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Antiulcerogenic activity of Lantana camara leaves on gastric and duodenal ulcers in experimental rats
Journal of Ethnopharmacology 134 (2011) 195–197
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Antiulcerogenic activity of Lantana camara leaves on gastric and duodenal ulcers in experimental rats R. Sathish a,∗ , Bhushan Vyawahare a , K. Natarajan b a b
Department of Pharmacology, Ultra College of Pharmacy, Madurai, India Department of Pharmaceutical Biotechnology, Ultra College of Pharmacy, Madurai, India
a r t i c l e
i n f o
Article history: Received 26 June 2010 Received in revised form 27 October 2010 Accepted 22 November 2010 Available online 1 December 2010 Keywords: Lantana camara Aspirin induced ulcerogenesis Pylorus ligated rats Duodenal ulcer Antioxidant
a b s t r a c t Ethnopharmacological relevance: Lantana camara L. (Verbenaceae), a widely growing shrub has been used in the traditional medicine for treating many ailments. The objective of the present study was to evaluate the effects of methanolic extract of Lantana camara leaves on gastric and duodenal ulcers. Materials and methods: The antiulcerogenic effect of methanolic extract of Lantana camara was evaluated in aspirin induced gastric ulcerogenesis in pyloric ligated rats, ethanol induced gastric ulcer, and cysteamine induced duodenal ulcer models. The extract was administered orally at two different doses of 250 mg/kg and 500 mg/kg. The lipid peroxidation, reduced glutathione levels of ethanol induced gastric ulcer model and inhibition zone in diameter against Helicobacter pylori also determined. Results: The L. camara extract significantly (P < 0.01) reduced ulcer index, total acidity and significantly (P < 0.01) increased the gastric pH of aspirin + pylorus-ligation induced ulcerogenesis and ethanol induced gastric ulcer models. The extract also significantly (P < 0.01) reduced the ulcer index of cysteamine induced duodenal ulcer. The L. camara showed significant (P < 0.01) reduction in lipid peroxidation and increase in reduced glutathione levels. The inhibition zone in diameter of extract against H. pylori was 20 mm. Conclusion: The methanolic extract of Lantana camara leaves shown healing of gastric ulcers and also prevents development of duodenal ulcers in rats. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The plants and herbs are used to treat different gastrointestinal illnesses, including peptic ulcers without side effects in ayurvedic medicinal system (Jaime et al., 2006). Peptic ulcer disease radically changed in 1983 by the discovery of H. pylori; most patients with ulcers are infected with H. pylori and eradicating the infection permanently cures the ulcers (John, 2003). Lantana camara (Verbenaceae) a widely growing evergreen shrub, the extracts are used in folk medicine for the treatment of cancers, chicken pox, measles, asthma, ulcers, swellings, eczema, tumors, high blood pressure, bilious fevers, catarrhal infections, tetanus, rheumatism, malaria and atoxy of abdominal viscera (Kirtikar and Basu, 1981; Ghisalberti, 2000; Day et al., 2003). In Asian countries, leaves were used to treat cuts, rheumatisms and ulcers (Ghisalberti, 2000; Lenika et al., 2005). Lantanoside, linaroside, and camarinic acid have been isolated and are being investigated as potential nematocides (Begun et al., 1995; Day et al., 2003). Verbascoside (phenylethanoid
∗ Corresponding author at: Department of Pharmacology, Ultra College of Pharmacy, Madurai, Tamilnadu, India. Tel.: +91 9865134949. E-mail address: [email protected] (R. Sathish). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.049
