin (6-HL) from the seeds of N. arbortristis. AT and 6-HL exhibited anti ulcer activity in experimentally induced ulcer models including cold restraint stress (CRU), alcohol (AL), pylorus ligationinduced gastric ulcer (PL) models and they also showed ulcer healing effect in chronic acetic acid-induced ulcer model (AC). © 2013 Elsevier GmbH. All rights reserved.
Introduction Nyctanthes arbortristis Linn (Oleaceae) is a Magnoliopsida class widely distributed in sub-Himalayan regions and southwards to Godavari (Kirtikar and Basu, 2000), commonly known as Harsingar and Night Jasmine. The indigenous people of Orissa state of India use N. arbortristis to cure various ailments. It has been also used extensively in Ayurveda, Sidha and Unani systems of medicines. The bioactive constituents such as polysaccharides, nyctanthoside, nyctanthic acid, -sitosterol, 6-hydroxyloganin, 7-O-Trans-cinnamoyl-6-hydroxyloganin, arbortristoside-A and arbortristoside-B (Purushothaman et al., 1985; Rathor et al., 1989; Stuppner et al., 1993) have been reported from N. arbortristis. Arbortristoside-A and arbortristoside-B have been reported to possess leishmanicidal (Tandon et al., 1991), antiplasmodial (Tuntiwahwuttikul et al., 2003), antispermatogenic (Gupta et al., 2006), antiallergic (Gupta et al., 1995), anti-inflammatory (Amrite et al., 2006; Patel et al., 1998; Saxena et al., 1984; Sanjita Das et al., 2008) antinociceptive (Sanjita Das et al., 2008) and analgesic activity (Saxena et al., 1987). It is evident from the literature and previous investigations that N. arbortristis also possesses significant anti-ulcer activity (Goyal et al., 2010). Peptic ulcer is one of the major gastrointestinal disorders which occur due to an imbalance between offensive and defensive factors.
∗ Corresponding authors. Tel.: +91 522 2612411; fax: +91 522 2623405. E-mail addresses: [email protected] (G. Palit), t [email protected], [email protected] (T. Narender). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.04.010
Major offensive factors are acid, pepsin, H. pylori and bile salts. Defensive factors mainly involve mucus-bicarbonate secretion and prostaglandins. Consequently reduction of gastric acid production as well as re-inforcement of gastric mucosal protection has been the major approaches for therapy of gastric ulcer disease (Hoogerwerf and Pasricha, 2006). Any potent anti-ulcer drug should possess both anti-ulcerogenic as well as ulcer-healing properties. Present study is done to establish the anti-ulcerogenic and ulcer-healing properties of the Arbortristoside-A (AT) (Fig. 1) and 7-O-trans-cinnamoyl-6hydroxyloganin (6-HL) (Fig. 1) in experimentally induced ulcer models including cold restraint stress (CRU), alcohol (AL) and pylorus ligation-induced gastric ulcer (PL). In addition, the healing effect of AT and 6-HL in chronic acetic acid-induced ulcer model (AC) was also examined to correlate the ethanopharmacological use of N. arbortristis. Materials and methods General chemistry IR spectra were recorded on Perkin-Elmer RX-1 spectrometer. Using either KBr pellets (or) in neat. 1 H NMR, 13 C NMR, DEPT-90 and DEPT-135 spectra were run on Bruker Advance DPX 300 MHz and 200 MHz in CDCl3 .Chemical shifts are reported as values in ppm relative to CHCl3 /DMSO with TMS as internal standard. ESI mass spectra were recorded on JEOL SX 102/DA-6000. Plates for thinlayer chromatography (TLC) were prepared from silica gel 60 GF254 (Merck) and activated by drying at 100 ◦ C for 2 h. Chromatography
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Fig. 1. Chemical structures of Arbortristoside-A (AT) and 7-O-trans-cinnamoyl-6-hydroxyloganin (6-HL) isolated from the Nyctanthes arbortristis.
