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Gastroprotective effect of Senecio candicans DC on experimental ulcer models
Journal of Ethnopharmacology 140 (2012) 145–150
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Gastroprotective effect of Senecio candicans DC on experimental ulcer models Lakshmanan Hariprasath ∗ , Raman Jegadeesh, Nanjian Raaman Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India
a r t i c l e
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Article history: Received 2 June 2011 Received in revised form 29 September 2011 Accepted 2 January 2012 Available online 10 January 2012 Keywords: Senecio candicans Antiulcerogenic Acute toxicity Aqueous extract
a b s t r a c t Ethnopharmacological relevance: Senecio candicans DC (Asteraceae) is used as a remedy for gastric ulcer and stomach pain in the Nilgiris district, Tamil Nadu for which no scientific evidence exists. Aim of the study: The present study was performed to evaluate the gastroprotective effects and acute oral toxicity of aqueous leaf extract of Senecio candicans (AESC) in experimental models. Materials and methods: The antiulcerogenic activity of AESC was performed in two different ulcer models viz., pylorus-ligated model and ethanol-induced model using Wistar albino rats. Acute toxicity study was also performed to get information on the admissible dose for treatment of ulcer. Preliminary phytochemical screening of AESC was performed to find the active principles present, which are thus responsible for the antiulcerogenic activity. DPPH assay was performed to confirm the antioxidant activity of AESC. Results: The acute toxicity study did not show any mortality up to 2500 mg/kg b.w. of AESC. Both the ulcer models showed gastroprotective effect comparable to that of the standard Omeprazole. The results of antioxidant enzymes, histopathology sections, ATPase and mucus content of gastric secretion showed that several mechanisms are involved in the gastroprotective effect. The preliminary phytochemical screening revealed the presence of alkaloids, flavonoids and steroids in AESC. The DPPH assay confirmed the antioxidant activity of AESC. Conclusion: The traditional consumption of AESC for the treatment of gastric ulcer is thus true, the antioxidant constituents present in the extract plays a major role in the gastroprotective activity, but since Senecio species are known for the presence of pyrrolizidine alkaloids, a detailed study in future is required to describe the safe dose for a prolonged period. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peptic ulcer disease (PUD) encompassing gastric and duodenal ulcer is the most prevalent gastrointestinal disorder (Valle, 2005). Duodenal, gastric ulcers and gastric cancer are common and serious diseases all over the world (Calam and Baron, 2001). These pathologies are among the most important causes of morbidity in the world population. Peptic ulcers are focal lesions of gastric or duodenal mucosa, occurring at sites where the mucosal epithelium is exposed to acid and pepsin and are characterized by gnawing or burning sensation in the abdomen (Dhuley and Naik, 1998). Traditionally peptic ulcers have been described as an imbalance between the luminal acid peptic attacks versus the mucosal defence (Mutra et al., 1996). This life time prevalence of PUD is about 10% (Brunton, 1996). An estimated 15,000 deaths occur each year as a consequence of PUD (Valle, 2005). Despite the availability of many orthodox medications for PUD, the morbidity and mortality toll is still very high.
∗ Corresponding author at: Centre for Advanced Studies in Botany, Life Sciences Building (2nd floor), University of Madras, Guindy Campus, Chennai 600 025. Tel.: +91 44 43833036. E-mail address: [email protected] (L. Hariprasath). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.002
In the United States, about 6500 deaths occur each year on ulcerrelated complications (Sonnenberg, 1994). Many pharmaceutical products have been employed for the treatment of gastroduodenal and peptic ulcer, but these products are still too expensive for the less wealthy population (Toma et al., 2004). Thus, in the foreseeable future, therapy for gastric and duodenal ulcers will be one of the major research goals. Medicinal plants are reservoirs for drugs and lead compounds for many therapeutic agents. The treatment of peptic ulcers with plant products used in folk medicine and the protection of induced gastric ulcer in laboratory animals using medicinal plants was well documented (Disi et al., 1998). There are avalanche of scientific support on the efficacy of medicinal plants in the management of ulcers of different aetiologies (Austin and Jegadeesan, 2000; Akah et al., 2001; Nwafor and Akah, 2003; Nwafor and Okoye, 2005). Senecio candicans DC (Asteraceae) is a sub-shrubby climber, endemic to the Western Ghats, India. Water boiled with leaves is used to treat gastric ulcer and stomach pain in various parts of Nilgiris district, Tamil Nadu, India. No evidence or detailed scientific investigations have been carried out to define the antiulcerogenic activities of Senecio candicans. Thus, the present work sets out to study the antiulcerogenic activity of aqueous leaf extract of Senecio candicans (AESC). The effect produced by the extract was compared
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with the standard antiulcerogenic drug Omeprazole. We investigated the acute toxicity of AESC and also performed a preliminary phytochemical screening to identify the active principles responsible for the gastroprotective effect. 2. Materials and methods 2.1. Chemicals Assay kits for the activity of catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (LPO), 1,1-diphenyl2-picrylhydrazyl (DPPH), quercetin were obtained from Sigma–Aldrich (Mumbai, India). All other chemicals used for the studies were of analytical grade. 2.2. Preparation of crude aqueous leaf extract Fresh leaves were collected from Kundah and Oddayaratty hills of the Nilgiris, India during the month of July and were authenticated by the Botanical Survey of India, Coimbatore, India. A herbarium has been submitted to the Centre for Herbal Sciences, University of Madras with accession number CHS-SC-203. The cleaned leaves were shade dried at room temperature and powdered. The dried powder of leaf (500 g) was boiled in 2 L of distilled water for 1 h and filtered. This was repeated for 2–3 times with the same powder. The filtrate was lyophilized and finally 68 g of aqueous extract was obtained. 