glycoside), umuhengerin (polymethoxylated flavones) and phytol (diterpene) has been isolated from methanolic extract of leaves (Herbert et al., 1991; Day et al., 2003). There is no scientific documentation about the antiulcer activity of L. camara. This prompted us to pursue the antiulcer and antioxidant effect of methanolic extract of L. camara leaves. 2. Materials and methods 2.1. Extraction and Phytochemical Screening L. camara leaves were collected from Azhagar kovil, Madurai, and was identified and authenticated by Dr. Stephen, American College, Madurai. The voucher specimen was kept in the herbarium of our college (UCP/09/021). 200 g of leaves powder were exhaustively extracted with methanol (1.5 L) using soxhlet apparatus and the yield was found to be 6.5% w/w. The methanolic extract of Lantana camara (LCME) was subjected to phytochemical screening for the presence of alkaloids, saponins, glycosides, carbohydrates, tannins, flavonoids, steroids and triterpenoids (Khandelwal, 2003). Thereafter, the LCME was loaded on TLC plate (Silica Gel 60, Merck) and developed by the following solvents CHCl3 :MeOH (9:1). TLC plate was sprayed with FeCl3 and visualized under UV light
196
R. Sathish et al. / Journal of Ethnopharmacology 134 (2011) 195–197
Table 1 Effect of methanolic extract of Lantana camara leaves on various ulcer models. Treatment group
pH of gastric juice APL
I II III IV
1.86 3.66 4.87 5.25
EtOH ± ± ± ±
0.02 0.0* 0.01* 0.02*
1.73 3.81 4.89 5.39
± ± ± ±
0.01 0.0* 0.01* 0.02*
Total acidity (Eq/100 g/4 h)
Ulcer index
APL
APL
88.19 55.91 40.01 28.46
EtOH ± ± ± ±
0.36 0.61* 0.83* 0.24*
82.91 60.16 43.85 30.69
± ± ± ±
0.61 0.40* 0.46* 0.52*
14.18 7.57 3.69 1.84
EtOH ± ± ± ±
0.36 0.30* (46.61%) 0.21* (73.97%) 0.16* (87.02%)
15.16 6.72 5.55 3.69
CYS ± ± ± ±
0.16 0.02* (55.60%) 0.22* (63.39%) 0.21* (75.65%)
2.83 1.66 0.88 0.39
± ± ± ±
0.01 0.04* (41.43%) 0.12* (68.90%) 0.22* (86.21%)
Values are mean ± SEM, n = 6 in each group, * P < 0.01 when compared with toxicant control group (one-way ANOVA followed by Dunnett’s test); APL – Aspirin induced ulcerogenesis in pyloric ligated model; EtOH – Ethanol induced gastric ulcer model; CYS – Cysteamine induced duodenal ulcer model.
(254–363 nm). The spot was observed on the TLC plate and Rf value was calculated. Standard was obtained from Sigma Chemical Company, St. Louis, MO, USA.
after the first dose of cysteamine and the duodenum was excised carefully and opened along the antimesenteric side (Deshpande et al., 2003). The ulcer index was determined based on the intensity of the ulcer (Khare et al., 2008).
2.2. Animals and drugs Wister albino rats of either sex weighing between 150 and 200 g were housed in standard cages at room temperature (25 ± 2 ◦ C) and relative humidity 45–55% at 12 h light and 12 h dark cycle. The animals were fed with commercial pellet diet and water ad libitum. The IAEC of Ultra College of Pharmacy approved this study (UCP/IAEC/2009/044). The dried LCME, famotidine, omeprazole and aspirin were suspended in water using 0.5% CMC and administered orally. 2.3. Experimental models The animals were divided into four groups, consisting of six rats each in all ulcer models. 2.3.1. Aspirin induced ulcerogenesis in pyloric ligated model (APL) Group I received aspirin (200 mg/kg); Group II and III received LCME 250 and 500 mg/kg respectively (Lenika et al., 2005) and Group IV received famotidine (20 mg/kg) for 7 days. From days 5 to 7 animals of the treatment groups (II, III, and IV) received aspirin orally (200 mg/kg), 2 h after the administration of respective drug treatment. On day 7 animals in all groups were fasted for 18 h after the respective assigned treatment. The abdomen was opened and pylorus portion of stomach was lifted out and ligated (Shay et al., 1945). After 4 h of surgery, rats were sacrificed and the stomach was dissected out. The accumulated gastric juice was collected and the ulcer index (UI) was determined (Dharmani et al., 2005; Umamaheshwari et al., 2007). 