was executed with silica gel (60–120 mesh) using mixtures of chloroform, methanol and hexane as eluants. Visualization was obtained under UV light and spraying with 10% sulphuric acid in methanol. Background of plant N. arbortristis Linn (Oleaceae), commonly known as Harsingar and Night Jasmine. It is a shrub growing to 10 m tall, with flaky gray bark. The fruit is a flat brown heart-shaped to round capsule 2 cm diameter, with two sections each containing a single seed. Which have been claimed to possess multiple pharmacological activities like antibacterial, antifungal, anti-influenza, anti-inflammatory, analgesic, antipyretic, antihistaminic, anti ulcer, hypnotic, tranquilizing, hepatoprotective, antidiabetic, antianemic, immunobioactivities, antioxidant, antispermatogenic etc. Nyctanthes has been used as traditional medicine for the treatment of various diseases. Collection of medicinal plant N. arbortristis Linn (seeds) was purchased from the local market of Lucknow, Uttar Pradesh, India and the authentification was done by Botany Division of Central Drug Research Institute, Lucknow. Extraction Powdered N. arbortristis Linn (seeds) (4 kg) were placed in glass percolator with 95% ethanol (10.l) and allowed to stand for 24 h at room temperature. The percolate was collected and these processes were repeated for four times. The combined percolate was evaporated under reduced pressure at 50 ◦ C to afford ethanol extract. The weight of extract was found to be 300 g. Fractionation The ethanol extract was macerated with hexane. The hexane soluble fraction was separated and evaporated under reduced pressure to afford hexane fraction (F001, 80 g). Chloroform was added to hexane insoluble portion, and the resultant solution was evaporated under reduced pressure to afford chloroform fraction (F002, 120 g). n-Butanol was added to chloroform insoluble portion, the n-butanol soluble fraction was evaporated under reduced pressure at 60 ◦ C afford n-butanol fraction (F003, 80 g). Isolation and purification of iridoids n-Butanol fraction (80 g) was chromatographed on a column of silica gel (60–120 mesh) and eluted with chloroform and
methanol in increasing polarity. Fractions were collected and then combined on the basis of TLC pattern to get two subfractions (A and B). Fraction A was rechromatographed on silica gel, eluting with chloroform–methanol (96:4); recrystallization from methanol afforded AT (compound 1; 80 mg). Fraction B was rechromatographed on silicagel (60–120 mesh), eluting with chloroform - methanol (90:10); recrystallization from methanol afforded 6HL (compound 2; 200 mg). The compounds visualization was done under UV light, also shown brown spot by spraying with 10% sulphuric acid in methanol. Arbortristoside-A (1): IR (KBr) 3433, 2924, 2857, 2099, 1637, 1458, 1377, 1286, 1218, 1076, 1014, 768, 671 cm−1 ; 1 H NMR (Pyridine, 300 MHz) ␦ 7.98 (d, 1H), 7.73 (s, 1H), 7.52 (d, 2H) 7.00 (d, 2H), 6.69 (d, 1H), 5.79 (d, 1H), 5.40 (d, 1H), 3.68 (s, OMe), 3.55 (s, OMe), 2.49 (d, 1H), 2.43 (d, 1H), 1.27–1.22(q, 1H), 1.14(d, 3H); 13 C NMR (Pyridine, 75 MHz) 167.92, 166.84, 161.63, 153.04, 144.43, 130.01, 129.90, 127.36, 116.26, 114.55, 109.39, 100.70, 96.71, 79.51, 78.77, 77.38, 76.58, 74.51, 71.23, 62.41, 55.10, 50.92, 45.71, 38.98, 38.10, 36.19, 14.97; ESI-MS: 566 [M+H]+ . 7-O-trans-cinnamoyl-6ˇ-hydroxyloganin (2): IR (KBr) 3435, 2087, 1636, 1458, 1380, 1219, 1163, 1068, 772, 673 cm−1 ; 1 H NMR (CD3 OD, 300 MHz) ␦ 7.65 (d, 1H), 7.59 (s, 1H), 7.44 (d, 2H) 6.78 (d, 2H), 6.38 (d, 1H), 5.30 (d, 1H), 5.15 (d, 1H), 3.64 (s, OMe), 2.49 (d, 1H), 2.43 (d, 1H), 1.27–1.22(q, 1H), 1.14(d, 3H); 13 C NMR(CD3 OD, 75 MHz) 168.76, 167.67, 159.82, 152.84, 145.38, 129.83, 125.87, 115.48, 113.81, 108.99, 98.71, 96.31, 76.97, 76.55, 76.43, 76.22, 73.28, 70.18, 61.38, 48.93, 38.20, 36.00, 29.33, 13.64; ESI - MS: 552 [M+H]+ . Biological study Animals Experimental protocols were approved by the Institutional Ethical and Usage Committee of Central Drug Research Institute (CDRI), Lucknow. Adult female Sprague–Dawley rats, weighing 180–220 g were used in the study. Animals were housed in raised bottom mesh cages to prevent coprophagy and were kept in environmentally controlled rooms with temperature regulated at 22 ± 2 ◦ C, 12/12 h light and dark cycle. Three rats were housed per cage in a room Animals were fed with reference laboratory food pellets and water was provided ad libitum, except during the experimental period when food or water deprivation was applied. Drugs and reagents Sucralfate was obtained from Menarini pharmaceuticals, India, whereas omeprazole and other chemicals were purchased from Sigma (St. Louis, MD, USA). All other reagents used were of analytical grade.