2.3. Experimental animals Healthy male and female Wistar albino rats weighing between 180 g and 200 g were used in this study. The female animals used were nulliparous and non-pregnant. The animals were fed pellet feed (purchased from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India). Food and water were provided ad libitum during acclimation and throughout the study. The animals were maintained at 22 ◦ C (±3 ◦ C) and relative humidity around 50–60%. Animals were housed in polypropylene cages over husk beddings and 12 h light and 12 h dark cycle was maintained throughout the experimental period. All the animal experiments were performed after getting necessary approval from the Institutional Animal Ethical Committee (IAEC No. 03/013/08, approval date 28.04.2008) of University of Madras, governed by the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India. All efforts were made to minimize both the number of animals used and unwanted stress or discomfort to the animals throughout experimental procedures. 2.4. Acute toxicity studies Animals were divided into six groups, each group containing six animals. Group 1 animals served as control which received normal saline. Group 2–Group 6 animals received AESC in single doses of 500, 1000, 1500, 2000 and 2500 mg/kg b.w., respectively. The AESC was suspended in normal saline (0.9% NaCl solution) and administered by gavage (P.O.). The animals were observed continuously for 1 h after treatment, then intermittently for 4 h, and thereafter for over a period of 14 days after administration (Silva et al., 1997) for behavioural changes, signs of toxicity and/or death. 2.5. Gastroprotective activity 2.5.1. Pylorus-ligated ulcer model The antiulcerogenic activity of AESC was performed in pylorusligated ulcer model as described by Oliveira et al., 2004. Animals
were divided into 4 groups each of six rats. Group 1 animals received normal saline and served as control. Group 2 animals received Omeprazole (40 mg/kg) orally and served as the reference drug for comparison. Groups 3 and 4 animals received 250 and 500 mg/kg b.w. of AESC, respectively. Rats in all the groups were fasted in individual cages for 24 h before the administration of AESC. All the above treatments were made 1 h prior to pyloric ligation. Pyloric ligation was performed in these animals under mild ether anaesthesia. The animals were deprived of food and water during post-operative period. The pyloric portion of the stomach was identified, slightly lifted out and ligated, avoiding traction to the pylorus or damage to the blood supply. The stomach was then replaced carefully and the abdominal wall closed by interrupted sutures. Four hours after ligation, all the animals were sacrificed. The stomach was cut along the greater curvature and separated from the surrounding tissues and thus brought out as a whole along with its contents. The content (gastric juice) was subjected to centrifugation at 3000 rpm for 10 min and the clear supernatant was then analysed for its volume, pH, the total acidity, free acidity, ulcer index and percent inhibition. The status of lipid peroxidation (LPO), catalase (CAT) and superoxide dismutase (SOD) were estimated by its appropriate kits (Sigma–Aldrich, Mumbai, India). A portion of the stomach was subjected to histopathology. The pH was estimated using pH strips with pH ranges of 2.0–4.5 and 5.0–8.5. 2.5.2. Ethanol-induced ulcer model The antiulcerogenic activity of AESC was performed in ethanolinduced ulcer model as described by Hollander et al. (1985). In this study, rats were divided into 4 groups. Group 1 animals received normal saline and served as control. Group 2 animals received Omeprazole (40 mg/kg) orally and served as the reference drug for comparison. Groups 3 and 4 animals received 250 and 500 mg/kg b.w. of AESC, respectively. Gastric lesions were induced by oral administration of 1 mL of absolute ethanol per rat. Test substances were given 30 min before the ulcerative agent and after 1 h animals were sacrificed by cervical dislocation and stomach was incised along the greater curvature and examined for ulcers in the glandular region. Erosions formed on the glandular portions of the stomach were counted and each given a severity rating on a 1–5 scale based on the diameter of the ulcer. The status of antioxidant enzymes SOD, CAT and LPO were determined. Stomach of all treated and control rats were subjected to visual macroscopic examination and ulcer score was calculated. 2.5.3. Determination of free and total acidity in gastric juice The free and total acidity of gastric juice collected from pylorusligated rats was determined by volumetric analysis as detailed by Hawk (1947). One mL of gastric juice was pipetted into a 100 mL conical flask and 2–3 drops of Topfer’s reagent (HiMedia Pvt., Ltd., Mumbai) was added and titrated with 0.01 N NaOH (which was previously standardized with 0.01 N oxalic acid) until all traces of red colour disappears and turned to yellowish orange. The volume of alkali was noted. This volume corresponds to free acidity. Then 2–3 drops of phenolphthalein solution was added and titration was continued until a definite red tinge reappears. Again, the total volume of alkali added was noted. This volume corresponds to total acidity. The free and total acidity of gastric juice was calculated by using the following formula: Acidity =
volume of NaOH × normality of NaOH 0.1 × 100 meq/L per 100 g
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2.5.4. Determination of ulcer index (UI) and percentage inhibition Ulcer index and percentage inhibition was calculated in both pylorus-ligated and ethanol-induced rats. For the determination of ulcer index, the stomach was cut open along the greater curvature and the inner surface was examined for ulceration with the help of a simple dissecting microscope. Usually, circular lesions were observed but, sometimes, linear lesions were also seen. The ulcer index was calculated according to the scoring method of Tan et al. (1996). Score 0 – no ulcer; Score 1 – vessel dilation and pointed ulcers; Score 2.5 – small ulcers <4 mm long; Score 5 – large ulcers >5 mm long. UI for each animal was calculated as mean ulcer score. The percentage of inhibition was calculated by the following formula: UI control − UI treated × 100 UI control 2.5.5. Histopathological studies The freshly excised stomachs were washed with saline and preserved in 10% formaldehyde solution for histopathological studies. The sections of stomachs were stained with haematoxylin and eosin and permanent mounts of the tissues were prepared (Bancroft and Cook, 1984) to investigate the histopathological changes. The microscopic slides were photographed. 