Anti-inflammatory effect of the hydralcoholic extract of Zingiber officinale rhizomes on rat paw and skin edema.

Summary

Plant extracts have been used for centuries as a popular mode of treatment for several health disorders. Over the last ten years, the study of those extracts has attracted attention in different fields of the biological sciences. Ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), is a commom constituent of diet worldwide and it has been reported that its extracts present some pharmacological activities. Here we investigate the effects of the crude hydraicoholic extract of ginger rhizomes on the classical models of rat paw and skin edema. The carrageenan-, compound 48/80- or serotonin-induced rat paw edema were inhibited significantly by the intraperitoneal administration of alcoholic ginger extract. Ginger extract was also effective in inhibiting 48/80-induced rat skin edema at doses of 0.6 and 1.8 mg/site. Rat skin edema induced by substance P or bradikinin was not affected by treatment with Z. officinalle extract. The intraperitoneai administration of ginger extract (186 mg/[kg.sup.-1] body wt.) 1 h prior to serotonin injections, reduced significantly the serotonin-induced rat skin edema. Our results demonstrated that crude extract of Zingiber officinale was able to reduce rat paw and skin edema induced by carrageenan, 48/80 compound and serotonin. The antiedematogenic activity seems to be related, at least partially, to an antagonism of the serotonin receptor.

Key words: Zingiber officinale, antiinflammatory activity, rat paw and shin edema

* Introduction

Plant extracts have been used for centuries as a popular method for treating several health disorders. Over the last ten years the study of those extracts has attracted attention in different fields of the biological sciences. Ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), originally from south-east Asia and introduced to South America and several countries of the African continent, is a common constituent of diet worldwide (Sertie et al. 1992). Rhizomes have been employed in cuisine as a condiment, and in folk medicine as a carminative, a diaphoretic, an antispasmodic against intestinal colic and as an anti-emetic (Yamahara et al. 1988; Phillips et al. 1993). Ginger was also reported be effective in reducing HCl/ethanol-induced gastric lesions in rats and in preventing ulcers (Sertie et al. 1992). A very potent anti-ulcerogenic compound was isolated from the Z. officinale rhizomes ((+)-angelicoidenol-2-o-D-glicopyranoside) (Yoshikawa et al. 1994). Cao et al. (1993) reported significant [O.sub.2.sub.-] scavenging activity of ginger. In 1994, Lumb demonstrated in a clinical study that dried ginger (up to 2 g/individual) was unable to affect platelet function. In contrast, Bordia et al. (1997) found that daily administration of 10 grams of ginger per individual was able to significantly reduce platelet aggregation induced by different stimuli, and also to reduce serum cholesterol levels of hypercholesterolemic patients. More recently Bhandari et al. (1998) demonstrated that ginger extract was able do reduce the plasma levels of cholesterol in rabbits fed a special, cholesterol-enriched diet. Treatment of rabbits with ginger extracts reduced aortic atherosclerosis significantly as compared to control.

In the present study, the anti-inflammatory activity of ginger was investigated using a crude hydroalcoholic rhizome extract in the classical models of rat paw and skin edema.

* Materials and Methods

Animals

All experiments were carried out in accordance with the guidelines of Vale do Paraiba University for animal care. The experiments were carried out on Male Wistar rats weighing between 150 and 200 g each, maintained under standard conditions of temperature (22-25[degrees]C), relative humidity (40-60%) and light/dark cycle, with access to food and water ad libitum. The animals were provided by the Central Animal House of the Research and Development Department of Vale do Paraiba University (UNIVAP). All rats were placed in a common box and divided randomly into groups of six animals

Preparation of the extract

Z. officinale extract was obtained as described previously (Sertie et al. 1992). Briefly, Z. officinale rhizomes were collected in March at Caraguatatuba City, State of Sao Paulo, and identified by the herbarium staff of the Department of Botany, Biosciences Institute--University of Sao Paulo (SP), Brazil, where the voucher specimen was deposited. Fresh material (200g) was peeled, finely cut and extracted using 1000 ml of 70% ethanol (ZO extract). After 2 days of maceration, the extract was filtered and concentrated in vacuo at 50[degrees]C. The viscous mass obtained from the extract was lyophilized (1.0 mg equivalent to 16.7 mg of fresh rhizome). The pharmacological trials were carried out with the dry material dissolved in 0.9% saline.