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Experimental procedure After 1 week of acclimatization, the rats were randomly divided into groups, each consisting of 6 animals. Ulcer was induced in rats as described above and given same vehicle solutions as used for the dissolution of respective drugs and used for the purpose of comparison accordingly. Treatment groups for antiulcer studies: Rats were divided into three groups Group I (ulcer control): Rats were treated with vehicle 45 min before to the induction of ulcer. Group II (AT or 6-HL): Treament schedule was same as in group I rats were 45 min prior pretreated with AT or 6-HL (20 mg/kg, p.o.), before the induction of ulcer. Group III (reference drug): Rats were treated with reference drug omeprazole (10 mg/kg, p.o.) in CRU, Aspirin and Pyloric ligiation model. Sucralfate (500 mg/kg, p.o.) was used as reference drug in AL model, 45 min before to the induction of gastric ulcer. Treatment groups for ulcer healing: Treament schedule was same as above for ulcer studies Rats were divided into three groups. Group I (ulcer control): Rats were treated with vehicle after 3rd day of induction of ulcer. Group II (AT or 6-HL): Rats were treated with AT or 6-HL (20 mg/kg) for 10 days after 3 days of ulcer induction. Group III (reference drug): Rats were treated with reference drug omeprazole (OMZ) at a dose of 10 mg/kg body weight p.o. for 10 days after 3 days of ulcer induction. Anti-ulcer studies Cold restraint stress (CRU) induced gastric ulcer model in rats. The rats were subjected to cold restraint stress after 45 min of treatment of AT, 6-HL and reference drug omperazole (OMZ). Rats were immobilized in restraint cage and kept at 4 ◦ C in an environmental chamber (Levine, 1971). 2 h later the animals were killed and stomachs were observed and scored under Magnascope for ulcers. Alcohol induced gastric ulcers in rats (AL). The animals were divided into three groups according to the treatment. Absolute alcohol (1 ml/200 g, body weight of animals) was induced gastric ulcer in rats by administering orally (Robert, 1979). The AT, 6-HL and reference drug sucralfate (SUC) were administered 45 min before alcohol treatment. After 1 h of alcohol administration, the animals were sacrificed and stomach was cut open along the greater curvature to observe the gastric lesions which appear as hemorrhagic bands along the mucosal ridges of the stomach. The lengths of the lesions were measured using Biovis image analyzer software and summated to give a total lesion score. Aspirin induced gastric ulcer model in rats (AS). Aspirin at a dose of 150 mg/kg was administered to induce ulcer after 45 min of treatment of AT, 6-HL and reference drug omeprazole (OMZ). The animals were sacrificed 5 h after aspirin treatment (Djahanguiri, 1969) and the stomach was dissected out, incised along the lesser curvature and the lesion was scored. Pyloric ligation induced ulcer model in rats (PL) This was done by ligating the pyloric end of stomach of rats under chloral hydrate anesthesia (300 mg/kg, i.p.), after 45 min of pre treatment with AT, 6-HL and reference drug omeprazole (OMZ) ulcer was induced in rats by pyloric ligation. The abdomen was opened and the pyloric end of the stomach was ligated avoiding any injury to the adjacent blood vessels (Shay et al., 1945). Animals were allowed to recover and stabilize in individual cage and were deprived of water during postoperative period. After 4 h of surgery, rats were sacrificed and lesions scored and gastric fluid was collected and centrifuged at 2000 rpm for 10 min. The collected