2.5.6. Estimation of H+ K+ ATPase activity The H+ K+ ATPase activity was assayed in ethanol-induced ulcer animals (Nagaya et al., 1987). Hundred L of the tissue homogenate was taken in centrifuge tube and incubated in the assay medium (70 mM Tris buffer, 5 mM MgCl2 , 10 mM KCl in a total volume of 1 mL; pH 6.8) for 1 h. The reaction was initiated by adding 2 mM ATP, incubated at 37 ◦ C for 20 min and the reaction was stopped by 10% TCA. The precipitates formed on addition of TCA in both the test and tissue control tubes were removed by centrifugation and their supernatants were transferred to fresh tubes. The reagent blank contained 1.8 mL of Tris–HCl buffer. The standard tubes containing Pi taken at a concentration range of 2–10 g/mL were placed in dist. water and were made up to 1.8 mL with Tris–HCl buffer. To all the above tubes, 0.5 mL of ammonium molybdate and 0.2 mL of 1amino-2-naphthol-4-sulphonic acid was added and left for 20 min for the blue colour to develop, which was read at 620 nm against the reagent blank using a spectrophotometer. Results are expressed as mmol of Pi liberated/min/mg protein. 2.5.7. Determination of mucus in gastric content The assay was performed in ethanol-induced ulcer animals according to the methodology described by Sun et al. (1991) with some modifications. The gastric contents of the stomach was immersed in 10 mL 0.02% Alcian blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8, and incubated for 24 h at 20 ◦ C. The Alcian blue binding extract was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured at 615 nm using a spectrophotometer. The free mucus in the gastric content was calculated from the amount of Alcian blue binding (mg/wet tissue (g)). 2.6. Preliminary phytochemical screening The crude AESC was subjected to qualitative chemical screening for the identification of major chemical constituents such as alkaloids (Dragendorff’s test: Waldi, 1965), phenolic constituents
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(Ferric chloride test: Mace, 1963), flavonoids and phytosterols (Asongalem et al., 2004). 2.7. In vitro antioxidant assay The scavenging activity of DPPH radicals by AESC was measured according to the method reported by Yen and Hsieh (1998). Assays were performed in 1.5 mL reaction mixtures containing 1.0 mL of 0.1 mM DPPH ethanol solution and 1.0 mL of different concentrations of crude extracts and fractions of Senecio candicans (500, 250, 125, 63, 32, 16 and 8 g/mL) or 0.5 mL ethanol (as control). After 30 min of incubation at room temperature under dark condition, the absorbance of the reaction mixtures were measured at 517 nm. Quercetin was used as the standard antioxidant. The inhibitory effect of DPPH was calculated according to the following formula:
Inhibition (%) =
absorbancecontrol − absorbancesample absorbancecontrol
× 100
A xy scatter graph was plotted to determine the inhibitory concentration (IC50 ) according to the regression equation. 2.8. Statistical analysis The data were subjected to One-way Analysis of Variance (ANOVA) and Tukey’s Multiple Comparison Test was done to evaluate the significance of difference of means of various treatment groups, using SPSS statistical software package (Version: 10). The values are presented as mean ± SD and *P < 0.05, **P < 0.01 and ***P < 0.001 were considered significant. 3. Results and discussion In acute toxicity study, mortality was not observed at any of the dose levels tested i.e. up to maximum dose (2500 mg/kg), whereas rats treated with 2000 and 2500 mg/kg b.w. showed symptoms of hypoactivity, loss of appetite and piloerection. The no-observed adverse effect level (NOAEL) was 1500 mg/kg. The lowest-observed adverse effect level (LOAEL) was 2000 mg/kg. These symptoms thus can be considered as the initial signs for toxicity. Hence, for further in vivo studies a NOAEL of 500 mg/kg b.w. was fixed as the highest dose. In pyloric-ligated animal model, Omeprazole pre-treatment caused a significant increase (P < 0.001) in gastric pH accompanied by a fall (P < 0.001) in gastric volume, free acidity and total acidity when compared to control rats. Similar changes were observed in rats pretreated with AESC in a dose-dependent manner. A significant decrease in the ulcer index (UI) was observed in Omeprazole pre-treated rats (2.17 ± 2.17) and AESC treatments (compared to control rats (31.75 ± 2.3)). Pre-treatment of 250 and 500 mg/kg b.w. of AESC prior to pylorus-ligation caused an effect similar to that of Omeprazole pre-treated group in a dose-dependent manner (16.58 ± 2.29 and 8.75 ± 0.94, respectively). The UI was significantly reduced from 31.75 ± 2.3 (control rats) to 8.75 ± 0.94 in rats pretreated with 500 mg/kg of AESC. Among the groups, pre-treatment with 500 mg/kg b.w. of AESC exhibited a percentage of ulcer inhibition (72.39%), whereas, pre-treatment with 250 mg/kg b.w. of AESC showed only 47.26% of ulcer inhibition (Table 1). In ethanol-induced model, the UI in control rats was 32.42 ± 2.82, whereas pre-treatment of rats with 250 and 500 mg/kg of AESC decreased the UI drastically to 10.75 ± 2.72 and 4.33 ± 0.52, respectively. Among the AESC pre-treated groups, rats with 500 mg/kg b.w. showed maximum percentage of inhibition (83.52), whereas 250 mg/kg b.w. pre-treatment showed only 66.73% of inhibition (Table 1).
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Table 1 Effect of aqueous leaf extract of Senecio candicans on various gastric parameters of pylorus-ligated and ethanol-induced rats. Treatment Pylorus ligated ulcer model Gastric volume (mL) pH Free acidity (mEq L−1 100 g−1 ) Total acidity (mEq L−1 100 g−1 ) Ulcer index Percent ulcer inhibition (%) Ethanol-induced ulcer model Ulcer index Ulcer inhibition (%)
Group 1 (control)
Group 2 (Omeprazole – 40 mg/kg)
Group 3 (AESC – 250 mg/kg)
Group 4 (AESC – 500 mg/kg)
8.25 ± 0.26 2.29 ± 0.2 56.67 ± 1.15 109.33 ± 3.06 31.75 ± 2.3 –
6.56 ± 0.22 a*** 4.75 ± 0.17 a*** 27.93 ± 1.47 a*** 75.33 ± 1.15 a*** 2.17 ± 2.17 a*** 93.18
7.82 ± 0.13 a**; b** 4.09 ± 0.33 a***; bns 42.67 ± 2.31 a***; b 99.33 ± 6.11 a**; b*** 16.58 ± 2.29 a***; b*** 47.26
7.27 ± 0.37 a***; bns 4.48 ± 0.32 a***; bns 38.53 ± 3.56 a***; b 82.53 ± 3.23 a***; bns 8.75 ± 0.94 a***; b*** 72.39
32.42 ± 2.82 –
2.42 ± 0.80 a*** 92.59
10.75 ± 2.72 a***; b*** 66.73
4.33 ± 0.52 a***; bns 83.52
Values presented are mean ± S.D. of six numbers of animals in each group (n = 6). Multiple comparisons between treatment groups were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. ns: not significant. *P < 0.05. **P < 0.01. ***P < 0.001.
Fig. 1. Status of antioxidant enzymes in gastric juice of pylorus-ligated rats treated with aqueous leaf extract of Senecio candicans. (A) Superoxide dismutase; (B) lipid peroxidation and (C) catalase. Values presented are mean ± S.D. of six numbers of animals in each group. Multiple comparisons were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant.