2.3.2. Ethanol induced ulcer model (EtOH) Group I received vehicle, Group II and III received LCME 250 and 500 mg/kg respectively and Group IV received famotidine (20 mg/kg) for 10 days. On 10th day, 1 h after the final dose of treatment, 90% ethanol (5 ml/kg, p.o.) was administered to overnight fasted rats of all groups. The animals were sacrificed after 1 h of ulcerogen and their stomachs were excised. The gastric contents were aspirated for pH and total acidity determination. The sum of areas (mm2 ) was expressed as ulcer index and percentage inhibition of UI was calculated (Balaraman and Bafana, 2005). The stomach tissues were further used for the determination of in vitro antioxidant activity. 2.3.3. Cysteamine induced duodenal ulcer model (CYS) Cysteamine hydrochloride (400 mg/kg, p.o.) was administered twice at an interval of 4 h to induce duodenal ulcer in rats. The LCME 250 mg/kg and 500 mg/kg and omeprazole (8 mg/kg) were administered 30 min before each dose of cysteamine HCl to Group II, Group III and Group IV respectively. Group I animals received vehicle with Cysteamine HCl. All the animals were sacrificed 24 h
2.3.4. Histopathological Evaluation Histopathological changes such as oedema, inflammation, infiltration and erosion in stomach or duodenum of ulcer models were determined (Umamaheshwari et al., 2007). 2.4. In vitro antioxidant and Antimicrobial activity The lipid peroxides (LPO) of stomach mucosa were determined indirectly by thiobarbituric acid reactive substance (TBARS) formation (Khalil et al., 2007). Reduced glutathione concentration was read off a standard curve and expressed as g GSH/g of wet tissue (Sedlak and Lindsay, 1968). Antimicrobial sensitivity was done by Kirby–Bauer disc method. The blood agar medium was sterilized and culture broth of H. pylori was swabbed on the medium. The paper discs impregnated with LCME 10 l of 100 mg/ml (dissolved in DMSO), tetracycline 30 g/disc and 10 l of DMSO were placed on the surface of the agar plate, and then incubated at 37 ◦ C, 10% v/v CO2 , 95% humidity for five days. The effectiveness is based on inhibition zone in diameter (IZD). 2.5. Statistical analysis The treated groups were compared with the toxicant control groups, all results were expressed as mean ± SEM of 6 animals in each groups. The results were analyzed statistically using one way ANOVA followed by Dunnett’s test, P < 0.05 was considered as significant. 3. Results and discussion Phytochemical screening of the LCME showed the presence of saponins, glycosides, carbohydrates, tannins, flavonoids, steroids and triterpenoids. In the present study TLC separation of LCME present a large number of compounds as revealed by fluorescent spots when visualized under UV light. One of the spot (Rf 0.52) was found to have similar Rf value as that of the standard verbascoside. Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity. Thus, verbascoside could act as a potent therapeutic agent against gastric ulcer disease (Neetu et al., 2010). Aspirin causes mucosal damage by interfering with prostaglandin synthesis, increasing acid secretion and back diffusion of H+ ions. Aspirin was administered to PL rats; thus, aspirin further aggravated the acidity and resistance of the gastric mucosa was decreased thereby causing extensive mucosal damage (Sanmugapriya and Venkataraman, 2007). The present study reveals that LCME treatment at both doses showed significant (P < 0.01) increase in gastric pH and reduction in total acidity when compared to toxicant control rats. LCME also inhib-
R. Sathish et al. / Journal of Ethnopharmacology 134 (2011) 195–197 Table 2 Effect of Lantana camara leaves on lipid peroxidase and reduced glutathione. Treatment group
Dose
LPO (nmol/g)
Ethanol (90%) LCME-I LCME-II Famotidine
5 ml/kg 250 mg/kg 500 mg/kg 20 mg/kg
39.71 29.23 27.7 26.3
± ± ± ±
0.22 0.35* 0.50* 0.32*
GSH (g/g) 82.83 181.52 202.9 220.5
± ± ± ±
1.35 0.83* 1.08* 0.38*
Values are mean ± SEM, n = 6 in each group, * P < 0.01 when compared with toxicant control (one-way ANOVA followed by Dunnett’s test). LCME – Methanolic extract of Lantana camara leaves.