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supernatant was used for the estimation of gastric secretion studies and mucin estimation and the gastric juice was collected for the estimation of free, total acids and mucin level. Gastric secretion analysis Free and total acidity was measured from the collected gastric juice by titrating against 0.01 N NaOH, using phenolphthalein as an indicator and expressed in terms of equiv./ml (Anoop and Jegadeesan, 2003). Mucin level in gastric juice was quantified by Crowther and Wetmore (1987). Evaluation of ulcer-healing activity Acetic acid-induced ulcer model in rats (AC). Acetic acid induced gastric ulcer on serosal surface of the stomach as described earlier by Okabe and Pfeiffer (1972) under chloralhydrate anesthesia. Stomach was exposed to 40% acetic acid for 90 s. Administartion with reference drug omeprazole and isolated compounds AT and 6-HL was started from the 3rd day of surgery, considering day 1 of treatment and was continued for another 3, 7 and 10 days. 10th day after the treatment, five rats of each group were sacrificed by cervical dislocation. 5 ml of 10% formalin was used to fixed ulcers. After 5 min, stomach was cut open along the greater curvature. The longitudinal and abscissal length of ulcer base was deliberated with micrometer and area (mm2 ) was calculated. Stomach was again dipped in formalin for 24 h. Paraffin blocks were prepared by embedding tissue for 4 h in an automatic block making machine. A section of 5 m was made with the help of automated microtome. Hematoxylin and eosin staining (HE) was done and stained slides were visualized with stereomicroscope under 40× magnification and photos were taken for respective slides. Measurement of ulcer lesion. Ulcers formed in stomach of rats in CRU, pyloric ligated, aspirin and acetic acid induced gastric ulcer models were scored according to the arbitrary scoring system and graded as following: (i) shedding of epithelium = 10; (ii) petechial and frank hemorrhages = 20; (iii) one or two ulcers = 30; (iv) more than two ulcers = 40; and (v) Perforated ulcers = 50 (Srivastava et al., 1991). In AL model after sacrificed the rats take images of stomach by Olympus trinocular zoom microscope and then length of the lesions were measured using Biovis image analyzer software (Expert Vision Lab Private Ltd., Mumbai, India) and summated to give a total lesion score. In vitro assay of H+ K+ -ATPase activity. Gastric microsomes isolated from normal fasted rat stomach (Berglindh, 1990). H+ K+ -ATPase activity was assayed in gastric microsomes incubated with or without different concentrations of AT and 6-HL as well as reference drug omeprazole (OMZ) for 10 min at 37 ◦ C, were added to an assay buffer containing (in mM) 150 KCl, 10 PIPES, 1 MgSO4 , 5 Mg ATP, 1 EGTA and 0.1 ouabain, at pH 7.2 and 10 g/ml valinomycin, 2.5 g/ml oligomycin. The reaction was carried out at 37 ◦ C for 20 min and was stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation (2000 g for 1 min), inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength (Sanui, 1974) and expressed as M/hr/mg protein. PGE2 estimation. Prostaglandin was determined in mucosal tissue samples obtained from control, treatment and reference drug groups. Briefly, mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH-7.4) containing 1 mM EDTA and 10 M indomethacin. The homogenate was centrifuged (10,000 rpm, 10 min, 4 ◦ C), and the supernatant was processed for PGE2 estimation using the Biotrak enzyme immunosorbent assay
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Fig. 2. Effect of graded dose of AT (10, 20 and 40 mg/kg, p.o.) and reference drugs omeprazole (OMZ) (10 mg/kg p.o.) on percentage protection of ulcer against cold restraint induced gastric ulcer models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