It is evident from both the ulcer model studies that AESC had a significant effect in controlling the ulcer. The highest dose i.e., 500 mg/kg b.w. of AESC showed a better reduction in UI and percentage of ulcer inhibition compared to 250 mg/kg b.w. pre-treated rats. Further, we studied the status of LPO, SOD and CAT in gastric juice of pylorus-ligated and ethanol-induced rats treated with AESC (Figs. 1 and 2). Pre-treatment of Omeprazole caused a significant decrease (P < 0.001) in the status of LPO and SOD accompanied by an increase (P < 0.001) in the status of CAT, when compared to the control rats. Pre-treatment of rats with AESC also caused a highly significant fall (P < 0.001) in the status of LPO and SOD accompanied by an increase (P < 0.001) in the status of CAT, when compared to control rats. A dose-dependent effect was found in AESC treatments in both the ulcer models. In view of these reports, it is suggested that the increase in LPO observed in gastric juice of pylorus-ligated and ethanol-induced rats could be due to the increase in the generation of O2 •− , H2 O2 − , OH• radical. The enhanced SOD activity could be due to increased synthesis of this enzyme for scavenging the superoxide radicals in the gastric juice of pylorus-ligated rats. The decrease in CAT in the gastric juice could be due to the over utilization of this enzyme in decreasing the increase in the generation of H2 O2 − . Pre-treatment AESC in pylorus-ligated and ethanol-induced rats prevented the increase in LPO and SOD accompanied by a decrease in CAT in a dose-dependent manner. It is likely that the extracts prevent gastric ulcer by markedly decreasing LPO and subsequent oxidative damage. AESC treatment might decrease lipid peroxidation by quenching free radicals in the gastric mucosa of pylorus-ligated rats to exhibit antiulcerogenic activity. It is clear from the results that antioxidant compounds present in AESC could play an active role in antiulcerogenic effect. Further, to confirm the antioxidant activity of AESC an in vitro antioxidant activity (DPPH free radical scavenging activity) was carried out. The AESC exhibited an IC50 value of 10.74 ± 0.34 g/mL, which is almost comparable to that of the standard quercetin (9.56 ± 0.24). The maximum percentage of free-radical scavenging activity (94.67 ± 0.98) was observed in quercetin followed by 91.67 ± 0.26 in AESC. The DPPH assay result strongly support that AESC has a rich source of antioxidant components. This is the first report on Senecio candicans. Previously, the in vitro DPPH scavenging activity have been reported in Senecio vulgaris and Senecio inaequidens by Conforti et al. (2006). The results of phytochemical screening showed the presence of phenols, flavonoids, steroids and alkaloids in AESC. Thus, the antioxidant activity could be due to presence of these constituents in the extract. The histopathology of stomach of pylorus-ligated rats pretreated with AESC and Omeprazole further supported our claim. The pylorus-ligated positive control rats showed dense inflammatory infiltrate of the mucus with submucosal oedema and loss of normal glandular architecture of the stomach. Pre-treatment of
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Fig. 3. Effect of aqueous leaf extract of Senecio candicans on H+ K+ ATPase activity in gastric mucosa and gastric wall mucus in ethanol-induced ulcer group.
Fig. 2. Status of antioxidant enzymes in gastric juice of ethanol-induced rats treated with aqueous leaf extract of Senecio candicans. (A) Superoxide dismutase; (B) lipid peroxidation and (C) catalase. Values presented are mean ± S.D. of six numbers of animals in each group. Multiple comparisons were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant.
Omeprazole (40 mg/kg) in pylorus-ligated rats showed restoration of normal glandular pattern with mild inflammatory infiltrate in the lamina propria. AESC pre-treatment at 250 mg/kg showed mucosal and submucosal oedema with reduction in inflammatory infiltrate in the glands and submucosa. AESC pre-treated at 500 mg/kg showed near normal architecture comparable to normal rats (Supp. Mat. Fig. 1). The H+ K+ ATPase activity observed in ethanol-induced control rats was found to decrease significantly in AESC pre-treatment rats in a dose-dependent manner. Pre-treatment of rats with 250 mg/kg of AESC showed significant inhibition (P < 0.05) in H+ K+ ATPase activity (1.03 ± 0.04) compared to the control rats (1.40 ± 0.03). The activity decreased to 0.62 ± 0.06 when treated with 500 mg/kg of
AESC. The gastric wall mucus was enhanced when treated with AESC in a dose-dependent manner. The effect caused by AESC at 500 mg/kg was comparable to the effect caused by the Omeprazole treatment (Fig. 3). The possible involvement of the extract on enhancing mucosal resistance could have offered gastroprotection and is regarded as a first line of defence against gastric ulcers. Not only antioxidants present in AESC, but also the mucosal barrier protection and restoration of mucous secretion could be a possible mechanism in gastroprotective action. This is the first report on gastroprotection action in Senecio candicans. Previously, the preventive activity of Senecio brasiliensis on gastric and duodenal induced ulcer was investigated by Toma et al. (2004). In conclusion, it is evident that the AESC exhibit antiulcerogenic activity in a dose-dependent manner and its efficacy is more at the maximum dosage (500 mg/kg). The present study provides additional support for the traditional use of this plant in the treatment of gastric ulcer. Preliminary phytochemical screening of the AESC gave positive results for the presence of phenols, flavonoids and phytosterols. It is possible that a combined effects of different bioactive constituents of the plant could be responsible for the antiulcerogenic action, which may be due to (i) decrease in gastric acid secretion, (ii) inhibition of free radical generation/prevention of lipid peroxidation, (iii) free radical scavenging/antioxidant properties and (iv) the protection of mucosal barrier and restoration of mucous secretion. The AESC appear to act in a non-specific manner hence, a detailed study on the isolation and characterization of active principles responsible for the antiulcerogenic activity is essential. On the other hand Senecio species are known for the presence of pyrrolizidine alkaloids, which are generally considered hepatotoxic. Since there is no report for the presence of pyrrolizidine alkaloids in Senecio candicans, a detailed study on the percentage of alkaloids present and their characterization is essential. This might bring more knowledge on the safety aspects of this plant when used for a long period. Conflict of interest None declared. Acknowledgement The authors are grateful to Professor Dr. Ramagopalan, Department of Pathology, University of Madras, Taramani Campus, Chennai, for histopathological examinations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2012.01.002.