ited the UI by 46.61% (250 mg/kg) and 73.97% (500 mg/kg) (Table 1). Ethanol is metabolized in the body and releases superoxide anion and hydroperoxy free radicals. It has been found that oxygenderived free radicals are implicated in the mechanism of acute and chronic ulceration in the gastric mucosa (Umamaheshwari et al., 2007). LCME significantly (P < 0.01) inhibited the UI by 55.60% and 63.39% at 250 and 500 mg/kg doses respectively (Table 1). Administration of LCME resulted the significant (P < 0.01) reduction in lipid peroxidation and increase in reduced glutathione levels (Table 2), which suggests its efficacy in preventing free radical-induced damage by the metabolism of ethanol and thereby heal the ulcers. Cysteamine hydrochloride inhibits alkaline mucus secretion from the Brunner’s gland in the proximal duodenum and stimulates the rate of gastric acid secretion (Khare et al., 2008). The significant (P < 0.01) protective effect of LCME (Table 1) against cysteamine induced duodenal ulcers may be due to the strengthening of duodenal mucosa or by increased gastric and duodenal alkaline secretion or by increased luminal prostaglandin levels. The results also displayed the potential antimicrobial activity of L. camara against H. pylori (IZD-20 mm) as it is the only microorganism associated with etiology of ulcers. DMSO alone did not produce any inhibition zone. Histopathology reports of APL and ethanol induced ulcer control rats shown perforated ulcer, deep ulceration of granular epithelium and almost reducing the sub-mucosa. The LCME at 250 mg/kg dose has shown the partial healing of ulcer with few inflammatory cells and the dose 500 mg/kg has shown the healed ulcer, normal mucosa and no inflammatory cells. Histopathology of rat duodenum treated with LCME showed the protection of lesions and few inflammatory cells at both dose levels. Thus our studies establish a significant antiulcer and cytoprotective effect of LCME. In conclusion, it is clear that methanolic extract of L. camara leaves have significant anti-ulcer, antioxidant activity in gastric and duodenal ulcer models and the leaves also proved as potent antimicrobial agent against H. pylori.
197
Acknowledgments The authors are thankful to Prof. K.R. Arumugam, Chairman, Dr. C. Vijaya, Professor & Dean and Dr. K.G. Lalitha, Professor, Ultra College of Pharmacy, Madurai for their valuable Suggestions. References Balaraman, R, Bafana, P.A., 2005. Antiulcer and antioxidant activity of pepticare, a herbomineral formulation. Phytomedicine 12, 264–270. Begun, S., Mohammad, B.S., Siddiqui, S., 1995. Triterpenoids from the aerial parts of Lantana camara. Journal of Natural Products 58, 1570–1574. Day, M.D., Wiley, C.J., Playford, J., Zalucki, M.P., 2003. Lantana: Current Management, Status and Future Prospects. Australian Centre for International Agricultural Research, Canberra. Deshpande, S.S., Shah, G.B., Parmar, N.S., 2003. Antiulcer activity of Tephrosia purpurea in rats. Indian Journal of Pharmacology 35, 168–172. Dharmani, P., Mishra, P.K., Maurya, R., Chauhan, V.S., Palit, G., 2005. Alloypus serratus: a plant with potential antiulcerogenic activity. Journal of Ethnopharmacology 99, 361–366. Ghisalberti, E.L., 2000. Lantana camara L. (Verbenaceae). Fitoterapia 71, 467–486. Herbert, J.M., Maffrand, J.P., Taoubi, K., et al., 1991. Verbascoside isolated from L. camara, an inhibitor of protein kinase. Journal of Natural Products 54, 1595–1600. Jaime, A.R., Cristina, T., Tania, Y., Jose, B., Guillermo, S.