Anti-inflammatory and antioxidant effects of Croton celtidifolius bark.

Summary

Croton celtidifolius Baill commonly known as "sangue-de-adave" is a tree found in the Atlantic Forest of south of Brazil, mainly in Santa Catarina. The bark and leaf infusions of this medicinal plant have been popularly used for the treatment of inflammatory diseases. In this study we evaluated the anti-inflammatory and antioxidant properties of crude extract (CE), aqueous fraction (AqF), ethyl acetate fraction (EAF), butanolic fraction (BuF) and catechin, gallocatechin and sub-fractions, 19SF, 35SF and 63SF that contained a mixture of proanthocyanidins and were derived from the EAF fraction. The CE, AqF, EAF, BuF, catechin and sub-fractions 35SF and 63SF reduced paw edema induced by carrageenan. The CE, fractions, sub-fractions and isolated compounds showed antioxidant properties in vitro, all were able to scavenge superoxide anions at a concentration of 100 [micro]g * [ml.sup.-1]. The EAF, catechin and gallocatechin were most effective in the deoxyribose assay, 1C50 0.69(0.44-1.06), 0.20 (0.11-0.39), 0.55(0 .28-1.08) [micro]g * [ml.sup.-1] respectively. The CE and other fractions and sub-fractions inhibited deoxyribose degradation up to 1 [micro]g * [ml.sup.-1]. In the hydrophobic system only AqF did not show lipid peroxidation inhibition. The CE, other fractions, sub-fractions and isolated compounds inhibited lipidid peroxidation only at a concentration of 100 [micro]g * [ml.sup.-1]. In summary, this study demonstrates that Croton celtidifolius bark has significant anti-inflammatory and antioxidant activity.

Key words: Croton ceitidifolius, paw edema, antioxidants, reactive oxygen species, proanthocyanidins, anti-inflammatory

Introduction

Medicinal plants have long been used for the treatment of many diseases. Recently, several experimental studies have contributed scientific evidence for the pharmacological effects of these medicinal plants observed in folk medicine. Croton celtidifolius Baill, commonly known as "sangue-de-adave", is a tree found in the Atlantic Forest, mainly in the south of Brazil, including the state of Santa Catarina. The bark and leaf infusions of this plant have been popularly used for the treatment of leukemia, ulcer rheumatism and other inflammatory diseases.

Inflammatory diseases are accompanied by the chronic release of cytokines and reactive oxygen and nitrogen species, which may be involved in increased tissue injury (Conner and Grisham, 1996; JanssenHeininger et al., 1999). It has been demonstrated that administration of specific antioxidants, such as superoxide dismutase (SOD), and/or catalase is effective in decreasing the tissue inflammation and injury observed in experimental models of ischemia and reperfusion, arthritis, chronic gut inflammation, and immune complex-induced pulmonary disease (Conner and Grisham, 1996). Because of the importance of reactive oxygen species as toxins, mediators and modulators of the inflammatory process, a number of natural and synthetic antioxidants have been studied as potential therapeutic agents (Salvemini et al., 2001). Many studies have shown that phenolic compounds are efficient scavengers of free radicals and blockers of lipid peroxidation (Rice-Evans and Miler, 1996). They are able to chelate transition metals, such as iron and copper ions and thereby prevent the redox cycle of these metals (Kuo et al., 1998).

Several compounds with pharmacological activity have been previously isolated from other Croton spp. It has been reported that the alkaloid tapsine may be the active principle responsible for the anti-inflammatory (Vaiberg et al., 1988) and anti-tumour activity (Chen et al., 1994) of latex of C. lechieri. Ninety per cent of the dried weight of C. lechleri is composed of catechin, epicatechin, gallocatechin, epigallocatechin and proanthocyanidins (Cai et al., 1991). Phytochemical study of the more known species of the genus Crcton, Crown cajucara, showed that the bark of this plant is rich in diterpenes and that the leaves possess diterpenes, flavonoids and steroids (Maciel et al., 2000).

However, studies about chemical constituents as well as pharmacological effects of C. celtid~fo1ius are still limited. There is only one report about the identification of cyclitols found in this species (Mukherjee and Axt, 1984), and we found no scientific reports describing the pharmacological activity of this plant. In addition, previous studies in our laboratory determined that 63SF from EAF are constituted by of proanthocyanidins (data unpublished).

The anti-inflammatory activity of C. celtidifolius bark established by popular medicine and the participation of oxygen free radicals in the inflammatory process lead us to investigate the anti-inflammatory activity and superoxide anion and hydroxyl radical scavenger properties, as well as inhibition of lipid peroxidation, of crude extract and fractions of this plant and to correlate these with the presence of chemical constituents.

Materials and Methods

Animals

Male Swiss mice (25-35 g) housed at 22 [+ or -] 2 [degrees]C under a 12 hr light/12 dark cycle and with free access to food and water, were used in the experiments. In all experiments, animals were managed following the principles and guidelines for the care of laboratory animals, according to Zimmerman (1983).

Plant material

Bark of Croton celtidifolius Baill was collected in March, 2000, at Orleans and the voucher specimen was identified with the collection documenting number 31272 at the Botany Department, UFSC, and deposited in the authors' laboratory.

Extraction and isolation

Air-dried bark (154 g) was chopped into small pieces and extracted three times with 250 ml of 80% aqueous EtOH at room temperature. The combined extracts were filtered; the solvent was evaporated in a vacuum to give 42.9 g of the crude extract. The aqueous suspension of this extract was successively partitioned with ether, ethyl acetate and n-butanol furnishing in ether (2.0 g), EtOAc (17.6 g), n-BuOH (22.2 g) and aqueous (6.9 g) fractions.