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chemical factor induced gastric and duodenal ulcers in experimental animals. Journal of Ethnopharmacology 52, 165–169. Nagaya, H., Satoh, H., Maki, Y., 1987. Actions of antisecretory agents on proton transports in hog gastric microsomes. Biochemical Pharmacology 36, 513–519. Nwafor, S.V., Okoye, C.F., 2005. Antiulcer properties of ethanol root extract of Cissampelos mucronata. Pharmaceutical Biology 43, 396–403. Nwafor, S.V., Akah, P.A., 2003. Effect of methanol leaf extract of Cissampelos mucronata A. rich against indomethacin induced ulcer in rats. Indian Journal of Medical Research 41, 181–183. Oliveira, F.A., Vieira@@Jr., G.M., Chaves, M.H., Almeida, F.R.C., Florencio, M.G., Lima@@Jr., R.C.P., Silva, R.M., Santos, F.A., Rao, V.S.N., 2004. Gastroprotective and anti-inflammatory effects of resin from Protium heptaphyllum in mice and rats. Pharmacological Research 49, 105–111. Silva, O., Daurk, A., Pimentel, M., Viegas, S., Barroso, H., Machado, J., Pires, I., Carbrita, J., Gomes, E., 1997. Antimicrobial activity of Terminilia macroptera root. Journal of Ethnopharmacology 57, 203–207. Sonnenberg, A., 1994. Peptic ulcer. In: Digestive Diseases in the United States: Epidemiology and Impact, No. 94-1447. US Department of Health and Human Services, Public Health Services, National Institute of Health, pp. 359–408. Sun, X.B., Matsumoto, T., Yamada, H., 1991. Effects of a polysaccharide fraction from the roots of Bupleurum falcatum L. on experimental gastric ulcer models in rats and mice. Journal of Pharmacy and Pharmacology 43, 699–704. Tan, P.V., Nditafon, G.N., Yewah, M.P., Dimo, T., Ayafor, J.F., 1996. Eremomastax speciosa: effects of leaf aqueous extract on ulcer formation and gastric secretion in rats. Journal of Ethnopharmacology 54, 139–142. Toma, W., Trigo, J.R., Bensuaski de Paula, A.C., Souza Brito, A.R.M., 2004. Modulation of gastrin and epidermal growth factor by pyrrolizidine alkaloids obtained from Senecio brasiliensis in acute and chronic induced gastric ulcers. Canadian Journal of Physiology and Pharmacology 82, 319–325. Valle, D.L., 2005. Peptic ulcer diseases and related disorders. In: Braunwald, E., Fauci, A.S., Kasper, D.L., Hauser, S.L., Longo, D.L., Jameson, J.L. (Eds.), Harrison’s Principles of Internal Medicine. , 16th ed. McGraw-Hill, New York, pp. 1746–1762. Waldi, D., 1965. Spray reagents for thin-layer chromatography. In: Egon Stahl (Ed.), Thin-Layer Chromatography – A Laboratory Handbook. Academic Press Inc., Publishers, New York, U.S.A., p. 491. Yen, G., Hsieh, C., 1998. Antioxidant activity of extracts from Duzhong (Eucommia ulmoides) towards various lipid peroxidation models in vitro. Journal of Agricultural Food Chemistry 46, 3952–3957.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Gastroprotective effect of Senecio candicans DC on experimental ulcer models Lakshmanan Hariprasath ∗ , Raman Jegadeesh, Nanjian Raaman Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 29 September 2011 Accepted 2 January 2012 Available online 10 January 2012 Keywords: Senecio candicans Antiulcerogenic Acute toxicity Aqueous extract
a b s t r a c t Ethnopharmacological relevance: Senecio candicans DC (Asteraceae) is used as a remedy for gastric ulcer and stomach pain in the Nilgiris district, Tamil Nadu for which no scientific evidence exists. Aim of the study: The present study was performed to evaluate the gastroprotective effects and acute oral toxicity of aqueous leaf extract of Senecio candicans (AESC) in experimental models. Materials and methods: The antiulcerogenic activity of AESC was performed in two different ulcer models viz., pylorus-ligated model and ethanol-induced model using Wistar albino rats. Acute toxicity study was also performed to get information on the admissible dose for treatment of ulcer. Preliminary phytochemical screening of AESC was performed to find the active principles present, which are thus responsible for the antiulcerogenic activity. DPPH assay was performed to confirm the antioxidant activity of AESC. Results: The acute toxicity study did not show any mortality up to 2500 mg/kg b.w. of AESC. Both the ulcer models showed gastroprotective effect comparable to that of the standard Omeprazole. The results of antioxidant enzymes, histopathology sections, ATPase and mucus content of gastric secretion showed that several mechanisms are involved in the gastroprotective effect. The preliminary phytochemical screening revealed the presence of alkaloids, flavonoids and steroids in AESC. The DPPH assay confirmed the antioxidant activity of AESC. Conclusion: The traditional consumption of AESC for the treatment of gastric ulcer is thus true, the antioxidant constituents present in the extract plays a major role in the gastroprotective activity, but since Senecio species are known for the presence of pyrrolizidine alkaloids, a detailed study in future is required to describe the safe dose for a prolonged period. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peptic ulcer disease (PUD) encompassing gastric and duodenal ulcer is the most prevalent gastrointestinal disorder (Valle, 2005). Duodenal, gastric ulcers and gastric cancer are common and serious diseases all over the world (Calam and Baron, 2001). These pathologies are among the most important causes of morbidity in the world population. Peptic ulcers are focal lesions of gastric or duodenal mucosa, occurring at sites where the mucosal epithelium is exposed to acid and pepsin and are characterized by gnawing or burning sensation in the abdomen (Dhuley and Naik, 1998). Traditionally peptic ulcers have been described as an imbalance between the luminal acid peptic attacks versus the mucosal defence (Mutra et al., 1996). This life time prevalence of PUD is about 10% (Brunton, 1996). An estimated 15,000 deaths occur each year as a consequence of PUD (Valle, 2005). Despite the availability of many orthodox medications for PUD, the morbidity and mortality toll is still very high.
∗ Corresponding author at: Centre for Advanced Studies in Botany, Life Sciences Building (2nd floor), University of Madras, Guindy Campus, Chennai 600 025. Tel.: +91 44 43833036. E-mail address: [email protected] (L. Hariprasath). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.002
In the United States, about 6500 deaths occur each year on ulcerrelated complications (Sonnenberg, 1994). Many pharmaceutical products have been employed for the treatment of gastroduodenal and peptic ulcer, but these products are still too expensive for the less wealthy population (Toma et al., 2004). Thus, in the foreseeable future, therapy for gastric and duodenal ulcers will be one of the major research goals. Medicinal plants are reservoirs for drugs and lead compounds for many therapeutic agents. The treatment of peptic ulcers with plant products used in folk medicine and the protection of induced gastric ulcer in laboratory animals using medicinal plants was well documented (Disi et al., 1998). There are avalanche of scientific support on the efficacy of medicinal plants in the management of ulcers of different aetiologies (Austin and Jegadeesan, 2000; Akah et al., 2001; Nwafor and Akah, 2003; Nwafor and Okoye, 2005). Senecio candicans DC (Asteraceae) is a sub-shrubby climber, endemic to the Western Ghats, India. Water boiled with leaves is used to treat gastric ulcer and stomach pain in various parts of Nilgiris district, Tamil Nadu, India. No evidence or detailed scientific investigations have been carried out to define the antiulcerogenic activities of Senecio candicans. Thus, the present work sets out to study the antiulcerogenic activity of aqueous leaf extract of Senecio candicans (AESC). The effect produced by the extract was compared
