Anticlastogenic activity exhibited by botryosphaeran, a new exopolysaccharide produced by Botryosphaeria rhodina MAMB-05

International Journal of Biological Macromolecules 42 (2008) 172–177

Anticlastogenic activity exhibited by botryosphaeran, a new exopolysaccharide produced by Botryosphaeria rhodina MAMB-05 Carolina C.B.O. Miranda a , Robert F.H. Dekker a,b , Juliana M. Serpeloni c , Eveline A.I. Fonseca a , Ilce M.S. C´olus c , Aneli M. Barbosa a,∗ a

Dept◦ de Bioqu´ımica e Biotecnologia – CCE, Universidade Estadual de Londrina, C.P. 6001, C.E.P. 86051-990, Londrina-PR, Brazil b Universidad de Castilla - La Mancha, Instituto de Regional Investigaci´ on Cient´ıfica Aplicada (IRICA), 13071 Cuidad Real, Spain c Dept◦ de Biologia Geral – CCB, Universidade Estadual de Londrina, C.P. 6001, C.E.P. 86051-990, Londrina-PR, Brazil Received 28 August 2007; received in revised form 8 October 2007; accepted 11 October 2007 Available online 18 October 2007

Abstract Biopolymers such as exopolysaccharides (EPS) are produced by microbial species and possess unusual properties known to modify biological responses, among them are antimutagenicity and immunomodulation. Botryosphaeran, a newly described fungal (1 → 3; 1 → 6)-␤-d-glucan produced by Botryosphaeria rhodina MAMB-05, was administered by gavage to mice at three doses (7.5, 15 and 30 mg/kg b.w.per day) over 15 days, and found to be non-genotoxic by the micronucleus test in peripheral blood and bone marrow. Botryosphaeran administered at doses of 15 and 30 mg EPS/kg b.w. decreased significantly (p < 0.001) the clastogenic effect of cyclophosphamide-induced micronucleus formation resulting in a reduction of the frequency of micronucleated cells of 78 and 82% in polychromatic erythrocytes of bone marrow, and reticulocytes in peripheral blood, respectively. The protective effect was dose-dependent, and strong anticlastogenic activity was exerted at low EPS doses. Variance analysis (ANOVA) showed no significant differences (p < 0.05) among the median body weights of the groups of mice treated with botryosphaeran during experiments evaluating genotoxic and protective activities of botryosphaeran. This is the first report on the biological activity attributed to botryosphaeran. © 2007 Elsevier B.V. All rights reserved. Keywords: Botryosphaeran; Botryosphaeria rhodina MAMB-05; Cyclophosphamide; Mouse; Anticlastogenicity; Micronucleus test

1. Introduction Exopolysaccharides (EPS) are carbohydrate macromolecules secreted extracellularly by several microorganisms including fungi. The biological activities attributed to EPS actively studied over the years include: host resistance to bacterial, viral, fungal and parasitic infections; inhibition of tumour growth and prevention of carcinogenesis; as a coadjutant in radiotherapy; and the immunomodulatory effects that increase the phagocytic and proliferative activity of the reticuloendothelial system among others [1,2]. Despite the wealth of knowledge regarding the effects of EPS and their therapeutic potential, the molecular mechanisms and cellular receptors mediating these responses are not well understood [3].

∗

Corresponding author. Tel.: +55 43 3371 4270; fax: +55 43 3371 4054. E-mail address: [email protected] (A.M. Barbosa).