-H., 2006. Gastroprotective and ulcer healing effect of ferruginol in mice and rats: Assessment of its mechanism of action using in vitro models. Life Sciences 78, 2503–2509. John, C., 2003. Peptic ulcer diseases. In: Warrell, D.A., Edward, J.B., John, D.F., Timothy, M.C. (Eds.), Oxford textbook of Medicine, vol. 2, fourth ed. Oxford University Press, New York, pp. 558–568. Khalil, H., Ismail, H., Taye, A., Kamel, M., 2007. Gastroprotective effect of Lippia nodiflora L. extracts in ethanol induced gastric lesions. Pharmacognosy Magazine 3, 259–262. Khandelwal, K.R., 2003. Practical Pharmacognosy techniques and experiments, tenth ed. Nirali Prakashan, Pune, pp. 140–157. Khare, S., Asad, M., Dhamanigi, S.S., Satya Prasad, V., 2008. Antiulcer activity of cod liver oil in rats. Indian Journal of Pharmacology 40, 209–214. Kirtikar, K.R., Basu, B.D., 1981. Indian medicinal plants. International Book Distributors, Derhadun, p. 984. Lenika, S., Rajesh, S., Sudarshan, O., 2005. Evaluation of antimotility effect of Lantana camara L. var. acuelata constituents on neostigmine induced gastrointestinal transit in mice. BMC Complementary and Alternative Medicine 5, 1–6. Neetu, S., Nivedita, S., Pratibha, S., Rolee, S., Rajendran, S.M., Rakesh, M., Gautam, P., 2010. Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity. Fitoterapia 81, 755–761. Sanmugapriya, E., Venkataraman, S., 2007. Antiulcerogenic potential of Strychnos potatorum Linn seeds on aspirin plus pyloric ligation-induced ulcers in experimental rats. Phytomedicine 14, 360–365. Sedlak, J., Lindsay, R.H., 1968. Estimation of total protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25, 1192–1205. Shay, H., Komarov, S.A., Fels, S.S., Meranze, D., Gruenstein, M., Siplet, H., 1945. A simple method for the production of gastric ulceration in the rat. Gastroenterology 5, 43–61. Umamaheshwari, M., Asokkumar, K., Rathidevi, R., Sivashanmugam, A.T., Subhadradevi, V., Ravi, T.K., 2007. Antiulcer and in vitro antioxidant activities of Jasminum grandiflorum L. Journal of Ethnopharmacology 110, 464–470.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Antiulcerogenic activity of Lantana camara leaves on gastric and duodenal ulcers in experimental rats R. Sathish a,∗ , Bhushan Vyawahare a , K. Natarajan b a b
Department of Pharmacology, Ultra College of Pharmacy, Madurai, India Department of Pharmaceutical Biotechnology, Ultra College of Pharmacy, Madurai, India
a r t i c l e
i n f o
Article history: Received 26 June 2010 Received in revised form 27 October 2010 Accepted 22 November 2010 Available online 1 December 2010 Keywords: Lantana camara Aspirin induced ulcerogenesis Pylorus ligated rats Duodenal ulcer Antioxidant
a b s t r a c t Ethnopharmacological relevance: Lantana camara L. (Verbenaceae), a widely growing shrub has been used in the traditional medicine for treating many ailments. The objective of the present study was to evaluate the effects of methanolic extract of Lantana camara leaves on gastric and duodenal ulcers. Materials and methods: The antiulcerogenic effect of methanolic extract of Lantana camara was evaluated in aspirin induced gastric ulcerogenesis in pyloric ligated rats, ethanol induced gastric ulcer, and cysteamine induced duodenal ulcer models. The extract was administered orally at two different doses of 250 mg/kg and 500 mg/kg. The lipid peroxidation, reduced glutathione levels of ethanol induced gastric ulcer model and inhibition zone in diameter against Helicobacter pylori also determined. Results: The L. camara extract significantly (P < 0.01) reduced ulcer index, total acidity and significantly (P < 0.01) increased the gastric pH of aspirin + pylorus-ligation induced ulcerogenesis and ethanol induced gastric ulcer models. The extract also significantly (P < 0.01) reduced the ulcer index of cysteamine induced duodenal ulcer. The L. camara showed significant (P < 0.01) reduction in lipid peroxidation and increase in reduced glutathione levels. The inhibition zone in diameter of extract against H. pylori was 20 mm. Conclusion: The methanolic extract of Lantana camara leaves shown healing of gastric ulcers and also prevents development of duodenal ulcers in rats. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The plants and herbs are used to treat different gastrointestinal illnesses, including peptic ulcers without side effects in ayurvedic medicinal system (Jaime et al., 2006). Peptic ulcer disease radically changed in 1983 by the discovery of H. pylori; most patients with ulcers are infected with H. pylori and eradicating the infection permanently cures the ulcers (John, 2003). Lantana camara (Verbenaceae) a widely growing evergreen shrub, the extracts are used in folk medicine for the treatment of cancers, chicken pox, measles, asthma, ulcers, swellings, eczema, tumors, high blood pressure, bilious fevers, catarrhal infections, tetanus, rheumatism, malaria and atoxy of abdominal viscera (Kirtikar and Basu, 1981; Ghisalberti, 2000; Day et al., 2003). In Asian countries, leaves were used to treat cuts, rheumatisms and ulcers (Ghisalberti, 2000; Lenika et al., 2005). Lantanoside, linaroside, and camarinic acid have been isolated and are being investigated as potential nematocides (Begun et al., 1995; Day et al., 2003). Verbascoside (phenylethanoid
∗ Corresponding author at: Department of Pharmacology, Ultra College of Pharmacy, Madurai, Tamilnadu, India. Tel.: +91 9865134949. E-mail address: [email protected] (R. Sathish). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.049
glycoside), umuhengerin (polymethoxylated flavones) and phytol (diterpene) has been isolated from methanolic extract of leaves (Herbert et al., 1991; Day et al., 2003). There is no scientific documentation about the antiulcer activity of L. camara. This prompted us to pursue the antiulcer and antioxidant effect of methanolic extract of L. camara leaves. 2. Materials and methods 2.1. Extraction and Phytochemical Screening L. camara leaves were collected from Azhagar kovil, Madurai, and was identified and authenticated by Dr. Stephen, American College, Madurai. The voucher specimen was kept in the herbarium of our college (UCP/09/021). 200 g of leaves powder were exhaustively extracted with methanol (1.5 L) using soxhlet apparatus and the yield was found to be 6.5% w/w. The methanolic extract of Lantana camara (LCME) was subjected to phytochemical screening for the presence of alkaloids, saponins, glycosides, carbohydrates, tannins, flavonoids, steroids and triterpenoids (Khandelwal, 2003). Thereafter, the LCME was loaded on TLC plate (Silica Gel 60, Merck) and developed by the following solvents CHCl3 :MeOH (9:1). TLC plate was sprayed with FeCl3 and visualized under UV light
196
R. Sathish et al. / Journal of Ethnopharmacology 134 (2011) 195–197
Table 1 Effect of methanolic extract of Lantana camara leaves on various ulcer models. Treatment group
pH of gastric juice APL
I II III IV
1.86 3.66 4.87 5.25
EtOH ± ± ± ±
0.02 0.0* 0.01* 0.02*
1.73 3.81 4.89 5.39
± ± ± ±
0.01 0.0* 0.01* 0.02*
Total acidity (Eq/100 g/4 h)
Ulcer index
APL
APL
88.19 55.91 40.01 28.46
EtOH ± ± ± ±
0.36 0.61* 0.83* 0.24*
82.91 60.16 43.85 30.69
± ± ± ±
0.61 0.40* 0.46* 0.52*
14.18 7.57 3.69 1.84
EtOH ± ± ± ±
0.36 0.30* (46.61%) 0.21* (73.97%) 0.16* (87.02%)
15.16 6.72 5.55 3.69
CYS ± ± ± ±
0.16 0.02* (55.60%) 0.22* (63.39%) 0.21* (75.65%)
2.83 1.66 0.88 0.39
± ± ± ±
0.01 0.04* (41.43%) 0.12* (68.90%) 0.22* (86.21%)
Values are mean ± SEM, n = 6 in each group, * P < 0.01 when compared with toxicant control group (one-way ANOVA followed by Dunnett’s test); APL – Aspirin induced ulcerogenesis in pyloric ligated model; EtOH – Ethanol induced gastric ulcer model; CYS – Cysteamine induced duodenal ulcer model.