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with the standard antiulcerogenic drug Omeprazole. We investigated the acute toxicity of AESC and also performed a preliminary phytochemical screening to identify the active principles responsible for the gastroprotective effect. 2. Materials and methods 2.1. Chemicals Assay kits for the activity of catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (LPO), 1,1-diphenyl2-picrylhydrazyl (DPPH), quercetin were obtained from Sigma–Aldrich (Mumbai, India). All other chemicals used for the studies were of analytical grade. 2.2. Preparation of crude aqueous leaf extract Fresh leaves were collected from Kundah and Oddayaratty hills of the Nilgiris, India during the month of July and were authenticated by the Botanical Survey of India, Coimbatore, India. A herbarium has been submitted to the Centre for Herbal Sciences, University of Madras with accession number CHS-SC-203. The cleaned leaves were shade dried at room temperature and powdered. The dried powder of leaf (500 g) was boiled in 2 L of distilled water for 1 h and filtered. This was repeated for 2–3 times with the same powder. The filtrate was lyophilized and finally 68 g of aqueous extract was obtained. 2.3. Experimental animals Healthy male and female Wistar albino rats weighing between 180 g and 200 g were used in this study. The female animals used were nulliparous and non-pregnant. The animals were fed pellet feed (purchased from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India). Food and water were provided ad libitum during acclimation and throughout the study. The animals were maintained at 22 ◦ C (±3 ◦ C) and relative humidity around 50–60%. Animals were housed in polypropylene cages over husk beddings and 12 h light and 12 h dark cycle was maintained throughout the experimental period. All the animal experiments were performed after getting necessary approval from the Institutional Animal Ethical Committee (IAEC No. 03/013/08, approval date 28.04.2008) of University of Madras, governed by the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India. All efforts were made to minimize both the number of animals used and unwanted stress or discomfort to the animals throughout experimental procedures. 2.4. Acute toxicity studies Animals were divided into six groups, each group containing six animals. Group 1 animals served as control which received normal saline. Group 2–Group 6 animals received AESC in single doses of 500, 1000, 1500, 2000 and 2500 mg/kg b.w., respectively. The AESC was suspended in normal saline (0.9% NaCl solution) and administered by gavage (P.O.). The animals were observed continuously for 1 h after treatment, then intermittently for 4 h, and thereafter for over a period of 14 days after administration (Silva et al., 1997) for behavioural changes, signs of toxicity and/or death. 2.5. Gastroprotective activity 2.5.1. Pylorus-ligated ulcer model The antiulcerogenic activity of AESC was performed in pylorusligated ulcer model as described by Oliveira et al., 2004. Animals
were divided into 4 groups each of six rats. Group 1 animals received normal saline and served as control. Group 2 animals received Omeprazole (40 mg/kg) orally and served as the reference drug for comparison. Groups 3 and 4 animals received 250 and 500 mg/kg b.w. of AESC, respectively. Rats in all the groups were fasted in individual cages for 24 h before the administration of AESC. All the above treatments were made 1 h prior to pyloric ligation. Pyloric ligation was performed in these animals under mild ether anaesthesia. The animals were deprived of food and water during post-operative period. The pyloric portion of the stomach was identified, slightly lifted out and ligated, avoiding traction to the pylorus or damage to the blood supply. The stomach was then replaced carefully and the abdominal wall closed by interrupted sutures. Four hours after ligation, all the animals were sacrificed. The stomach was cut along the greater curvature and separated from the surrounding tissues and thus brought out as a whole along with its contents. The content (gastric juice) was subjected to centrifugation at 3000 rpm for 10 min and the clear supernatant was then analysed for its volume, pH, the total acidity, free acidity, ulcer index and percent inhibition. The status of lipid peroxidation (LPO), catalase (CAT) and superoxide dismutase (SOD) were estimated by its appropriate kits (Sigma–Aldrich, Mumbai, India). A portion of the stomach was subjected to histopathology. The pH was estimated using pH strips with pH ranges of 2.0–4.5 and 5.0–8.5. 2.5.2. Ethanol-induced ulcer model The antiulcerogenic activity of AESC was performed in ethanolinduced ulcer model as described by Hollander et al. (1985). In this study, rats were divided into 4 groups. Group 1 animals received normal saline and served as control. Group 2 animals received Omeprazole (40 mg/kg) orally and served as the reference drug for comparison. Groups 3 and 4 animals received 250 and 500 mg/kg b.w. of AESC, respectively. Gastric lesions were induced by oral administration of 1 mL of absolute ethanol per rat. Test substances were given 30 min before the ulcerative agent and after 1 h animals were sacrificed by cervical dislocation and stomach was incised along the greater curvature and examined for ulcers in the glandular region. Erosions formed on the glandular portions of the stomach were counted and each given a severity rating on a 1–5 scale based on the diameter of the ulcer. The status of antioxidant enzymes SOD, CAT and LPO were determined. Stomach of all treated and control rats were subjected to visual macroscopic examination and ulcer score was calculated. 2.5.3. Determination of free and total acidity in gastric juice The free and total acidity of gastric juice collected from pylorusligated rats was determined by volumetric analysis as detailed by Hawk (1947). One mL of gastric juice was pipetted into a 100 mL conical flask and 2–3 drops of Topfer’s reagent (HiMedia Pvt., Ltd., Mumbai) was added and titrated with 0.01 N NaOH (which was previously standardized with 0.01 N oxalic acid) until all traces of red colour disappears and turned to yellowish orange. The volume of alkali was noted. This volume corresponds to free acidity. Then 2–3 drops of phenolphthalein solution was added and titration was continued until a definite red tinge reappears. Again, the total volume of alkali added was noted. This volume corresponds to total acidity. The free and total acidity of gastric juice was calculated by using the following formula: Acidity =
volume of NaOH × normality of NaOH 0.1 × 100 meq/L per 100 g