0141-8130/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2007.10.010

The most studied fungal EPS are those produced by the basidiomycetes, mostly ligninolytic, and identified as non-cellulosic ␤-glucans with various linkages, but chiefly of the (1 → 3)-␤glucosidic type [4]. Besides participating in the composition of the fungal cell wall, ␤-glucans are frequently excreted into the culture media when cultured in liquid medium. However, the functions of ␤-glucans are not well understood in fungal metabolism. Studies on ligninolytic basidiomycetes indicate that the ␤-glucans often constitute a sheath around the fungal hyphae that protects against desiccation and free radical damage by reactive oxygen species. They are further thought to play a role in lignin degradation by providing an indirect source for hydrogen peroxide through extracellular glucose generated and regulated through the specific enzymatic hydrolysis of the ␤-glucan itself, and can also act as an immobilizing matrix for secreted enzymes [5]. There are no known reports indicating toxicity of fungal EPS of the ␤-glucan type.

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Actually, ␤-glucans extracted from Agaricus brasiliensis [6] and Saccharomyces cerevisiae [7], besides not exhibiting genotoxic effects in mammalian cells in vitro, were protective against damage caused by different mutagens. Botryosphaeria rhodina MAMB-05 is an endophytic ascomyceteous fungus isolated from a eucalypt stem canker [8] and ligninolytic [9]. Isolate MAMB-05 produces an EPS described as a ␤-d-glucan [10], and structurally characterized as a (1 → 3; 1 → 6)-␤-d-glucan named botryosphaeran [11]. Botryosphaeran consists of a main chain comprising glucose residues linked through ␤(1 → 3) bonds with 22% ramification consisting of branches of ␤-1,6-linked glucose and gentiobiose residues [11]. Until now, only studies on the microbial physiology of botryosphaeran production [12], and some structural characteristics of the botryosphaerans produced by B. rhodina when grown on different carbohydrate substrates, have been reported [12,13]. It is important that when assessing new compounds for potential biomedical applications, they first be screened for mutagenic activity, as this property reflects if DNA damage occurred [14]. The objective of this work, therefore, was to evaluate the genotoxic activity in vivo of botryosphaeran and its effect on the clastogenicity induced by cyclophosphamide (CP) in mice using the micronucleus test (MNT) in bone marrow and peripheral blood cells. Micronuclei (MN) are small, extra-nuclear bodies that arise in dividing cells from accentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase [15]. Thus, the use of MNT indicates possible clastogenic or aneugenic effects of botryosphaeran, and this will establish its biotechnological applications as a complementary and alternative medicine. Therefore, we report herein for the first time the biological effects of botryosphaeran in relation to its protective effect of cyclophosphamide-induced clastogenicity in mice. 2. Materials and methods 2.1. Microorganism and growth conditions Botryosphaeria rhodina isolate MAMB-05 was cultivated according to Barbosa et al. [9]. The fungus was maintained on potato agar dextrose slants at 4 ◦ C with successive transfers each 3 months. Pre-inoculum was prepared from fungal mycelial mats on agar plates (Vogel minimal salts medium (VMSM; [16]), 20 g/L agar and 10 g/L glucose), and transferred to liquid medium (VMSM and 5 g/L glucose) as previously described [11,12]. Fungal cultures were grown in Erlenmeyer flasks containing VMSM and glucose (50 g/L), and left for 72 h at 28 ◦ C in an orbital shaker (180 rpm). 2.2. Production of botryosphaeran Following cultivation, flasks were harvested by centrifugation (1250 × g/15 min) to remove mycelium, and the supernatant collected and dialysed (48 h) against several changes of deionised water. The dialysate was treated with three volumes