kit (Caymen EIA kit), following the manufacturer’s instructions. Results were expressed as pg PGE2 /mg protein. Estimation of alteration in gene expression by RT-PCR. Total RNA was extracted from gastric samples using TRIZOL Reagent (Sigma, USA). CDNA was generated from 5 g of total RNA using RETRO script kit (fermentos, USA) following manufacturer’s instructions. Genes for TNF-a, IL-1 and -Actin were amplified with specific primer sets. cDNA samples were annealed at 94 ◦ C (5 min) and amplified for 35 cycles with the following cycling conditions: 94 ◦ C for 1 min; respective annealing temperature for COX-1 70 ◦ C, COX-2 63 ◦ C, TNF-a 70 ◦ C, IL-1 65 ◦ C, and -Actin 55 ◦ C for 1 min; 72 ◦ C for 1 min followed by a final extension at 72 ◦ C for 10 min and was run on Bioer XP Cycler. PCR products were electrophoresed on a 1.0% agarose gel using 100-bp ladder (Amersham Biosciences, Buckinghamshire, UK) and intensity was measured using Biovis gel documentation software and expressed as relative intensity of PCR-product/-actin ratio. Statistical analysis. All values shown in the figures and tables represent the means ± S.E.M. IC50 values with 95% confidence limits were estimated using Maximum Likelihood Iterative Procedure (Finney, 1952). Statistical analysis was performed with Prism version 3.0 software using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. p < 0.05 was considered to be statistically significant. Results and discussion Effect of AT and 6-HL against cold restraint ulcer in rats In our pilot study, we have screened AT and 6-HL at graded doses (10, 20 and 40 mg/kg, p.o.) in CRU model exhibited (29.17%, 50.0%, 58.5%) and (37.5%, 62.5%, 66.67%) respectively compared with reference drug omeprazole (OMZ) (77.74%). From our preliminary study we found that 20 mg/kg dose as effective and selected for further studies (Figs. 2 and 3). Gastric ulcer caused by CRU in which more acid secretion and peripheral sympathetic activation carry
Fig. 3. Effect of graded dose of 6-HL (10, 20 and 40 mg/kg, p.o.) and reference drug omeprazole (OMZ) (10 mg/kg, p.o.) on percentage protection of ulcer against CRU models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
main function (Djahanguiri et al., 1973). AT and 6-HL imparted significant protection in this model. This interesting finding in CRU model intrigued us to additional explores its effect on other gastric ulcer models in rats. Effect of AT and 6-HL against alcohol induced gastric ulcer in rats AT and 6-HL showed significant anti-ulcer activity against ethanol induced ulcer having 72.41% (p < 0.01) and 80.67% (p < 0.001) protection whereas the reference drug, sucralfate (SUC), showed 65.67% protection (p < 0.01) as depicted in Fig. 4. AT and 6-HL exerted a protective effect against ethanol in contrast to reference drug. The necrotizing agents produce ulceration in gastric mucosa by depleting gastric mucus and breaking the mucosal barrier (Devenport, 1967). Alcohol is necrotizing agent causes abruption of gastric mucosa and inhibits the release of mucosal prostaglandins (Miller and Henagan, 1984). Hence a cytoprotective agent, who increases mucus secretion, will be effective in this model. In our studies we have observed that AT and 6-HL has significantly reduced the ulcer in AL model. 6-HL is more significantly protects the gastric mucosa as comparison with AT. Effect of AT and 6-HL against aspirin induced gastric ulcer in rats To confirm the cytoprotective effect of AT and 6-HL we further checked AT and 6-HL in aspirin induced ulcer model. AT and 6-HL exhibited 48.45% and 52.5% protection respectively (Fig. 4). Reference drug omeprazole (OMZ) showed 58.50% protection in comparison to control. AT and 6-HL reduced ulcer incidence, which further supports cytoprotective effect, which may be mediated by prostaglandins. Effect of AT and 6-HL against pyloric ligation induced gastric ulcer models in rats Anti-ulcer effect of AT and 6-HL were also studied against pyloric ligation induced ulcer model in rats where they showed protection of 50.0% and 62.50% (p < 0.01) respectively whereas
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Fig. 5. Effect of AT, 6-HL and reference drug omeprazole (OMZ) on H+ K+ -ATPase activity in the rat gastric microsomes. Dots and lines are mean ± S.E.M. of experiments performed in triplicates (n = 3). *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