The ether fraction was chromatographed on silica gel using a solvent gradient system from hexane to EtOAc to give three fractions (fr. A, fr. B, fr. C). Fractions A and B were analysed by high-resolution gas chromatography/mass spectrometer (HRGC/MS) showing hydrocarbons, esters, acid waxes and sterols. Recrystalisation of the fraction C yielded colourless crystals (52 mg), mp 130[degrees]C identified as 13-sitosterol.

The EAF (9.0 g) was chromatographed over a column of silica gel water (20%) inactivated and eluted with hexane/EtOAc (4:1) and increasing the polarity by gradual addition of EtOAc and MeOH. After thin layer cromatography (TLC) analysis, four sub-fractions were obtained, named 11SF, 19SF, 35SF and 63SF.

Sub-fraction 11SF was further purified by flash chromatography using hexane 40%/EtOAc 59%/AcOH 1% as isocrastic solvent to give Catechin and Gallocatechin.

Paw edema Induced by Carrageenan in Mice

Carrageenan (300 [micro]g/paw; 20 p1) was injected into the plantar surface of the right hind paw in male Swiss mice (25-35 g) (Henriques et al., 1987), 30 mm after the intraperitoneal (i.p.) administration of the EAF, BuF, AqF, ether fraction at doses of 3-100 mg.[kg.sup.1]; catechin, gallocathechin, sub-fractions 19SF, 35SF, 63SF derived from the EAF, at the dose of 30 mg . [kg.sup.1] and indomethacin (5 mg*[kg.sup.-1]) as positive control. The oral administration (p.o.) of the crude extract (CE), EAF, BuF, AqF, ether fraction at doses 10-300 mg*[kg.sup.-1] and indomethacin (15 mg*[kg.sup.-1]), occurred 1 hr prior to carrageenan. All fractions, sub-fractions and isolated compounds were diluted in phosphate buffered saline (PBS), except for the Ether fraction that was diluted in 5% dimethyl sulfoxide (DMSO) and indomethacin in Tween 10%. Control animals received only vehicle (0.1 ml/10g body w.). The contralateral paw received the same volume of PBS (20 [micro]l). Edema was measured plethysmographically 30, 60, 120 and 240 min later. Results were expressed as the difference in volume between the carrageenanpaw and PBS-paw.

Superoxide Scavenger Activity

The non-enzymatic generation of superoxide anion was measured in samples which contained 10 [micro]M phenazine methosulphate, 100 [micro]M nicotinamide nucleotide diphosphate in its reduced form (NADH) and 600 [micro]M nitro blue tetrazolium (NBT) in 0.1 M phosphate buffer ([K.sub.2][HPO.sub.4]-[KH.sub.2][PO.sub.4]) pH 7.8 (Robak and Griglewiski, 1988). After 2 min of incubation at room temperature the reaction was stopped with 0.1 M HCl, and spectrophotometric measurement was performed at 560 nm against control samples, which contained no phenazine methosulphate. Different concentrations of CE, fractions, sub-fractions and isolated compounds from C. celtidifolius bark (0.1 to 100 [micro]g*[ml.sup.-1]) were added to test tubes before the addition of the phenazine methosulphate. Superoxide Dismutase (SOD - 100 U/ml; 10 [micro]l) was utilized as positive control.

Oxidative Damage to Biomolecules

Free radical-mediated degradation of deoxyribose and methyl linoleate was measured as the production of 2-thiobarbituric acid-reactive substances. The ability of the CE, fractions, sub-fractions and isolated compounds from Croton celtidifolius to inhibit the iron-induced decomposition of deoxyribose was assessed (Halliwell and Gutteridge, 1981). The reaction mixture contained the following reagents (final concentrations in brackets) in a final volume of 1.2 ml: deoxyribose (2.8 mM), ferric chloride [FeCl.sub.3] (25 [micro]M), nitrilotriacetic acid (NTA; 100 [micro]M) (NTA and [Fe.sup.3+] ions are premixed at the ratio given, prior to the addition of deoxyribose), phosphate buffer ([K.sub.2][HPO.sub.4]-[KH.sub.2][PO.sub.4]) pH 7.4 (10 mM), different concentrations of CE, fractions, sub-fractions and isolated compounds (0.1 to 100 [micro]g*[ml.sup.-1]) and hydrogen peroxide ([H.sub.2][O.sub.2] 2.8 mM). The test tubes were incubated at 37 [degrees]C for 1 hr. Products of [degrees]OH attack upon deoxyribose was a ssessed by reaction with thiobarbituric acid (TBA) in acid solution. 1 ml of 1% (w/v) TBA and 1 ml of 2.8% trichloroacetic acid (TCA) was added to the tubes. After heating for 15 minutes at 100 [degrees]C and cooling, the absorbance of the pink chromophore was measured at 532 nm. Manitol (50 mM), the known hydroxyl radical scavenger, was utilized as positive control (Bhat and Madyastha, 2001).

Lipid peroxidation was assayed using rat liver homogenate as the target substrate (Chen and Tappel, 1996). In this system the lipid peroxidation was induced by t-butyl-hydroperoxide. The reagents were added in the following order: 10 mM phosphate buffer ([K.sub.2][HPO.sub.4]-[KH.sub.2][PO.sub.4]) pH 7.4, different concentrations of the CE, fractions, sub-fractions and isolated compounds (0.1 to 100 [micro]g*[ml.sup.-1]), or butylated hydroxytoluene (BHT; 1 mM) as positive control, rat liver homogenate (150 [micro]l) and 100 [micro]M t-butyl-hydroperoxide. The mixture was incubated at 37 [degrees]C, under aerobic conditions, for 2 h. After the incubation, 1 ml of 1% (w/v) TBA and 1 ml of 2.8% trichloroacetic acid (TCA) was added to the tubes and heated to 100 [degrees]C for 15 min. After removing the precipitates by centrifugation, the quantity of TBA reactive substances was spectrophotometrically determined at a wavelength of 532 nm.

