Research Article
Effects of Anethum chryseum Leaves Extracts on Gastric Irritation
School of Veterinary Sciences, Ilam University Ilam, Iran
Valadi Ali
School of Veterinary Sciences, Ilam University Ilam, Iran
Nature has served as a rich repository of medicinal plants for thousands of years and an impressive number of modern drugs have been isolated from natural sources, notably of plant origin (Cowan, 1999).
Anethum chryseum is a sparse looking plant with feathery leaves and tiny yellow flowers. Some pharmacological effects have been reported, such as: antimicrobial (Chaurasia and Jain, 2003; Delaquis et al., 2002) and antihypercholesterolaemic activities (Yazdanparast and Alav, 2001). Cancer-preventive actions (Block, 2003), stimulation of phagocytotic function of macrophages and lymphocyte proliferation (Tadi et al., 2000). The cardiovascular-protective effects of A. chryseum have been evaluated extensively in recent years (Rios and Recios, 2005). In animal experiments, garlic extracts have been shown antibacterial and phytochemical effect (Kaur and Arora, 2008). As a folk remedy, A. chryseum is considered for some gastrointestinal ailments such as flatulence, indigestion, stomachache and colic (Duke, 2001). Anethum chryseum fruit has an antispasmodic effect on the smooth muscles of the gastrointestinal tract (Fleming, 2000). For that purpose, we planned to research about the antisecretory and mucosal protective effects of A. chryseum leaves extracts were evaluated in mice.
This project was conducted in Medical Research Laboratory, University of Ilam, during August 2008-May 2009.
Animals: Male albino BALB/c mice 25-35 g were obtained from the animal house of School of Pharmacy, Ilam University of Medical Sciences. Animals were housed in colony room 12/12 h light/dark cycle at 24±1°C. After 24 h fasting, the mice were used for the experiments but were allowed drinking water during the 24 h fasting period. All animal experiments were carried out in accordance with Ilam University of Medical Sciences, Ethical Committee acts.
Plant material: The seed was collected at Ilam (a town in Ilam Province, the Western of Iran). All samples collected were dried in shade and then powdered. Ilam University properly identified the plant and voucher samples were preserved for reference in the herbarium of Department of Pharmacognosy, School of Pharmacy, Tehran (293-0107-18).
The preparation of extracts: The seed powder was extracted using maceration with ethanol (80 v/v) or water for 3 days and, subsequently, the mixture was filtered and concentrated under reduced pressure (by a rotaevaporator) at 40°C. The yield (w/w) of the aqueous and ethanolic extracts was 6.46 and 8.5%, respectively.
HCl or ethanol-induced mucosal membrane lesions: Gastric mucosal lesions were induced by the modified method of Mizui and Doteuchi (2006). The mice were divided into groups of 6 animals. After 24 h fasting, the extracts and drugs were administered orally to the mice. Thirty minutes thereafter, each mouse received 0.2 mL of 1 N HCl or absolute ethanol by oral administration. The 60 min after administration of the necrotizing agent, each animal was killed by ether and the stomach was excised, inflated by injecting 2 mL of normal saline and then fixed for 30 min in 5% formalin solution. After opening along the greater curvature, HCl induced gastric damage was observed in the gastric mucosa as elongated black-red lines parallel to the long axis of the stomach of the mice. The lesion index was determined as the sum of erosion length per mouse (Sun et al., 1991). Ethanol induced lesion was assessed and scored for severity according to, (0) absence of lesion, (1) superficial 1-5 hemorrhagic points, (2) superficial 6-10 hemorrhagic points, (3) submucosal hemorrhagic lesions with small erosions and (4) severe hemorrhagic lesion and some invasive lesions.
Antisecretory study: One hour after extract or test drug treatment, mice were anesthetized (xylazine 10 mg kg-1 plus ketamine 100 mg kg-1, i.p.) and the pylorus was ligated. The animals were killed 3 h later and their stomach content was drained into a tube which was centrifuged 2000 rpm for 10 min. The pH was recorded with a digital pH meter. Total acid content of gastric secretion was determined by titration against 0.05 N NaOH (Sertie et al., 2000).
ED50 values: ED50 values and the corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4).
Acute toxicity: Different doses of extracts were injected intraperitoneally into groups of six mice. The number of death was counted at 24 h after treatment. LD50 values and the corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 6).
Statistical analysis: The data were expressed as mean values±SEM and tested with analysis of variance followed by the multiple comparison test of Tukey-Kramer. Ethanol induced lesion was assessed by Dunn's test.
The maximum non-fatal doses of aqueous and ethanolic extracts were 0.47 and 4 g kg-1 (i.p.), respectively. LD50 values of the aqueous and ethanolic extracts were 2.94 g kg-1, i.p. (1.5-6.16) and 6.98 g kg-1, i.p. (5.69-8.56), respectively.
The aqueous of A. chryseum leaves significantly decreased the occurrence of gastric lesions induced by HCl [ED50 values were 0.12 g kg-1 (0.18-7.88) (Table 1) and ethanolic extracts (1.12 g kg-1 (0.81-1.55)], (Table 2) and induced by ethanol (ED50 values were 0.34 g kg-1 (0.26-0.43) (Table 3) and 1.73 g kg-1 (0.43-7.05), respectively) (Table 4).
The potency ratio of the aqueous extract versus the ethanolic extract was significant (p<0.05). At a high dose (4 g kg-1), the protection of the ethanolic extract against ethanol induced lesion was also equal to sucralfate.
All the extracts showed anti-ulcer activity in a dose-dependent manner.
The oral and intraperitoneal administrations of both extracts induced a significant decrease in total gastric acid together with an increase in pH values. These effects were dose-dependent.