of PGE2 . The PGE2 generation in the ulcer control group was 2551 ± 222.9 pg/mg tissue protein. The PGE2 value of AT, 6-HL and reference drug omeprazole (OMZ) treated group was found to be 3857 ± 141.2 (Pb < 0.05), 4724 ± 441.8 (Pb < 0.001), 4168 ± 320.8 (Pb < 0.01) respectively (Fig. 6). Fig. 4. Effect of AT and 6-HL (20 mg/kg, p.o.) and reference drugs (SUC and OMZ) on percentage protection of ulcer against alcohol, aspirin and pyloric ligation induced gastric ulcer models in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by one way ANOVA followed by Dunnett’s multiple comparison test. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
reference drug omeprazole (OMZ) showed 69.42% (p < 0.01) protection (Fig. 4). In this model, auto-digestion of mucosa by gastric acid results in the development of ulcers (Goel and Bhattacharya, 1991). AT and 6-HL significantly reduced free and total acidity in this model, which suggests its anti-secretory potency. Effect of AT and 6-HL on gastric secretion The effect of AT and 6-HL on offensive factors like free and total acidity, and defensive factors like mucin secretion, which play a crucial role in the pathogenesis of gastric ulcers, was studied by collecting gastric juice from stomachs in PL model. As shown in Table 1, treatment with AT and 6-HL at a dose of 20 mg/kg body weight has significantly reduced the free 40.28 and 47.56%, respectively and total acidity by 18.61 and 28.83%, respectively (Table 1). On the other side, AT and 6-HL at a dose of 20 mg/kg body weight has increased the mucin secretion by 54.90 and 64.06%, respectively in comparison to control (Table 1).
After acetic acid induced ulcer, healing effect of AT and 6-HL AT and 6-HL (20 mg/kg, p.o.) showed ulcer healing effect (55.94% and 59.97%) respectively after 10 days of treatment (Fig. 7). Histopathology after chronic treatment of AT and 6-HL Control rats showed sharply defined mucosal ulcer in the stomach at the site of exposure to 40% acetic acid (Fig. 8). After intraluminal application of acetic acid injured mucosal cells, glands, inflammatory exudates, proliferated fibroblasts, mixed leukocytic infiltrate and cellular debris were found in the ulcerated wall of the stomach. The control rats showed mucosal ulcer in the stomach at the site of acetic acid exposure. After 10 days control rats groups exhibited decreased inflammatory exudates along with mucosal regeneration, glandular organization and reduced size of the ulcer. AT and 6-HL treated rats at this time point showed much clearer evidences of restoration of mucosal epithelium (i.e., re-epitheliazation), continued clearance of inflammatory exudates and better secretory activity of normally arranged glands (Fig. 9) reference drug omeprazole (OMZ) also showed restoration of mucosal epithelium (Fig. 10).
Effect of AT and 6-HL on H+ K+ -ATPase activity The gastroprotective activity of the compounds AT and 6-HL is not well established. Thus, we investigated the effect of AT and 6-HL on H+ K+ -ATPase inhibitory activity in isolated gastric microsomes from rat stomach. AT and 6-HL inhibited the proton pump activity with an IC50 103.86 g/ml and 65.01 g/ml respectively comparable to reference drug omeprazole with an IC50 30.24 g/ml. it was signifying the anti-secretory activity of the AT and 6-HL (Fig. 5). Effect of AT, 6-HL and omeprazole on PGE2 level AT and 6-HL significantly reduced ulcer, which further supports cytoprotective effect of AT and 6-HL, which may be mediated by prostaglandins. Especially PGE2 , have been paid much attention as major defensive factors. AT and 6-HL been identified as a major defensive factor by increasing the level
Fig. 6. Effect of AT, 6-HL and reference drug omeprazole (OMZ) on gastric PGE2 level in comparison to ulcer control group. *Statistically significant at p < 0.05 and **p < 0.01, in comparison to control n = 6 in each group.