Drugs

Carrageenan, phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), thiobarbituric acid (TBA), nitro blue tetrazolium (NBT), Superoxide Dismutase (SOD) nitrilotriacetic acid (NTA), butylated hydroxytoluene (BHT), t-butyl-hydroperoxide, Phenazine Methosulphate, nicotinamide nucleotide diphosphate (NADH), Deoxyribose and Indomethacin were obtained from Sigma Co. (USA), trichloroacetic acid (TCA, Aldrich, USA), Mannitol, [FeCl.sub.3] (Reagem, Brazil) and [H.sub.2][O.sub.2] (Quimex, Brazil).

Statistical Analysis

All experiments "in vitro" were performed at least three times. The results were expressed as mean [+ or -] standard deviation (S.D.). The statistical significance of the data was analysed using Student t-test or ANOVA with post hoc comparison by Tukey, *p < 0.05 was considered statistically significant. All experiments in vivo were expressed as mean [+ or -] standard error of mean (S.E.M.). Statistically significant differences between groups were measured using one-way analysis of variance (ANOVA) followed by Dunnet's test, *p < 0.05 or **p < 0.01 was considered statistically significant. The geometric mean [IC.sub.50] values, accompanied by their respective 95% confidence limits, were determined by non-linear regression from individual experiments using "Graph Pad Prism Software".

Results

The crude extract (CE) was fractionated into ether, ethyl acetate (EAF), n-butanolic (BuF) and aqueous (AqF) fractions by partitioning. Catechin, gallocatechin and a mixture of condensed proanthocyanidins 35SF and 63SF were obtained from the EAF fraction. The identification was performed by mass spectrometer (MS), Infra-Red (IR), [blank.sup.1]H Nuclear Magnetic Resonance ([blank.sup.1]H NMR), [blank.sup.13]C Nuclear Magnetic Resonance ([blank.sup.13]C NMR), with spectral data in agreement with previously published data (Agrawal, 1989).

Catechin and Gallocatechin identification by IR, [blank.sup.1]H NMR and [blank.sup.13]C NMR

Catechin (543 mg) 1. Amorphous, m.p. 143-144 [degrees]C, [M.sup.+] 290, IR [V.sub.max] [cm.sup.-1]: 3382, 1612, 1518, 1464. The NMR data were identical with those reported in the literature (Agrawal, 1989).

Gallocatechin (98 mg) 2. Amorphous, m.p. 167-170 [degrees]C, [M.sup.+] 306, JR [V.sub.max] [cm.sup.-1]: 3374, 1626, 1520, 1462, 1352. The NMR data were identical with these reported in the literature (Agrawal, 1989).

Anti-inflammatory Activity

CE (Table 1), BuF (Table 3) and AqF (Table 4) at the doses of 10, 30, 100 and 300 mg * [kg.sup.-1] oral route (p.o.), caused a reduction in paw edema of 13.7; 8.1; 27.7; 35.6% CE; 4.71; 18.7; 24.6; 38.6% BuF and 7.83; 19.6; 30.1; 47.5% AqF, respectively. Similarly, BuF and AqF, when administered by intraperitoneal (i.p.) route (3, 10, 30 and 100 mg * [kg.sup.-1]), reduced by 4.12; 38.1; 56.8; 74.3% BuF and 22.2; 35.2; 60.4; 6 1.0% AqF, respectively, the paw edema induced by carrageenan in mice. The area under the curve was calculated the percentage of edema reduction. The indomethacin and EAF also significantly inhibited the carrageenan-induced paw edema, by i.p. and oral route (Table 2).

Only sub-fractions 35SF and 63SF, and Catechin derived from the EAF fraction reduced the paw edema. Catechin only reduced the paw edema after 60 and 120 mm (Table 5). The ether fraction, gallocatechin and 19SF did not reduce paw edema.

Antioxidant Activity

The CE, fractions, sub-fractions and isolated compounds showed antioxidant properties in vitro. The superoxide scavenger capacity was evaluated by the NET reduction test. In this system, the inhibition of NBT reduction was observed only at the concentration of 100 [micro]g * [ml.sup.-1]. The positive control SOD (100U/ml; 75 [+ or -] 2.3%), CE (55.0 [+ or -] 4.3%), fractions BuF (36.3 [+ or -] 0.3%), AqF (57.8 [+ or -] 3.1%), EAF (44.5 [+ or -] 4.4%), sub fractions 19SF (41.0 [+ or -] 1.3%), 35SF (35.0 [+ or -] 1.5%), 63SF (50.7 [+ or -] 5.2%) and isolated compound Gallocatechin (32.5 [+ or -] 4.9%), inhibited NBT reduction (Fig. 1). Catechin, showed no effect in this system.

The CE, fractions sub-fractions and isolated compounds, were evaluated for their ability to scavenge hydroxyl radicals and/or to chelate iron. The results showed that all were able to inhibit deoxyribose degradation. The EAF was the most effective among fractions and catechin and gallocatechin, inhibited deoxyribose degradation. The CE, other fractions and subfractions inhibited the deoxyribose degradation up to 1 [micro]g [ml.sup.-1] (Table 6).