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2.5.4. Determination of ulcer index (UI) and percentage inhibition Ulcer index and percentage inhibition was calculated in both pylorus-ligated and ethanol-induced rats. For the determination of ulcer index, the stomach was cut open along the greater curvature and the inner surface was examined for ulceration with the help of a simple dissecting microscope. Usually, circular lesions were observed but, sometimes, linear lesions were also seen. The ulcer index was calculated according to the scoring method of Tan et al. (1996). Score 0 – no ulcer; Score 1 – vessel dilation and pointed ulcers; Score 2.5 – small ulcers <4 mm long; Score 5 – large ulcers >5 mm long. UI for each animal was calculated as mean ulcer score. The percentage of inhibition was calculated by the following formula: UI control − UI treated × 100 UI control 2.5.5. Histopathological studies The freshly excised stomachs were washed with saline and preserved in 10% formaldehyde solution for histopathological studies. The sections of stomachs were stained with haematoxylin and eosin and permanent mounts of the tissues were prepared (Bancroft and Cook, 1984) to investigate the histopathological changes. The microscopic slides were photographed. 2.5.6. Estimation of H+ K+ ATPase activity The H+ K+ ATPase activity was assayed in ethanol-induced ulcer animals (Nagaya et al., 1987). Hundred L of the tissue homogenate was taken in centrifuge tube and incubated in the assay medium (70 mM Tris buffer, 5 mM MgCl2 , 10 mM KCl in a total volume of 1 mL; pH 6.8) for 1 h. The reaction was initiated by adding 2 mM ATP, incubated at 37 ◦ C for 20 min and the reaction was stopped by 10% TCA. The precipitates formed on addition of TCA in both the test and tissue control tubes were removed by centrifugation and their supernatants were transferred to fresh tubes. The reagent blank contained 1.8 mL of Tris–HCl buffer. The standard tubes containing Pi taken at a concentration range of 2–10 g/mL were placed in dist. water and were made up to 1.8 mL with Tris–HCl buffer. To all the above tubes, 0.5 mL of ammonium molybdate and 0.2 mL of 1amino-2-naphthol-4-sulphonic acid was added and left for 20 min for the blue colour to develop, which was read at 620 nm against the reagent blank using a spectrophotometer. Results are expressed as mmol of Pi liberated/min/mg protein. 2.5.7. Determination of mucus in gastric content The assay was performed in ethanol-induced ulcer animals according to the methodology described by Sun et al. (1991) with some modifications. The gastric contents of the stomach was immersed in 10 mL 0.02% Alcian blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8, and incubated for 24 h at 20 ◦ C. The Alcian blue binding extract was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured at 615 nm using a spectrophotometer. The free mucus in the gastric content was calculated from the amount of Alcian blue binding (mg/wet tissue (g)). 2.6. Preliminary phytochemical screening The crude AESC was subjected to qualitative chemical screening for the identification of major chemical constituents such as alkaloids (Dragendorff’s test: Waldi, 1965), phenolic constituents
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(Ferric chloride test: Mace, 1963), flavonoids and phytosterols (Asongalem et al., 2004). 2.7. In vitro antioxidant assay The scavenging activity of DPPH radicals by AESC was measured according to the method reported by Yen and Hsieh (1998). Assays were performed in 1.5 mL reaction mixtures containing 1.0 mL of 0.1 mM DPPH ethanol solution and 1.0 mL of different concentrations of crude extracts and fractions of Senecio candicans (500, 250, 125, 63, 32, 16 and 8 g/mL) or 0.5 mL ethanol (as control). After 30 min of incubation at room temperature under dark condition, the absorbance of the reaction mixtures were measured at 517 nm. Quercetin was used as the standard antioxidant. The inhibitory effect of DPPH was calculated according to the following formula:
Inhibition (%) =
absorbancecontrol − absorbancesample absorbancecontrol
× 100
A xy scatter graph was plotted to determine the inhibitory concentration (IC50 ) according to the regression equation. 2.8. Statistical analysis The data were subjected to One-way Analysis of Variance (ANOVA) and Tukey’s Multiple Comparison Test was done to evaluate the significance of difference of means of various treatment groups, using SPSS statistical software package (Version: 10). The values are presented as mean ± SD and *P < 0.05, **P < 0.01 and ***P < 0.001 were considered significant. 3. Results and discussion In acute toxicity study, mortality was not observed at any of the dose levels tested i.e. up to maximum dose (2500 mg/kg), whereas rats treated with 2000 and 2500 mg/kg b.w. showed symptoms of hypoactivity, loss of appetite and piloerection. The no-observed adverse effect level (NOAEL) was 1500 mg/kg. The lowest-observed adverse effect level (LOAEL) was 2000 mg/kg. These symptoms thus can be considered as the initial signs for toxicity. Hence, for further in vivo studies a NOAEL of 500 mg/kg b.w. was fixed as the highest dose. In pyloric-ligated animal model, Omeprazole pre-treatment caused a significant increase (P < 0.001) in gastric pH accompanied by a fall (P < 0.001) in gastric volume, free acidity and total acidity when compared to control rats. Similar changes were observed in rats pretreated with AESC in a dose-dependent manner. A significant decrease in the ulcer index (UI) was observed in Omeprazole pre-treated rats (2.17 ± 2.17) and AESC treatments (compared to control rats (31.75 ± 2.3)). Pre-treatment of 250 and 500 mg/kg b.w. of AESC prior to pylorus-ligation caused an effect similar to that of Omeprazole pre-treated group in a dose-dependent manner (16.58 ± 2.29 and 8.75 ± 0.94, respectively). The UI was significantly reduced from 31.75 ± 2.3 (control rats) to 8.75 ± 0.94 in rats pretreated with 500 mg/kg of AESC. Among the groups, pre-treatment with 500 mg/kg b.w. of AESC exhibited a percentage of ulcer inhibition (72.39%), whereas, pre-treatment with 250 mg/kg b.w. of AESC showed only 47.26% of ulcer inhibition (Table 1). In ethanol-induced model, the UI in control rats was 32.42 ± 2.82, whereas pre-treatment of rats with 250 and 500 mg/kg of AESC decreased the UI drastically to 10.75 ± 2.72 and 4.33 ± 0.52, respectively. Among the AESC pre-treated groups, rats with 500 mg/kg b.w. showed maximum percentage of inhibition (83.52), whereas 250 mg/kg b.w. pre-treatment showed only 66.73% of inhibition (Table 1).
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Table 1 Effect of aqueous leaf extract of Senecio candicans on various gastric parameters of pylorus-ligated and ethanol-induced rats. Treatment Pylorus ligated ulcer model Gastric volume (mL) pH Free acidity (mEq L−1 100 g−1 ) Total acidity (mEq L−1 100 g−1 ) Ulcer index Percent ulcer inhibition (%) Ethanol-induced ulcer model Ulcer index Ulcer inhibition (%)
Group 1 (control)
Group 2 (Omeprazole – 40 mg/kg)
Group 3 (AESC – 250 mg/kg)
Group 4 (AESC – 500 mg/kg)
8.25 ± 0.26 2.29 ± 0.2 56.67 ± 1.15 109.33 ± 3.06 31.75 ± 2.3 –
6.56 ± 0.22 a*** 4.75 ± 0.17 a*** 27.93 ± 1.47 a*** 75.33 ± 1.15 a*** 2.17 ± 2.17 a*** 93.18
7.82 ± 0.13 a**; b** 4.09 ± 0.33 a***; bns 42.67 ± 2.31 a***; b 99.33 ± 6.11 a**; b*** 16.58 ± 2.29 a***; b*** 47.26
7.27 ± 0.37 a***; bns 4.48 ± 0.32 a***; bns 38.53 ± 3.56 a***; b 82.53 ± 3.23 a***; bns 8.75 ± 0.94 a***; b*** 72.39
32.42 ± 2.82 –
2.42 ± 0.80 a*** 92.59
10.75 ± 2.72 a***; b*** 66.73
4.33 ± 0.52 a***; bns 83.52
Values presented are mean ± S.D. of six numbers of animals in each group (n = 6). Multiple comparisons between treatment groups were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. ns: not significant. *P < 0.05. **P < 0.01. ***P < 0.001.
