Institute of Biological Sciences, Federal University of Juiz de Fora MG, Minas Gerais, Brazil.

Effect of Calendula officinalis infusion on indomethacin-induced gastric lesions in Wistar rats.

MD. Luiz Carlos Bertges,1 MD. Ângela Maria Gonçalves Felga,2 MD. João Batista Piccinini Teixeira,2 Carolina Frade Magalhães Girardin Pimentel 3 y Priscilla Ornella Neves3

Summary

Calendula officinalis is a phytopharmaceutical believed to inhibit gastric emptying and to have hypoglycemic and gastroprotective effects. We aimed to study the putative effect of an infusion of Calendula officinalis on indomethacin-induced gastric lesions. 30 adult male Wistar rats, divided in 2 groups, received indomethacin 20 mg/kg through gavage. Group A (Study) further received 1 mL of 10 % doses of Calendula officinalis, and group B (Control) received saline, for 2 days. After this period, the stomachs were extracted and examined for lymphocytic and neutrophylic infiltrates, and for mucosal lesions such as ulcers and erosions. We concluded that Calendula officinalis did not significantly influence the appearance of such alterations in the indomethacin-injured gastric mucosa.

Key words: Calendula officinalis, indomethacin, gastric injury, rats.

Of the two cyclo-oxigenase isoenzymes, COX-1 maintains gastric mucosal integrity while COX-2 plays a role in the pathophysiology of the inflammatory response.1

It has been recently demonstrated that selective COX-1 inhibitors considerably suppress prostaglandin production by healthy stomachs, without causing gastric injury.2

Conversely, simultaneous COX-1 and COX-2 inhibition leads to the development of important gastric lesions, i.e. in the normal mucosa both cyclo-oxigenases have to be inhibited so that ulcerations are provoked.2,3

The non-selective cyclo-oxigenase inhibitor indomethacin inhibits the production of E₂ prostaglandins (PGE₂), enhances gastric motility, and produces important lesions in the gastric mucosa.4,5

Some plant extracts have been studied as regards their lipo-oxigenase and cyclo-oxigenase inhibition potential, a property that affords anti-inflammatory activity. Other medicinal plants, such as Calendula officinalis L., have a hypoglycemic effect, inhibit gastric emptying, and have active components with gastroprotective properties.6

We aimed to verify if the administration of Calendula officinalis infusion protects the stomach against indomethacin-induced lesions.

METHODs

Thirty male Wistar rats, aged 4 months, and with a mean weight of 292,8 g, were studied. The study sample was divided in two groups of 15 rats (A and B), kept at room temperature and with light exposure following a 12/12h light/dark rhythm. In the 24h preceding the experiment, all the rats fastened and received only 5 % glucose ad libitum .

The gastric lesions, in all animals, were provoked by gavage-administered indomethacin in a single 20 mg/Kg dose. The indomethacin solution was prepared in a dedicated pharmacy according to the following composition: 0,1 g indomethacin, 0,2 g methyl-paraben, 10 g CMC, 10 g sorbitol, and 100 ml simple syrup. Group A animals received 1ml of 10% Calendula officinalis infusion, while group B animals received 1ml saline.

Both the active infusion and saline were administered 10 hours after the indomethacin dose, and repeated every 12 hours for 2 days.

The plant, obtained from the plant nursery of the School of Pharmacy and Biochemistry of the Federal University of Juiz de Fora, through locally obtained spontaneous generation seedling transplantation, was cultivated in mild, temperate climate, with full solar exposure. Harvesting was carried out during florescence period, when the flowers were in full bloom.

The flowers were dried in a wooden incubator disinfected with 70 % alcohol, at 35°C for 5 hours, during 5 days. Once dried, the flowers were kept in well-sealed, transparent, plastic bags.

The plant sample for the experiment was sent for quality control testing, carried out in the Pharmacognosy Laboratory of the School of Pharmacy and Biochemistry of the Federal University of Juiz de Fora, by Dr Magda Narciso Leite (herbarium number: CESJ 471595).