Experiments were also carried out to determine whether CE, fractions, sub-fractions and isolated compounds could inhibit rat liver homogenate degradation induced by t-butyl-hydroperoxide. The positive control BHT (1 mM; 58,6 [+ or -] 11.7%) and the treatment groups CE (45.6 [+ or -] 2.6%), BuF (46.8 [+ or -] 3.5%), EAF (46.0 [+ or -] 2.9%), 19SF (48.0 [+ or -] 4.7%), 35SF (63.5 [+ or -] 8.5%), Catechin (54.0 [+ or -] 5.5%) and Gallocatechin (49.3 [+ or -] 3.8%), showed some inhibition of lipid peroxidation, only at the concentration of 100 mg [ml.sup.-1] (Fig. 2). In this hydrophobic system, only AqF and 63 SF failed to inhibit lipid peroxidation.

Discussion

The present study demonstrated that CE, fractions, sub-fractions and isolated compounds from C. celtidifolius demonstrate an anti-inflammatory and antioxidant properties, since they were able to reduce paw edema, protect deoxyribose and rat liver homogenate from oxidative damage, in addition to inhibiting [O.sup.*-.sub.2] induced NBT reduction.

The injection of carrageenan into the sub plantar area produces edema with plasma exudation and neutrophils were the predominant cell until 24 hr after carrageenan injection (Henriques et al., 1987).

CE, EAF, BuF, AqF inhibited paw edema, while the Ether fraction had no effect. These results lead to us believe that not one but several compounds from Croton celtidifolius may be responsible for the anti-inflammatory activity.

In the same manner, sub-fractions 35SF and 63SF, derived from the EAF fraction, that contains a mixture of condensed proanthocyanidins and catechin, demonstrated significant anti-inflammatory activity. Reports have showed an important anti-inflammatory activity in the carrageenan rat paw edema model for proanthocyanidins, isolated from Polypodium feei (Subarnas and Wagner, 2000).

There is a large amount of evidence to show that the production of reactive species such as superoxide anion radical ([O.sub.2.sup.*-], Hydrogen peroxide ([H.sub.2][O.sub.2]), hydroxyl radical ([OH.sup.*]) and peroxinitrite occurs at the site of inflammation and contributes to tissue damage (Salvemini et al., 1996; Cuzzocrea et al., 1997). In acute and chronic inflammation, the production of [O.sub.2.sup.*-] is increased at a rate that overwhelms the capacity of the endogenous SOD enzyme defence system to remove them. The imbalance results in O{ mediated damage (Salvemini et al., 2001). Some important pro-inflammatory roles for [O.sub.2.sup.*-] include endothelial cell damage and increased microvascular permeability (Droy-Lefaix et al., 1991), formation of chemotactic factors such as leukotriene [B.sub.4] (Fantone and Ward, 1982; Deitch et al., 1990), lipidic peroxidation and oxidation and DNA single strand damage (Dix et al.,1996).

Another most reactive radical is [OH.sup.*], which, on reacting with biomolecules, has been shown to peroxidize lipids, oxidize proteins, and promote DNA strand breakage. In fact, much of the [H.sub.2][O.sub.2]-induced alterations in DNA and other organic substrates have been suggested to result from the site-specific metal catalysed production of [OH.sup.*] via the Fenton reaction (Grisham, 1992). Thus, substances that scavenge this reactive oxygen species can modulate the inflammatory process.

We evaluated the [O.sub.2.sup.*-] scavenger ability of C. celtidifolius in the phenazine-methosuiphate system. The scavenging capacity of the CE, fractions, sub-fractions and isolated compounds was only evident at the concentration of 100 [micro]g. [mL.sup.-1]. In a study employing a non-enzymatic system to generate superoxide radical, Hanasaki and colleagues (1994), showed that flavonoids including epicatechin, miricetin, rutin, catechin, epigalocatechin, quercetin, galangin and morin were able to scavenge [O.sub.2.sup.*-]. Since the preliminary phytochemistry study of the C. celtidifolius, disclosed the presence of a great amount of flavonoids in CE and fractions, our results towards to the described in literature.

Free radical scavenging is the principal mechanism of the antioxidant function of flavonoids, due to their capacity to donate electrons (Rice-Evans et al., 1996). In this study, the CE, fractions, sub-fractions and isolated compounds were able to inhibit deoxyribose degradation in a concentration-dependent manner; the majority were effective at concentrations as low as 1 [micro]g*[ml.sup.-1] This result could suggest that these substances possess antioxidant properties. Several flavonoids and other phenolic compounds are considered to be antioxidants not only because of their free radical-scavenging activity (Cotelle et al., 1996; Cops et al., 1998), but also because they chelate metals, contributing to increased antioxidant capacity (Rice-Evans et al., 1996; Moini et al., 1999). The effect of CE, fractions and isolated compounds could be either due to scavenging activity properties or their ability to chelate iron. Chelation of catalytic metal ions may prevent their involvement in Fenton-type reactions, whic h can generate highly reactive hydroxyl radicals (Croft, 1998).

In the lipidic system our results demonstrated that the CE, EAF, BuF, 19SF, 35SF, catechin and gallocatechin decreased the oxidation of rat liver homogenate induced by t-butyl-hydroperoxide. Some reports have shown that the inhibitory effect of the phenolic compounds on lipidic system (LDL oxidation) is time-dependent on sample concentration and the pro-oxidant used (Yamanaha et al., 1997; Bourne and Rice-Evans, 1997; Croft, 1998; Kondo et al., 1999). Presumably flavonoids can reduce [Cu.sup.2+] to [Cu.sup.+] and hence allow the formation of initial radicals (Cao et al., 1997). Chemically, the lipidic system used in our experiments is considered biphasic, where there is an aqueous phase and a lipidic phase. Thus compounds with the most hydroxyl groups apparently exert the greatest antioxidant activity in aqueous phase (Castelluccio et al., 1995; RiceEvans and Miler, 1996). These data corroborate the lack of effect observed for AqF in the inhibition of rat liver homogenate oxidation.