Fig. 1. Status of antioxidant enzymes in gastric juice of pylorus-ligated rats treated with aqueous leaf extract of Senecio candicans. (A) Superoxide dismutase; (B) lipid peroxidation and (C) catalase. Values presented are mean ± S.D. of six numbers of animals in each group. Multiple comparisons were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant.
It is evident from both the ulcer model studies that AESC had a significant effect in controlling the ulcer. The highest dose i.e., 500 mg/kg b.w. of AESC showed a better reduction in UI and percentage of ulcer inhibition compared to 250 mg/kg b.w. pre-treated rats. Further, we studied the status of LPO, SOD and CAT in gastric juice of pylorus-ligated and ethanol-induced rats treated with AESC (Figs. 1 and 2). Pre-treatment of Omeprazole caused a significant decrease (P < 0.001) in the status of LPO and SOD accompanied by an increase (P < 0.001) in the status of CAT, when compared to the control rats. Pre-treatment of rats with AESC also caused a highly significant fall (P < 0.001) in the status of LPO and SOD accompanied by an increase (P < 0.001) in the status of CAT, when compared to control rats. A dose-dependent effect was found in AESC treatments in both the ulcer models. In view of these reports, it is suggested that the increase in LPO observed in gastric juice of pylorus-ligated and ethanol-induced rats could be due to the increase in the generation of O2 •− , H2 O2 − , OH• radical. The enhanced SOD activity could be due to increased synthesis of this enzyme for scavenging the superoxide radicals in the gastric juice of pylorus-ligated rats. The decrease in CAT in the gastric juice could be due to the over utilization of this enzyme in decreasing the increase in the generation of H2 O2 − . Pre-treatment AESC in pylorus-ligated and ethanol-induced rats prevented the increase in LPO and SOD accompanied by a decrease in CAT in a dose-dependent manner. It is likely that the extracts prevent gastric ulcer by markedly decreasing LPO and subsequent oxidative damage. AESC treatment might decrease lipid peroxidation by quenching free radicals in the gastric mucosa of pylorus-ligated rats to exhibit antiulcerogenic activity. It is clear from the results that antioxidant compounds present in AESC could play an active role in antiulcerogenic effect. Further, to confirm the antioxidant activity of AESC an in vitro antioxidant activity (DPPH free radical scavenging activity) was carried out. The AESC exhibited an IC50 value of 10.74 ± 0.34 g/mL, which is almost comparable to that of the standard quercetin (9.56 ± 0.24). The maximum percentage of free-radical scavenging activity (94.67 ± 0.98) was observed in quercetin followed by 91.67 ± 0.26 in AESC. The DPPH assay result strongly support that AESC has a rich source of antioxidant components. This is the first report on Senecio candicans. Previously, the in vitro DPPH scavenging activity have been reported in Senecio vulgaris and Senecio inaequidens by Conforti et al. (2006). The results of phytochemical screening showed the presence of phenols, flavonoids, steroids and alkaloids in AESC. Thus, the antioxidant activity could be due to presence of these constituents in the extract. The histopathology of stomach of pylorus-ligated rats pretreated with AESC and Omeprazole further supported our claim. The pylorus-ligated positive control rats showed dense inflammatory infiltrate of the mucus with submucosal oedema and loss of normal glandular architecture of the stomach. Pre-treatment of
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Fig. 3. Effect of aqueous leaf extract of Senecio candicans on H+ K+ ATPase activity in gastric mucosa and gastric wall mucus in ethanol-induced ulcer group.
Fig. 2. Status of antioxidant enzymes in gastric juice of ethanol-induced rats treated with aqueous leaf extract of Senecio candicans. (A) Superoxide dismutase; (B) lipid peroxidation and (C) catalase. Values presented are mean ± S.D. of six numbers of animals in each group. Multiple comparisons were performed by Tukey’s test. a: Group 1 compared with Groups 2–4; b: Group 2 compared with Groups 3 and 4. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant.
Omeprazole (40 mg/kg) in pylorus-ligated rats showed restoration of normal glandular pattern with mild inflammatory infiltrate in the lamina propria. AESC pre-treatment at 250 mg/kg showed mucosal and submucosal oedema with reduction in inflammatory infiltrate in the glands and submucosa. AESC pre-treated at 500 mg/kg showed near normal architecture comparable to normal rats (Supp. Mat. Fig. 1). The H+ K+ ATPase activity observed in ethanol-induced control rats was found to decrease significantly in AESC pre-treatment rats in a dose-dependent manner. Pre-treatment of rats with 250 mg/kg of AESC showed significant inhibition (P < 0.05) in H+ K+ ATPase activity (1.03 ± 0.04) compared to the control rats (1.40 ± 0.03). The activity decreased to 0.62 ± 0.06 when treated with 500 mg/kg of
AESC. The gastric wall mucus was enhanced when treated with AESC in a dose-dependent manner. The effect caused by AESC at 500 mg/kg was comparable to the effect caused by the Omeprazole treatment (Fig. 3). The possible involvement of the extract on enhancing mucosal resistance could have offered gastroprotection and is regarded as a first line of defence against gastric ulcers. Not only antioxidants present in AESC, but also the mucosal barrier protection and restoration of mucous secretion could be a possible mechanism in gastroprotective action. This is the first report on gastroprotection action in Senecio candicans. Previously, the preventive activity of Senecio brasiliensis on gastric and duodenal induced ulcer was investigated by Toma et al. (2004). In conclusion, it is evident that the AESC exhibit antiulcerogenic activity in a dose-dependent manner and its efficacy is more at the maximum dosage (500 mg/kg). The present study provides additional support for the traditional use of this plant in the treatment of gastric ulcer. Preliminary phytochemical screening of the AESC gave positive results for the presence of phenols, flavonoids and phytosterols. It is possible that a combined effects of different bioactive constituents of the plant could be responsible for the antiulcerogenic action, which may be due to (i) decrease in gastric acid secretion, (ii) inhibition of free radical generation/prevention of lipid peroxidation, (iii) free radical scavenging/antioxidant properties and (iv) the protection of mucosal barrier and restoration of mucous secretion. The AESC appear to act in a non-specific manner hence, a detailed study on the isolation and characterization of active principles responsible for the antiulcerogenic activity is essential. On the other hand Senecio species are known for the presence of pyrrolizidine alkaloids, which are generally considered hepatotoxic. Since there is no report for the presence of pyrrolizidine alkaloids in Senecio candicans, a detailed study on the percentage of alkaloids present and their characterization is essential. This might bring more knowledge on the safety aspects of this plant when used for a long period. Conflict of interest None declared. Acknowledgement The authors are grateful to Professor Dr. Ramagopalan, Department of Pathology, University of Madras, Taramani Campus, Chennai, for histopathological examinations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2012.01.002.
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