Table 1: | Effects of the oral administration of Anethum chryseum leaves aqueous extract and sucralfate on the cytoprotective action against HCl-induced (1 N) gastric lesions in mice |
Values are the Mean±SEM of 6 mice, ***p<0.001 |
Table 2: | Effects of the oral administration of Anethum chryseum leaves ethanolic extract and sucralfate on the cytoprotective action against HCl-induced (1 N) gastric lesions in mice |
Values are the Mean±SEM of 6 mice, ***p<0.001 |
Table 3: | Effects of the oral administration of Anethum chryseum leaves aqueous extract and sucralfate on the cytoprotective action against absolute ethanol-induced gastric lesions in mice |
Values are the Mean±SEM of 6 mice, **p<0.01 |
Table 4: | Effects of the oral administration of Anethum chryseum leaves ethanolic extract and sucralfate on the cytoprotective action against absolute ethanol-induced gastric lesions in mice |
Values are the Mean±SEM of 6 mice, **p<0.01, ***p<0.001 |
Table 5: | Effects of the oral administration of Anethum chryseum leaves aqueous extract and cimetidine on the pH and total gastric acid from pylorus-ligated mice |
Values are the Mean±SEM of 6 mice, ***p<0.001, Compared to control, Tukey-Kramer |
Table 6: | Effects of the oral administration of Anethum chryseum leaves ethanolic extract and cimetidine on the pH and total gastric acid from pylorus-ligated mice |
Values are the Mean±SEM of 6 mice, ***p<0.001, compared to control, Tukey-Kramer |
Table 7: | Effects of the intraperitoneal administration of Anethum chryseum leaves aqueous extract and cimetidine on the pH and total gastric acid from pylorus-ligated mice |
Values are the Mean±SEM of 6 mice, ***p<0.001, compared to control, Tukey-Kramer |
Table 8: | Effects of the intraperitoneal administration of Anethum chryseum leaves ethanolic extract and cimetidine on the pH and total gastric acid from pylorus-ligated mice |
Values are the Mean±SEM of 6 mice, ***p<0.001, compared to control, Tukey-Kramer |
Both extracts with higher doses showed antisecretory activity as effective as cimetidine. The ED50 values of the aqueous and ethanolic extracts oral treatment were 0.17 g kg-1 (0.43-2.04) (Table 5) and 0.07 g kg-1 (0.03-0.15), respectively (Table 6).
The ED50 values of the aqueous and ethanolic extracts intraperitoneal treatment were 0.02 g kg-1 (0.01-0.03) (Table 7) and 0.03 g kg-1 (0.01-0.08), respectively (Table 8). The potency ratio of the aqueous extract versus the ethanolic extract was not significant in these tests.
These results indicate that the extracts of A. chryseum leaves have effective antisecretory and anti-ulcer activity against HCl- and ethanol-induced stomach lesions.
In respect to LD50 values, the aqueous extract was more toxic than the ethanolic extract. Compare with a toxicity classification (Loomis, 1968; Kaur and Arora, 2008), the aqueous and ethanolic extracts are relatively and little toxic, respectively. As high doses of ethanolic extract was used in this study, in clinical trial the toxicity of A. chryseum should be considered.
The oral administration of the extracts diminished HCl-induced gastric lesions in mice. This may be related to an antacid effect or cytoprotective properties in gastric mucus. The cytoprotective action against ethanol showed that the effects of extracts are not a simple acid neutralizing activity but the leaves extracts have a cytoprotective effect against the gastric mucosa in ethanol-induced gastric lesion in mice.
It is possible that the inhibitory effects of extracts are due, at least partly, to the presence of terpenes in A. chryseum (Bouwmeester et al., 1995; Faber et al., 1997; Derman et al., 2009). Terpenes were associated to antiulcerogenic activity in other plants (Matsunaga et al., 2000; Hiruma-Lima et al., 2001). Some triterpenes are known as antiulcer drugs and their action has been suggested to be due to the activation of cellular protection, reduction of mucosal prostaglandins metabolism-cytoprotective action and reduction of gastric vascular permeability (Sertie et al., 2000).
Flavonoids have antiulcer and gastroprotective activities (Alvarez et al., 1999). The aqueous extracts of Phoradendron crassifolium and Franserio artemisiodes that contain polyphenolic agents exerted cytoprotective activity in rats (Gonzales et al., 2000). Two flavonoids have been isolated from A. chryseum leaves, quercetin 3-O-beta-D-glucuronide and isoharmentin 3-O-beta-D-glucuronide, have antioxidant activity and could counteract with free radicals. This effect may help to prevent ulcer peptic (Moehle et al., 1985; Mahran et al., 1999).
The extracts provoked a marked decrease in total gastric acid together with an increase in pH values. As the fruit of A. chryseum has an antispasmodic effect on the smooth muscles of the gastrointestinal tract (Fleming, 2000), it is possible that both extracts act via cholinergic system (an anticholinergic activity) and block release of HCl. At this stage, other mechanisms such as H2 receptor antagonist effect or the inhibition of gastric H+, K+-ATPase can not be excluded.
In respect to the ED50 values, the aqueous extract was more potent than the ethanolic extract in HCl- or ethanol-induced stomach lesions. However, both extracts showed similar potency in reduction total gastric acid together with an increase in pH values. This may be related to the extracts different mechanisms of action. The ED50 values of extracts were much lower than their LD50 values.
In conclusion, A. chryseum leaves markedly inhibits acid secretion and the occurrence of lesions in stomach but exact mechanisms are not clear yet and need further investigations.
The results suggest that A. chryseum leaves extracts have significant mucosal protective and antisecretory effects of the gastric mucosa in mice.
This research has been done by supporting from the Ilam university under financial contract No. 32456778 in Mar 2008.