The results show that EAF, BuF and 35 SF have significant antioxidant and anti-inflammatory activity. On the other hand, AqF and 63 SF showed an anti-inflammatory activity more evident than antioxidant activity. According to Winyard and Blake (1997), many antioxidant compounds with anti-inflammatory properties upon inflammatory cellular messenger systems, inhibiting activation of NF[kappa]B, thus directly advise free radical-mediated damage to biomolecules.

Our hypothesis is that the anti-inflammatory effect of Croton celtidifolius, results from the direct action of compounds from CE, BuF, AqF, and proanthocianidins from the EAF, on specific molecular targets, such as cyclooxygenase. A recent report showed that the mechanism of action for the reduction of edema by proanthocianidin isolated from Polypodium feei, might be due to the inhibition of prostaglandin biosynthesis (Subarnas and Wagner, 2000), but also that the presence of antioxidant compounds can modulate the inflammatory process by reduction of reactive oxygen species. A similar synergic effect between the compounds may be responsible for the anti-inflammatory action of Croton celtidifolius.

In summary, the results presented here indicate that Croton celtidifolius bark has significant anti-inflammatory and antioxidant activity. Further studies are required to identify the other compounds present in the bark extract and fractions.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Table 1

Anti-inflammatory effect of the oral (p.o.) administration of crude
extract (CE) of Croton celtidifolius in the mouse paw edema model.

                       Edema induced by carrageenan (mean [+ or
                       -] S.E.M.) [10.sup.-3] (ml)
Samples   Dose mg .
          [kg.sup.-1]  30 min               60 min

Control   p.o.         46.7 [+ or -] 6.1    50.0 [+ or -] 5.2

CE        10           32.0 [+ or -] 6.6    50.0 [+ or -] 3.2
          30           34.0 [+ or -] 6.8    50.0 [+ or -] 7.1
          100          25.0 [+ or -] 6.6    42.5 [+ or -] 6.3
          300          26.0 [+ or -] 2.4 *  32.0 [+ or -] 6.6

          Edema induced by carrageenan (mean [+ or -]
          S.E.M.) [10.sup.-3] (ml)
Samples
          120 min                  240 min

Control   78.3 [+ or -] 6.5        78.3 [+ or -] 4.0

CE        64.0 [+ or -] 10         72.0 [+ or -] 10
          72.0 [+ or -] 4.9        74.0 [+ or -] 5.1
          57.5 [+ or -] 2.5 *      55,0 [+ or -] 2.9 *
          56.0 [+ or -] 2.4 *      44.0 [+ or -] 6.8 **


Samples   Edema Inhibition
          (%) #

Control   0.0

CE        13.7
          8.1
          27.7
          35.6

Values are expressed as mean [+ or -] S.E.M. Statistically. significant
differences between groups were measured using ANOVA with post hoc
comparison by Dunett's test.

* p < 0.05

**  p < 0.01, n = 6-10 animals.

# The area under the curve determined the edema inhibition.

Table 2

Anti-inflammatory effect of the oral (p.o.) and intraperitoneal (i.p.)
administration of Indomethacin (INDO) and Ethyl Acetate fraction (EAF)
of Croton celtidifolius in the mouse paw edema model.

                       Edema induced by carrageenan (mean [+ or -]
                       S.E.M.) [10.sup.-3] (ml)
Samples
          Does mg .
          [kg.sup.-1]  30 min                60 min

Control   (i.p.)       34.3 [+ or -] 4.8     52.0 [+ or -] 6.1
INDO        5          18.3 [+ or -] 1.7 **  25.0 [+ or -] 5.6 **

EAF         3          30.0 [+ or -] 4.5     68.0 [+ or -] 10.1
           10          23.3 [+ or -] 2.1     36.6 [+ or -] 3.3
           30          21.0 [+ or -] 4.0     28.3 [+ or -] 4.2 *
          100          10.0 [+ or -] 0.5 **  20.0 [+ or -] 3.2 **

Control   (p.o.)       50.0 [+ or -] 5.5     54.4 [+ or -] 5.3
INDO       15          21.7 [+ or -] 4.0 **  28.3 [+ or -] 4.0 **

EAF        10          40.0 [+ or -] 4.5     48.0 [+ or -] 6.6
           30          30.0 [+ or -] 7.7     45.0 [+ or -] 3.4
          100          31.0 [+ or -] 5.4     36.7 [+ or -] 4.9
          300          23.3 [+ or -] 4.2 **  26.7 [+ or -] 3.3 **

          Edema induced by carrageenan (mean [+ or -]
          S.E.M.) [10.sup.-3] (ml)
Samples                                               Edema
                                                      Inhibition
          120 min               240 min               (%) #

Control   61.4 [+ or -] 3.4     94.2 [+ or -] 7.2      0.0
INDO      26.7 [+ or -] 7.6 **  18.3 [+ or -] 7.O **  64.7

EAF       50.0 [+ or -] 8.2     84.0 [+ or -] 4.0     16.5
          41.0 [+ or -] 4.9 *   66.0 [+ or -] 4.5 **  31.1
          23.3 [+ or -] 4.2 **  46.6 [+ or -] 4.5 **  52.7
          12.0 [+ or -] 2.0 **  28.0 [+ or -] 37 **   72.6

Control   74.4 [+ or -] 4.7     76.7 [+ or -] 3.7      0.0
INDO      26.6 [+ or -] 4.9 **    35 [+ or -] 4.3 **  57.86

EAF       64.0 [+ or -] 7.5     78.0 [+ or -] 5.8      9.5
          66.6 [+ or -] 8.8     68.3 [+ or -] 8.7     14.5
          53.3 [+ or -] 3.3 *   53.3 [+ or -] 6.1 *   30.4
          40.0 [+ or -] 6.3 **  51.7 [+ or -] 7.9 **  43.3

Values are expressed as mean [+ or -] S.E.M. Statistically significant
differences between groups were measured using ANOVA with post hoc
comparison by Dunett's test.