Induction of rat hind-paw edema

Rats received a subplantar injection of carrageenan (0.1 ml of a 1% suspension in 0.85% saline; Sigma Chemical Co., St Louis, MO, USA), compound 48/80 (10 [micro]g/paw--Sigma Chemical Co., St Louis, MO, USA) or serotonin (240 ng/paw) in the left hind paw under a brief (1-min) anesthesia with halothane. The total volume injected was always 0.1 ml.

Volumetric measurements of the rat paw edema

The insertion of the inflamed paw in a tube of fluid elevates the fluid level, and test and control levels can be compared. The method offers a precise and efficient technique when using a hydroplethysmometer (Milanino, 1988), particularly if two platinum electrodes immersed in an electrolyte are used to determine fluid level changes (Freidoni et al. 2000).

The volume of paw edema (ml) was measured in each animal using a plethysmometer (plethysmometer 7150, Ugo Basile, Italy) to a precision of two decimal places. The measurements were made immediately before and 1, 2, 3, and 4 h after injection. The increase in paw volume (ml) was calculated by subtracting the basal volume from the final volume. The area under the time-course curve (AUC; ml.h) was also calculated using the trapezoidal rule (Giraldello et al. 1994).

Measurement of rat skin edema

Local skin edema formation induced by 48/80 compound (500 ng/site) (Giraldelo et al. 1994; Palframan et al. 1996;) or serotonin (120, 240 and 500 ng/site) (Newbold et al. 1993; Kajekar et al. 1995) was measured in male Wistar rats (150-200 g each) as the local accumulation of i.v.-injected [sup.125]I-human serum albumin into skin sites as described previously (Williams, 1979; Brain and Williams, 1985). Rats were anesthetized by intraperitoneal injection of sodium pentobarbitone (Sagatal, 30-40 mg/kg body wt.). [sup.125]I-human serum albumin (10 mCi/kg) and Evans Blue dye (1.5 ml/kg body wt., 2.5% w/v) were injected via the tail vein. Test agents were made up in Tyrode bicarbonate solution and injected in volumes of 0.1 ml into the shaved dorsal skin according to a balanced site pattern with two replicates per each agent. After a 15 min-accumulation period, a 5 ml cardiac blood sample was taken into heparin and the animal killed by sodium pentobarbital overdose.

The dorsal skin was removed and the injection sites punched out (15-mm diameter) and radioactivity was counted using a gamma counter. Edema formation at each site was expressed as plasma volume, calculated from the counts in 1 ml plasma.

Statistical Analysis

Results are expressed as mean [+ or -] s.e./mean of n experiments. Analysis of variance and Student's unpaired t test were employed to evaluate the data. P < 0.05 was taken as significant.

Chemicals and Reagents

Carrageenan, serotonin, Substance P, Bradykinin and 48/80 compound were purchased from Sigma Chemical Co. (St Louis, MO, USA).

* Results

Effect of the Hydroalcoholic Extract of Zingiber officinale on Rat Paw edema

Rat paw edema induced by the injection of carrageenan (1 mg/paw), compound 48/80 (10 [micro]g/paw) or serotonin (240 ng/paw) was affected significantly by the intraperitoneal administration of the alcoholic ginger extract (Figs. 1, 2 and 3). Ginger extract significantly reduced the area under the time vs. edema curves (from 0 to 4 h) by 49.4 [+ or -] 3% and 37 [+ or -] 2.7% at doses of 186 and 310 mg/[kg.sup.-1] body wt. (p < 0.05; Fig. 1) for carrageenan-induced rat paw edema.

[FIGURES 1-3 OMITTED]

The administration of the extract 1 h before 48/80 injection reduced significantly the area under the time vs. edema curves (from 0 to 180 min) by 29.8 [+ or -] 6, 31.1 [+ or -] 5 and 30 [+ or -] 2% at all doses (18.6, 62 and 186 mg/[kg.sup.-1] body wt.; Fig. 2).

The administration of ginger extratct 1 h prior to serotonin injection (240 ng/paw) reduced significantly the area under the time vs. the edema curves (from 0 to 180 min) only at the highest dose of 310 mg/[kg.sup.-1] reduced by 27 [+ or -] 2% (Fig. 3).

Effect of the Hydroalcoholic Extract of Zingiber officinale on Rat Skin Edema Induced by 48/80 Compound or Serotonin

Hydroacoholic extract of Zingiber officinale reduced significantly 48/80-induced rat skin edema (500 ng/site) from 105.2 [+ or -] 8 to 85.8 [+ or -] 14 and 69.8 [+ or -] 7 ([micro]l of plasma) at doses of 0.6 and 1.8 mg/site (Fig. 4). Rat skin edema induced by substance P or bradikinin was not affected by the treatment with Z. officinale extract (Data not shown).