After 2 days, the rats were sacrificed with an ethyl ether overdose. Their stomachs were removed and opened through the greater curvature, and then washed and submitted to a lesion search.

The material was fixed and sent to the Department of Histology of the Juiz de Fora Federal University, where it was handled according to the usual technique. 3 to 7 parallel longitudinal sections were made (M 7,3), so as to allow for the examination of both the proximal squamous gastric mucosa and the distal glandular one. All fragments were stained with hematoxylin and eosin and examined for the presence of ulcers, erosions, and neutrophylic and lymphocytic infiltrates. Neutrophylic infiltrate was rated as mild (+) when there were few cells, moderate (++) when fibers were evident among the cells, and severe (+++) when fibers were not evident among the cells. The lymphocytic infiltrate was recorded as present or absent. The fundus squamous mucosa, the columnar-squamous transition, and the body glandular mucosa were analyzed.

Mann Whitney 's test was used, with significance set at p < 0,05.

RESULTS

In group A, single erosion in the gastric body of only one of the rats was observed, while in group B there was an antral ulcer in one rat and 5 erosions of the gastric body, in 5 different rats. In group A, a mild neutrophylic infiltrate was found in 9 animals, a moderate infiltrate in 4, and a severe one in 1, with a lymphocytic infiltrate absent in 12 and present in 2 rats. 3 erosions and 1 ulcer were identified, all in different animals (Table 1).

Table 1. Histopathology of group A animals

Rat	Neutrophils	Lymphocytes	Lesions
1	Mild	Absent	Absent
2	Mild	Absent	Absent
3	Mild	Absent	Absent
4	Mild	Absent	Absent
5	*	*	*
6	Mild	Absent	Absent
7	Moderate	Absent	Erosion
8	Moderate	Absent	Absent
9	Mild	Absent	Absent
10	Mild	Absent	Absent
11	Moderate	Present	Erosion
12	Moderate	Absent	Erosion
13	Mild	Absent	Absent
14	Mild	Absent	Absent
15	Severe	Absent	Ulcer

*Case ruled out due to autolysis.

In group B, there was a mild neutrophylic infiltrate in 11 rats, a moderate infiltrate in 3, and a severe one in 1. A lymphocytic infiltrate was present in 3 and absent in 12 animals. There was an ulcer in 1 and erosions in 3, all in different animals (Table 2).

Table 2. Histopathology of group B animals

Rat	Neutrophyls	Lymphocytes	Lesions
1	Mild	Absent	Absent
2	Mild	Present	Absent
3	Moderate	Absent	Erosion
4	Mild	Absent	Absent
5	Mild	Present	Absent
6	Moderate	Absent	Erosion
7	Mild	Absent	Absent
8	Mild	Absent	Absent
9	Mild	Absent	Absent
10	Moderate	Absent	Erosion
11	Severe	Absent	Ulcer
12	Mild	Absent	Absent
13	Mild	Present	Absent
14	Mild	Absent	Absent
15	Mild	Absent	Absent

Statistical analysis

There was no statistically significant difference as regards the presence of neutrophylic infiltrate between groups A and B (p = 0,371).

Likewise, there was no statistically significant difference between the 2 groups as regards the lymphocytic infiltrate (p = 1,00).

The occurrence of lesions such as ulcers or erosions (alterations) was also similar in both groups (p = 0,469).

Discussion

The use of medicinal plants to treat gastroduodenal disorders has been investigated for a long time. Chakurski et al. (1981), investigated the use of an herbal combination, with and without antacids, in the treatment of duodenal ulcers and gastroduodenitis.7 A relief of spontaneous pain in 90 % of the patients, regardless of antacid association, was found.

Other substances have also been under investigation. Guzel et al. (1995) compared the administration of fish and olive oils with an ordinary diet, in an attempt to promote cicatrisation of indomethacin-induced gastric lesions. 8 Fish oil proved to have a powerful cicatrizing effect on gastric erosions and ulcers besides increasing the mucous lining.

Calendula officinalis has reputedly showed a beneficial effect on the treatment of ulcers and gastritis.