* < 0.05

** p < .01, n = 6-10 animals.

# The area under the curve determined the edema inhibition.

Table 3

Anti-inflammatory effect of the oral (p.o.) and intraperitoneal (i.p.)
administration of butanolic fraction (BuF) of Croton celtidifolius in
the mouse paw edema model.

                       Edema induced bycarrageenan (mean [+ or -]
                       S.E.M.) [10.sup.-3] (ml)
Samples   Dose mg .
          [kg.sup.-1]
                       30 min                60 min

Control   p.o.         38.0 [+ or -] 5.0     47.4 [+ or -] 5.0

BuF       10           36.0 [+ or -] 4.3     45.0 [+ or -] 2.2
          30           29.6 [+ or -] 6.0     41.5 [+ or -] 6.2
          100          26.8 [+ or -] 6.4     43,3 [+ or -] 6.1
          300          17.0 [+ or -] 3.0     34.3 [+ or -] 4.1

Control   i.p.         45.5 [+ or -] 3.7     48.3 [+ or -] 3.4

BuF       3            36.7 [+ or -] 3.3     45.0 [+ or -] 7.2
          10           25.6 [+ or -] 5.3 **  28.0 [+ or -] 4.8 **
          30           18.3 [+ or -] 4.8 **  21.7 [+ or -] 3.1 **
          100          11.7 [+ or -] 3.1 **  13.3 [+ or -] 4.2 **

          Edema induced bycarrageenan (mean [+ or -]
          S.E.M.) [10.sup.-3] (ml)
Samples                                               Edema
                                                      Inhibition
          120 min               240 min               (%) #

Control   72.0 [+ or -] 3.3     90.0 [+ or -] 7.1      0.0

BuF       69.0 [+ or -] 3.4     85.0 [+ or -] 6.5      4.71
          63.8 [+ or -] 11      63.8 [+ or -] 3.8 **  18.7
          57.7 [+ or -] 6.2     57.7 [+ or -] 5.1 **  24.6
          37.5 [+ or -] 6.6 **  64.0 [+ or -] 1.8 **  38.6

Control   57.3 [+ or -] 5.1     80.0 [+ or -] 5.8      0.0

BuF       55.5 [+ or -] 5.0     80.0 [+ or -] 7.1      4.12
          30.0 [+ or -] 3.3 **  60.7 [+ or -] 5.3 **  38.1
          21.7 [+ or -] 4.0 **  38.3 [+ or -] 4.0 **  56.8
          16.7 [+ or -] 4.2 **  16.7 [+ or -] 2.1 **  74.3

Values are expressed as mean [+ or -] S.E.M. Statistically significant
differences between groups were measured using ANOVA with post hoc
comparison by Dunett's test.

* p < 0.02

** p < 0.01, n = 6-10

# The area under the curve determined the edema inhibition.

Table 4

Anti-inflammatory effect of the oral (p.o.) and intraperitoneal (i.p.)
administration of aqueous fraction (AqF) of Croton celtidifolius in the
mouse paw edema model.

                       Edema induced by carrageenan (mean [+ or -]
                       S.E.M) [10.sup.-3] (ml)
Samples   Dose mg .
          [kg.sup.-1]
                       30 min               60 min

Control   p.o.         46.9 [+ or -] 4.4    52.9 [+ or -] 4.4

AqF         10         50.0 [+ or -] 3.7    51.7 [+ or -] 6.0
           30          40.0 [+ or -] 3.7    48.3 [+ or -] 4.8
          100          38.3 [+ or -] 6.0    38.3 [+ or -] 7.0
          300          33.3 [+ or -] 6.1    36.7 [+ or -] 4.2

Controle  i.p.         46.9 [+ or -] 4.4    52.9 [+ or -] 4.4

AqF         3          34.0 [+ or -] 2.4    45.0 [+ or -] 7.2
           10          36.7 [+ or -] 1.6    45.0 [+ or -] 5.5
           30          23.3 [+ or -] 5.0**  23.3 [+ or -] 4.9 **
          100          15.0 [+ or -] 3.4**  15.0 [+ or -] 3.4 **

          Edema induced by carrageenan (mean [+ or -]
          S.E.M) [10.sup.-3] (ml)
Samples                                                Edema
                                                       Inhibition
          120 min                240 min               (%) #

Control   71.4 [+ or -] 4.0      99.2 [+ or -] 4.6      0.0

AqF       70.0 [+ or -] 4.5      83.4 [+ or -] 6.1     7.83
          63.3 [+ or -] 6.1      68.3 [+ or -] 6.0 **  19.6
          58.3 [+ or -] 8.7      55.0 [+ or -] 8.5 **  30.1
          40.0 [+ or -] 3.7 **   38.3 [+ or -] 6.0 **  47.5

Controle  71.4 [+ or -] 4.0      99.2 [+ or -] 4.6      0.0

AqF       58.3 [+ or -] 4.0      72.0 [+ or -] 7.3     22.2
          48.3 [+ or -] 7.5      50.0 [+ or -] 3.7 **  35.2
          20.0 [+ or -] 3.7 **   46.7 [+ or -] 8.0 **  60.4
          28.0 [+ or -] 2.0 **   43.3 [+ or -] 3.3 **  61.0

Values are expressed as mean [+ or -] S.E.M. Statistically significant
differences between groups were measured using ANOVA with post hoc
comparison by Dunett's test.