[FIGURE 4 OMITTED]

The intraperitoneal administration of the hydroalcoholic extract of Z. officinale (186 mg/[kg.sup.-1] body wt.) 1 h prior to serotonin injections (120, 240 and 500 ng/site), reduced significantly serotonin-induced rat skin edema from 72 [+ or -] 7 to 22 [+ or -] 8, 86 [+ or -] 12 to 39 [+ or -] 14 and [+ or -] 12 to 28 [+ or -] 4 (ml of plasma), respectively (p < 0.05; Fig. 5).

[FIGURE 5 OMITTED]

* Discussion

Previous studies (see Introduction) demonstrated the ability of Zingiber officinale extract to prevent gastric ulcerations induced by HCl and ethanol. The antiulcerogenic activity of this plant suggests that the extract could interfere with the production or action of eicosanoids. Ginger also demonstrates significant activity in reducing aortic atheroma formation in rabbits and humans. Here we studied a possible role for ginger extract as a potential anti-inflammatory drug and thus decided to study ginger extract, using for this purpose the classical models of rat paw and skin edema.

It is well-established that carrageenan-induced rat paw edema is associated with three distinct phases. The first phase is early mediated by mast cell degranulation and histamine and serotonin release (first h), the second phase (60 to 150 min) is characterized by bradykinin release and pain, and further eicosanoid production in the late phase (third-fourth h) (Di Rosa and Willoughby, 1971; Di Rosa, 1972; Goetzl, 1980). At the highest dose, hydroalcoholic extract of Z. officinale was able to inhibit carrageenan-induced rat paw edema from the first hour. These results suggest that ginger extract could inhibit rat paw edema through the inhibition of eicosanoid production. Because the inhibition was persistent up to the fourth hour of experiments, however, mediated by mast cell degranulation and serotonin release, we started using the model of rat paw edema induced by 48/80, a strong mast-cell-degranulating synthetic compound. Rat paw edema induced by the 48/80 compound was reduced significantly by the ginger hydroalcoholic extract at all doses. The rat mast cells present high amounts of serotonin, which is released by degranulation induced by the 48/80 compound. In this way, the Z. officinale extract could be acting either as an inhibitor of mast cell degranulation or as a serotonin antagonist. Indeed, the hydroalcoholic extract of Z. officinale reduced significantly the rat paw edema induced by exogenous serotonin, and unpublished data showed that the ginger extract could not inhibit mast cell degranulation induced by the 48/80 compound, suggesting a possible mechanism involving the antagonism of serotonin receptors.

On the other hand, one short screening using the rat skin edema model demonstrated a relative specificity of the ginger extract that only inhibited rat skin edema induced by the 48/80 compound (Fig. 4) and by serotonin (Fig. 5), without affecting that induced by Substance P or bradykinin. The crude hydroalcoholic extract of Z. officinale was able to inhibit serotonin-induced rat skin edema, even at doses of 240 and 500 ng per site of injection.

Our results demonstrated that crude extracts of Zingiber officinale were able to reduce rat paw and skin edema induced by carrageenan, 48/80 compound and serotonin. The mechanism of this inhibition remains unclear, but evidence indicates that it could be due to serotonin receptor antagonism. The purification of extracts has been made in our laboratory, in order to elucidate the mechanisms of action of this extract, which deserve further investigation.

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* Address

R. A. B. Lopes-Martins, Laboratorio de Farmacologia e Experimentacao Animal, Instituto de Pesquisa e Desenvolvimento--IP&D, Universidade do Vale do Paraiba--UNIVAP. Avenida Shishima Hifumi, 2911-Urbanova, Sao Jose dos Campos--SE 12244-000. Tel.: [phone]; Fax: [phone]; e-mail: [e-mail]

S. C. Penna (2), M. V. Medeiros (2), F. S. C. Aimbire (1), H. C. C. Faria-Neto (3), J. A. A. Sertie (2), and R. A. B. Lopes-Martins (1)

(1) Laboratorio de Farmacologia e Experimentacao Animal, Instituto de Pesquisa e Desenvolvimento--IP&D, Universidade do Vale do Paraiba--UNIVAP, Urbanova, Sao Jose dos Campos 

(2) Departamento de Farmacologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil

(3) Departamento de Fisiologia e Farmacodinamica, Fundacao Oswaldo Cruz--Av. Brasil--Manguinhos