(254–363 nm). The spot was observed on the TLC plate and Rf value was calculated. Standard was obtained from Sigma Chemical Company, St. Louis, MO, USA.
after the first dose of cysteamine and the duodenum was excised carefully and opened along the antimesenteric side (Deshpande et al., 2003). The ulcer index was determined based on the intensity of the ulcer (Khare et al., 2008).
2.2. Animals and drugs Wister albino rats of either sex weighing between 150 and 200 g were housed in standard cages at room temperature (25 ± 2 ◦ C) and relative humidity 45–55% at 12 h light and 12 h dark cycle. The animals were fed with commercial pellet diet and water ad libitum. The IAEC of Ultra College of Pharmacy approved this study (UCP/IAEC/2009/044). The dried LCME, famotidine, omeprazole and aspirin were suspended in water using 0.5% CMC and administered orally. 2.3. Experimental models The animals were divided into four groups, consisting of six rats each in all ulcer models. 2.3.1. Aspirin induced ulcerogenesis in pyloric ligated model (APL) Group I received aspirin (200 mg/kg); Group II and III received LCME 250 and 500 mg/kg respectively (Lenika et al., 2005) and Group IV received famotidine (20 mg/kg) for 7 days. From days 5 to 7 animals of the treatment groups (II, III, and IV) received aspirin orally (200 mg/kg), 2 h after the administration of respective drug treatment. On day 7 animals in all groups were fasted for 18 h after the respective assigned treatment. The abdomen was opened and pylorus portion of stomach was lifted out and ligated (Shay et al., 1945). After 4 h of surgery, rats were sacrificed and the stomach was dissected out. The accumulated gastric juice was collected and the ulcer index (UI) was determined (Dharmani et al., 2005; Umamaheshwari et al., 2007). 2.3.2. Ethanol induced ulcer model (EtOH) Group I received vehicle, Group II and III received LCME 250 and 500 mg/kg respectively and Group IV received famotidine (20 mg/kg) for 10 days. On 10th day, 1 h after the final dose of treatment, 90% ethanol (5 ml/kg, p.o.) was administered to overnight fasted rats of all groups. The animals were sacrificed after 1 h of ulcerogen and their stomachs were excised. The gastric contents were aspirated for pH and total acidity determination. The sum of areas (mm2 ) was expressed as ulcer index and percentage inhibition of UI was calculated (Balaraman and Bafana, 2005). The stomach tissues were further used for the determination of in vitro antioxidant activity. 2.3.3. Cysteamine induced duodenal ulcer model (CYS) Cysteamine hydrochloride (400 mg/kg, p.o.) was administered twice at an interval of 4 h to induce duodenal ulcer in rats. The LCME 250 mg/kg and 500 mg/kg and omeprazole (8 mg/kg) were administered 30 min before each dose of cysteamine HCl to Group II, Group III and Group IV respectively. Group I animals received vehicle with Cysteamine HCl. All the animals were sacrificed 24 h
2.3.4. Histopathological Evaluation Histopathological changes such as oedema, inflammation, infiltration and erosion in stomach or duodenum of ulcer models were determined (Umamaheshwari et al., 2007). 2.4. In vitro antioxidant and Antimicrobial activity The lipid peroxides (LPO) of stomach mucosa were determined indirectly by thiobarbituric acid reactive substance (TBARS) formation (Khalil et al., 2007). Reduced glutathione concentration was read off a standard curve and expressed as g GSH/g of wet tissue (Sedlak and Lindsay, 1968). Antimicrobial sensitivity was done by Kirby–Bauer disc method. The blood agar medium was sterilized and culture broth of H. pylori was swabbed on the medium. The paper discs impregnated with LCME 10 l of 100 mg/ml (dissolved in DMSO), tetracycline 30 g/disc and 10 l of DMSO were placed on the surface of the agar plate, and then incubated at 37 ◦ C, 10% v/v CO2 , 95% humidity for five days. The effectiveness is based on inhibition zone in diameter (IZD). 2.5. Statistical analysis The treated groups were compared with the toxicant control groups, all results were expressed as mean ± SEM of 6 animals in each groups. The results were analyzed statistically using one way ANOVA followed by Dunnett’s test, P < 0.05 was considered as significant. 