* p < 0.05

** p < 0.01, n = 6-10 animals.

# The area under the curve determined the edema inhibition.

Table 5

Anti-inflammatory effect of the intraperitoneal (i.p.) injection of
Catechin and sub-fractions 35SF and 63SF of Croton celtidifolius, in the
mouse paw edema model.


                           Edema induced by carrageenan (mean [+ or -]
                           S.E.M) [10.sup.-3] (ml)
Samples       Dose mg.
              [kg.sup.-1]  30 min                60 min

Control                    48.7 [+ or -] 4.1     60.0 [+ or -] 3.9
Galocatechin  30           44.0 [+ or -] 6.8     64.0 [+ or -] 4.0
19SF          30           31.7 [+ or -] 3.1     45.0 [+ or -] 2.2
Catechin      30           40.0 [+ or -] 3.7     35.5 [+ or -] 2.9 **
35SF          30           25.0 [+ or -] 1.9 **  32.5 [+ or -] 4.1 **
63SF          30           13.7 [+ or -] 3.2 **  27.5 [+ or -] 2.5 **


              Edema induced by carrageenan (mean [+ or -]
              S.E.M) [10.sup.-3] (ml)
Samples
              120 min               240 min

Control       82.0 [+ or -] 3.9     78.7 [+ or -] 2.6
Galocatechin  62.0 [+ or -] 4.9     82.0 [+ or -] 9.1
19SF          65.0 [+ or -] 5.0     81.7 [+ or -] 4.8
Catechin      50.0 [+ or -] 4.7 **  70.0 [+ or -] 3.3
35SF          31 2 [+ or -] 4.4 **  33.7 [+ or -] 3.2 **
63SF          31.3 [+ or -] 3.9 **  33.8 [+ or -] 3.7 **


              Edema Inhibition
Samples       (%) #


Control        0.0
Galocatechin   9.91
19SF          15.8
Catechin      28.9
35SF          56.3
63SF          59.9

Values are expressed as mean [+ or -] S.E.M. Statistically significant
differences between groups were measured using ANOVA with post hoc
comparison by Dunett's test.

* p < 0.05

** p < 0.01, n = 6-10 animals.

# The area under the curve determined the edema inhibition.

Table 6

Comparison of IC50 values, from inhibition of deoxyribose degradation of
crude extract, fractions sub-fractions and isolated compounds of Croton
celtidifolius.

Deoxyribose assay  IC50 (mg. [ml.sup.-1])  Maximal
                                           Inhibition (%)

Mannitol *         -                       85.62 [+ or -] 3.52
Crude Extract      4.32 (3.03-6.15)        81.3 [+ or -] 2.91
EAF                0.69 (0.44-1.06)        86.0 [+ or -] 12.8
BuF                1.45 (0.73-2.89)        81.0 [+ or -] 2.47
AqF                5.07 (3.15-8.11)        81.2 [+ or -] 6.21
Catechin           0.20 (0.11-0.39)        87.0 [+ or -] 7.70
Gallocatechin      0.55 (0.28-1.08)        77.0 [+ or -] 1.94
19 SF              3.09 (1.78-5.37)        83.3 [+ or -] 11.5
35SF               1.11 (0.48-2.57)        82.0 [+ or -] 4.56
63SF               5.53 (3.05-8.72)        82.5 [+ or -] 3.04

IC50 are presented as geometric means accompanied by 95% confidence
limits. The IC50 values were determined by non-linear regression using
"Graph Pad Prism Software".

* Positive control.

Acknowledgements

This study was supported by CNPq and CAPES - Brazil.

References

Arawal PK (1989) Carbon-13 NMR of Flavonoids. Elsevier, Chapter 6: 432-496

Bhat VB and Madyastha KM (2001) Antioxidant and Radical Scavenging Properties of 8-Oxo Derivatives of Xanthine Drugs Pentoxifylline and Lisofylline. Biochem Biophys Res 288: 1212-1217

Bourne LC, Rice-Evans CA (1997) The effect of the phenolic antioxidant ferulic acid on the oxidation of low density lipoprotein depends on the pro-oxidant used. Free Radical Res 27: 337-344

Cai Y, Evans F, Roberts M, Phillipson JD, Zenk M, Gleba Y (1991) Phenolic compounds from Croton Lechieri. Phytochemistry 30: 2033-2040

Cao G, Sofic E, Prior RE (1997) Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radical Biol Med 22: 749-760

Castelluccio C, Paganga G, Melikian N, Bolwell GP, Pridham J, Sampson J, Rice-Evans CA (1995) Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Lett 368: 188-192

Chen H, Tappel A (1996) Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate. Lipids 31 (1): 47-50

Chen Z, Cai Y, Phillipson JD (1994) Studies on the anti-tumour, anti-bacterial and wound-healing properties of dragon's blood. Planta Med 60: 541-545

Conner EM, Grisham MB (1996) Inflammation, free radicals and antioxidants. Nutrition 12: 274-277

Cops P, Ying L, Calomme M, Hu JP, Cimanga K, Poel BV, Pieters L, Vlietinck AJ, Bergue DV (1998) Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J Nat Prod 61: 71-76

Cotelle N, Bemier JL, Catteau JP, Wallet JC, Gaydan EM (1996) Antioxidant properties of hydroxy-flavones. Free Radical Biol Med 20: 35-43

Croft KD (1998) The chemistry and biological effects of flavonoids and phenolic acids. Ann. N.Y Acad Sci 20: 435-442