Marukami et al. (2001), have isolated 2 glycosides, A and B officinosides, besides 2 sesquiterpene oligoglycosides, C and D officinosides, from the flowers of Egyptian Calendula officinalis.9 Their studies aimed to characterize the hypoglycemic and gastroprotective effects of this phototherapeutic, along with its inhibitory effect on gastric emptying. Yoshikawa et al. (2001), have discussed a possible relation between the structure of these glycosides and their hypoglycemic, gastroprotective, and gastric-emptying inhibitory effects.6

Matev et al. (1981), tested 19 patients with duodenal ulcer and 19 patients with gastroduodenitis, all of whom with constipation. 10 An association of Symphitum officinalis and Calendula officinalis was used to treat the duodenal ulcers and the gastroduodenitis. A combination of Rhamus frangula and Citrus aurantium was used to treat constipation. 100 % of the patients had relief of their constipation but improvement or cicatrisation of ulcers and gastritis was not as successful.

Some authors have shown that Calendula officinalis has antibacterial, sedative, hypotensive, and fungicide effects.11-13

In our study, a single dose of indomethacin (20 mg/kg) was administered in an attempt to produce gastric lesions and an inflammatory infiltrate. Calendula officinalis was subsequently administered for 2 days, no significant difference as regards the study variables in both groups being found.

We conclude that the oral administration of Calendula officinalis infusion did not reduce the indomethacin-induced neutrophylic and lymphocytic infiltrates or gastric lesions in the studied period.

Sumario

Effecto de la infusion de Calendula officinalis sobre las lesions gástricas provocadas por indometacina

La Calendula officinalis es un fitofármaco que se considera inhibe el vaciamiento gástrico y tiene efectos hipoglicémicos y gastroprotectores. En el presente trabajo se estudia el posible efecto de una infusión de Calendula officinalis sobre las lesiones gástricas provocadas por indometacina (dosis de 20 mg/kg) a través de un lavado. El grupo A (estudio) recibió además 1 ml de infusión de Calendula officinalis y al grupo B (control) se le administró solución salina al 10 % durante dos días. Después de este período, se procedió a la extracción de los estómagos y se examinaron en busca de infiltrados linfocíticos y neutrofílicos y de lesiones mucosas, tales como úlceras y erosiones. Se concluye que la Calendula officinalis no influye de manera significativa en la aparición de estas alteraciones en la mucosa gástrica dañada por indometacina.

Palabras claves: Calendula officinalis , indometacina, lesión gástrica, ratas.

Referencias bibliográficas

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7. Chakurski I, Matev M, Stefanov G, Koichev A, Angelova I. Treatment of duodenal ulcers and gastroduodenitis with a herbal combination of Symphitum officinalis and Calendula officinalis with and without antacids. Vutr Boles. 1981;20:44-7.

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9. Marukami T, Kishi A, Yoshikawa M. Medicinal flowers. IV. Marigold. (2): Structures of new ionone and sesquiterpene glycosides from Egyptian Calendula officinalis. Chem Pharm Bull (Tokyo). 2001;49:974-8.

10. Matev M, Chakurski I, Stefanov G, Koichev A, Angelov I. Use of an herbal combination with laxative action on duodenal peptic ulcer and gastroduodenitis patients with a concomitant constipation syndrome. Vutr Boles. 1981;20:48-51.

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13. Bojadjiev C. On the Sedative and Hypotensive Effect of preparations from the plant Calendula officinalis. Nauchni Tr Vissh Med Inst Sofiia. 1964;43:15 -20.

Recibido: 28 de agosto de 2006. Aceptado: 11 de septiembre de 2006.
MD.Luiz Carlos Bertges, Campus Universitário, Departamento de Fisiologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora MG, CEP 36.036-900, e- mail: [e-mail]

1Professor at the Department of Physiology, Institute of Biological Sciences, Federal University of Juiz de Fora, MG, Brazil.
2Lecturer at the Department of Morphology, Institute of Biological Sciences, Federal University of Juiz de Fora, MG, Brazil.
3Medical Undergraduate, School of Medicine, Federal University of Juiz de Fora, MG, Brazil.