3. Results and discussion Phytochemical screening of the LCME showed the presence of saponins, glycosides, carbohydrates, tannins, flavonoids, steroids and triterpenoids. In the present study TLC separation of LCME present a large number of compounds as revealed by fluorescent spots when visualized under UV light. One of the spot (Rf 0.52) was found to have similar Rf value as that of the standard verbascoside. Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity. Thus, verbascoside could act as a potent therapeutic agent against gastric ulcer disease (Neetu et al., 2010). Aspirin causes mucosal damage by interfering with prostaglandin synthesis, increasing acid secretion and back diffusion of H+ ions. Aspirin was administered to PL rats; thus, aspirin further aggravated the acidity and resistance of the gastric mucosa was decreased thereby causing extensive mucosal damage (Sanmugapriya and Venkataraman, 2007). The present study reveals that LCME treatment at both doses showed significant (P < 0.01) increase in gastric pH and reduction in total acidity when compared to toxicant control rats. LCME also inhib-
R. Sathish et al. / Journal of Ethnopharmacology 134 (2011) 195–197 Table 2 Effect of Lantana camara leaves on lipid peroxidase and reduced glutathione. Treatment group
Dose
LPO (nmol/g)
Ethanol (90%) LCME-I LCME-II Famotidine
5 ml/kg 250 mg/kg 500 mg/kg 20 mg/kg
39.71 29.23 27.7 26.3
± ± ± ±
0.22 0.35* 0.50* 0.32*
GSH (g/g) 82.83 181.52 202.9 220.5
± ± ± ±
1.35 0.83* 1.08* 0.38*
Values are mean ± SEM, n = 6 in each group, * P < 0.01 when compared with toxicant control (one-way ANOVA followed by Dunnett’s test). LCME – Methanolic extract of Lantana camara leaves.
ited the UI by 46.61% (250 mg/kg) and 73.97% (500 mg/kg) (Table 1). Ethanol is metabolized in the body and releases superoxide anion and hydroperoxy free radicals. It has been found that oxygenderived free radicals are implicated in the mechanism of acute and chronic ulceration in the gastric mucosa (Umamaheshwari et al., 2007). LCME significantly (P < 0.01) inhibited the UI by 55.60% and 63.39% at 250 and 500 mg/kg doses respectively (Table 1). Administration of LCME resulted the significant (P < 0.01) reduction in lipid peroxidation and increase in reduced glutathione levels (Table 2), which suggests its efficacy in preventing free radical-induced damage by the metabolism of ethanol and thereby heal the ulcers. Cysteamine hydrochloride inhibits alkaline mucus secretion from the Brunner’s gland in the proximal duodenum and stimulates the rate of gastric acid secretion (Khare et al., 2008). The significant (P < 0.01) protective effect of LCME (Table 1) against cysteamine induced duodenal ulcers may be due to the strengthening of duodenal mucosa or by increased gastric and duodenal alkaline secretion or by increased luminal prostaglandin levels. The results also displayed the potential antimicrobial activity of L. camara against H. pylori (IZD-20 mm) as it is the only microorganism associated with etiology of ulcers. DMSO alone did not produce any inhibition zone. Histopathology reports of APL and ethanol induced ulcer control rats shown perforated ulcer, deep ulceration of granular epithelium and almost reducing the sub-mucosa. The LCME at 250 mg/kg dose has shown the partial healing of ulcer with few inflammatory cells and the dose 500 mg/kg has shown the healed ulcer, normal mucosa and no inflammatory cells. Histopathology of rat duodenum treated with LCME showed the protection of lesions and few inflammatory cells at both dose levels. Thus our studies establish a significant antiulcer and cytoprotective effect of LCME. In conclusion, it is clear that methanolic extract of L. camara leaves have significant anti-ulcer, antioxidant activity in gastric and duodenal ulcer models and the leaves also proved as potent antimicrobial agent against H. pylori.
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