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Table 1 Effect of AT, 6-HL reference drug omeprazole (OMZ) on free acidity, total acidity and mucin contents in pyloric ligation model (n = 6 in each group). Treatment
Free acid (equiv./ml)
Total acid (equiv./ml)
Mucin (g/ml)
Control AT(20 mg/kg) 6-HL(20 mg/kg) Omz(10 mg/kg)
74.19±4.234 44.3±8.167* 38.9±5.408** 36.24±8.197**
127.43±6.310 103.71±9.215* 90.68±2.890** 90.75±3.247**
2245.174±205.2 4979.127±341.8* 6248.248±419.6** 4643.294±192.4*
* **
Statistically significant at p < 0.05. p < 0.01, in comparison to control n = 6 in each group.
Fig. 7. Images of ulcerated stomach obtained from rats of control groups and treated with AT, 6-HL and reference drug omeprazole (OMZ) in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group).
Effect of on AT and 6-HL cyclo-oxygenase 1 and 2 We investigated AT and 6-HL effect on the gene expression of COX-1 and COX-2. After 10 day of treatment the mRNA
expression levels of COX-2 was significantly increased in the ulcer control group in comparison to sham. COX-1 was constitive gene and an expression level is normal in all groups (Figs. 11 and 12).
Fig. 8. Sections of ulcerated stomach obtained from rats of control groups and sham in acetic acid-induced ulcer model in rats (n = 6 in each group). HE 40×.
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Fig. 9. Sections of ulcerated stomach obtained from rats of treated with AT and 6-HL in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group). HE 40×.
Effect of on AT and 6-HL pro-inflammatory cytokines (TNF-˛ & IL-1ˇ)
Fig. 10. Sections of ulcerated stomach obtained from rats of treated with OMZ in acetic acid-induced ulcer model in rats after 10 days of treatment (n = 6 in each group). HE 40×.
Fig. 11. Values are expressed as mean ± SEM. **p < 0.01 in respect to control.
To verify whether AT and 6-HL is imparting any effect in controlling the inflammatory events initiated by ulcer we investigated its effect on the levels of gene expression of proinflammatory cytokines like TNF-␣, IL-1. The mRNA expression levels of TNF␣, IL-1 were significantly increased in the ulcer control group in comparison to sham. As evident from Fig. 13, induction of ulcer caused a significant increase in the levels of TNF-␣ (p < 0.001), IL-1 (p < 0.01) signifying the proinflammatory nature of ulcer. Administration of AT and 6-HL at a dose of 20 mg/kg, p.o. suppressed the levels of expression of these cytokines significantly after 10 day of treatment (Figs. 13 and 14). In the gastric ulcer healing, the proinflammatory response, which results in an increase in the level of TNF-a and IL-1B. Thus after AT and 6-HL treatment it reduces the expression levels of TNF-a and IL-1B (10 days treatment after the 3rd day of formation of ulcer formation of a gastric ulcer) the necrotic cell death associated with gastric ulcer formation to activate proinflammatory, resulting in the stimulation of gastric ulcer. This notion is consistent with the idea that after AT and 6-HL orally adminstration it accelerated the healing process.
Fig. 12. Values are expressed as mean ± SEM. ***p < 0.001 in comparison to sham.
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Acknowledgments Authors are thankful to Dr. T.K. Chakraborty, Director, CSIR-CDRI for constant encouragement for the program on natural products of biological importance. Vaibhav Mishra gratefully acknowledge CSIR, New Delhi, for providing financial support. We also thank the ICMR, New Delhi for providing research grant and fellowship. SAIF, CDRI for spectral data. This is CDRI communication No. 8448.
References
Fig. 13. Values are expressed as mean ± SEM. **p < 0.01 in comparison to control.
Fig. 14. Values are expressed as mean ± SEM. ***p < 0.001 in comparison to control.
Conclusions For the treatment of gastric ulcers it is important not only to prevent further ulcer formation, but also to enhance ulcer healing. In our current studies AT and 6-HL isolated from N. arbortristis exhibited both properties. They not only prevented the formation of irritant-induced gastric ulcers but also enhanced gastric ulcer healing. Conflict of interest The authors have no conflict of interest. Disclosure statement The authors have nothing to disclose.
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