Cuzzocrea S, Zingarelli B, Constantino G, Szabo A, Salzman, AL, Caputi AP, Szabo C (1997) Beneficial effects of 3-aminobenzamide, na inhibitor of poly (ADP-ribose) synthetase in the rat model of splanchic artery occlusion and reperfusion. Brit J Pharmacol 121: 1065-1074

Deitch EA, Briges W, Berg R, Specian RD, Granger N (1990) Hemorrhagic Shock-induced Bacterial translocation: The role of neutrophils and hydroxyl radicals. J Traumatol 30: 942-951

Dix TA, Hess KM, Medina MA, Sullivan RW, Tilly SL, Webb TLL (1996) Mechanism of site-selective DNA nicking by the hydrodioxyl (perhydroxyl) radical. Biochemistry 35: 4578-4583

Droy-Lefaix MT, Drouet Y Geraud G, Hosfod D, Braquet P (1991) Superoxide dismutase (SOD) and the PAF-antagonist (BN 52021) reduced small intestinal damage induced by ischemia-reperfusion. Free Radical Res Comm 12: 725-726

Fantone JC, Ward PA (1982) A review: role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 107: 395-418

Grisham MB (1992) Reactive oxygen metabolism. In: Grisham MB., ed. Reactive metabolites of oxygen and nitrogen in biology and medicine. Austin: R. G. Landers Company: 39

Halliwell B, Gutteridge JMC (1981) Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts. FEBS Lett 128: 347-352

Hanasaki Y, Ogawa S, Fukui S (1994) The correlation between active oxygen scavenging and antioxidative effects of flavonoids. Free Radical Biol Med 16: 845-850

Henriques MG, Silva PM, Martins MA, Flores CA, Cunha FQ, Assreuy-Filho, J, Cordeiro RS (1987) Mouse paw edema. A new model for inflammation? Braz J Med Biol Res 20(2): 243-9

Janssen-Heininger YMW, Macara L, Mossman B (1999) Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kB. Am J Resp Cell Mol Biol 20: 942-952

Kondo K, Kurihara M, Miyata N, Suzuki T, Toyoda M (1999) Mechanistic studies of catechins as antioxidants against radical oxidation. Arch Biochem Biophys 362: 79-86

Kuo SM, Leavitt PS, Lin CP (1998) Dietary flavonoids interact with trace element metals and affect metallothionein level in human intestinal cells. Biol Trace Elem Res 62: 135-153

Maciel MAM, Pinto A, Arruda AC, Pamplona SGSR, Vanderlinde FA, Lapa AJ, Echevarria A, Grynberg NF, Colus IMS, Farias RAF, Costa AMI, Rao V (2000) Ethnopharmacology, phytochemistry and pharmacology: a successful combination in the study of Croton cajucara. J Ethnopharmacol 70: 41-55

Moini H, Arroyo A, Vaya J, Packer L (1999) Bioflavonoids effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state. Redox Rep 4: 35-41

Mukherjee R, Axt EM (1984) Cytelitols from Croton celtidifolius. Phytochemistry 23: 2682-2684

Rice-Evans CA, Miler NJ (1996) Antioxidant activities of flavonoids as bioactive components of food. Biochem Soc Trans 24: 790-795

Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol Med 20: 933-956

Robak J, Griglewiski RJ (1988) Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 37: 837-841

Salvemini D, Mazzon E, Dugo I, Riley DP, Serriano L, Caputi AP, Cuzzocrea S (2001) Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403. Brit. J Pharmacol 132: 815-827

Salvemini D, Wang ZQ, Bourdon DM, Stern MK, Currie MG, Manning, PT (1996) Evidence of peroxinitrite involvement in the carrageenan-induced rat paw edema. Eur J Pharmacol 303: 217

Subarnas A, Wagner H (2000) Analgesic and anti-inflammatory activity of the proanthocyanidin shellegueain A from Polypodium feei METT. Phytomedicine 7 (5): 401-405

Vaiberg A, Milla M, Planas M, Cordova J, de Agusti E, Ferreyra R, Mustiga M, Carlin L, Hammond G (1988) Taspine is the cicatrizant in Sangre de Drago extracted from Croton lechleri. Planta Med 55: 140-143

Winyard PG, Blake DR (1997) Antioxidant, redox-regulated transcript factors and inflammation. Adv Pharmacol 18: 403-421

Yamanaha N, Oda O, Nagao S (1997) Green tea catechins such as (-) -epicatechin and (-) epigallocatechin accelerate [Cu.sup.2+]-induced low density lipoprotein oxidation in propagation phase. FEBS Lett 401: 230-234

Zimmermann M (1983) Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16: 109-110

G.M. Nardi, (1) R. Felippi, (1) S. DalBo, (1) J.M. Siqueira-Junior, (1) D.C. Arruda, (1) F. Delle Monache, (2) A.K. Timbola, (3) M.G. Pizzolatti, (3) K. Ckless, (4) R.M. Ribeiro-do-Valle (1)

(1.) Departamento de Farmacologia, CCB, UFCS, Florianopolis, Brazil

(2.) Lnstituto di Biochimica e Biochimica Clinica, CNR, Universitd Cattolica Del Sacro Cuore, Rome, Italy

(3.) Departamento de Quimica, CFM, UFSC, Plorian6polis, SC, Brazil

(4.) Departamento de Bioquimica, CCB, UFCS. Florian6polis, Brazil. [Present address: Department of Pathology, University of Vermont, HRSF Building, Burlington, USA]

Address

R. M. Ribeiro-do-Valle, PhD, Departamento de Farmacologia, CCB, UFSC, Rua Ferreira Lima, n[degrees] 82, CEP: 880 10-970 - Florianopolis - SC, Brazil

Tel.: ++55-48-33 1-9491; Fax: ++55-48-331-9191; e-mail: [e-mail]