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of absolute ethanol and left at 4 ◦ C for 24 h, and the resulting precipitated material (botryosphaeran) filtered off, re-dissolved in water, and dialysed against de-ionised water (48 h). The dialysate was lyophilized and the dried material (EPS) was stored at −20 ◦ C. 2.3. Preparation of botryosphaeran solutions and analyses Stock solutions of botryosphaeran for biological assays were prepared of the following concentrations: 0.75, 1.5 and 3.0 g/L in isotonic saline solution and autoclaved at 121 ◦ C for 20 min. Aliquots of each EPS solution were reserved for the determination of reducing sugars and total sugars by the methods of Somogyi [17], and Dubois et al. [18], respectively, to confirm the concentrations used during the animal experiments. 2.4. Treatment with cyclophosphamide Cyclophosphamide (CP, Sigma) was used to induce clastogenicity, and was administered at a final dose of 50 mg/kg of mouse body weight (b.w.). CP was diluted in de-ionised water and administered once only to the animals through intraperitoneal injection (i.p.). 2.5. Experimental design for genotoxic and protective activities Swiss adult mice weighing 27–32 g were obtained from the mouse breeding colony at Universidade Estadual de Londrina, and maintained individually in polyethylene cages under controlled conditions [temperature (23 ± 2 ◦ C), relative humidity (50 ± 10%), a 12-h light–dark cycle, ad-libitum access to a rodent diet (Nuvilab, Apucarana-PR, Brazil) and water]. The animals were randomized into eight groups each of 10 mice (5 males and 5 females). The first and the second groups were used respectively, as the negative and positive controls, with each animal of the first group receiving a placebo comprising only isotonic saline solution as treatment. On the final day of treatment, the positive control group received CP (50 mg/kg) by i.p. to induce micronucleus formation. Animals of groups 3–5 each received equal doses of EPS administered by gavage at three different doses: 7.5, 15 and 30 mg botryosphaeran/kg b.w. per day over 15 days for the analysis of genotoxic activity of botryosphaeran. Doses of EPS chosen were based on its solubility limit of 3 g/L in isotonic saline solution. In groups 6–8, botryosphaeran was administered by gavage over 15 days at the same doses as groups 3–5, but on day 15 of treatment received an intraperitoneal injection of CP (50 mg/kg). The CP injection was applied 1 h after the animals were treated with the EPS solutions, thus simulating a simultaneous treatment in the anticlastogenicity assay. Animals of all the groups were weighed at the beginning and at the end of each treatment to evaluate possible variations in body weight caused by the administration of botryosphaeran, and to evaluate if the EPS was capable of serving as an energy source. All animals were euthanised 30 h after the 15th day of

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treatment, and peripheral blood was taken and bone marrow collected for the micronucleus tests.

ulocytes (MNRETs) analyzed in 1000 reticulocytes (RETs) was recorded for each animal.

2.6. Micronucleus test

2.7. Calculation for reduction of micronucleus frequency

2.6.1. Micronucleus test in bone marrow cells Genotoxic effects were evaluated by the micronucleus test in mouse bone marrow cells according to the protocol of Schmid [19]. After the animals were euthanised, both femurs were immediately removed and the bone marrow washed out with 1 mL of fetal bovine serum into centrifuge tubes containing 1 mL of fetal bovine serum. The cellular suspension was mixed and centrifuged at 350 × g/10 min, and the supernatant discarded. The pellet (bone marrow cells) was mixed with 0.5 mL of fetal bovine serum and a small aliquot applied to a glass slide and smeared. After the smears dried (24 h at ambient temperature), they were fixed in methanol for 10 min and stained with 5% (w/v) Giemsa diluted in 0.06 M phosphate buffer (Na2 HPO4 and KH2 PO4 ) pH 6.8 for 8 min. Two slides were prepared for each animal with material from two femurs and used for scoring. From each slide, 1000 polychromatic erythrocytes (PCEs) were scored for the presence of micronuclei under oil immersion at 100× magnification using a binocular light microscope [20,21], and the number of micronucleated polychromatic erythrocytes (MNPCEs) in 2000 PCEs recorded for each animal.

The reduction in frequency of MN after treatment with botryosphaeran was calculated according to Manoharan and Banerjee [23] and Waters et al. [24] from the equation:

2.6.2. Micronucleus test in peripheral blood The micronucleus test in mouse peripheral blood was conducted using slides stained with acridine orange according to Hayashi et al. [22]. Before euthanasia, 5 ␮L of venous blood was taken from the tail of each animal and placed on a set of slides with cover slips. Once dry, the slides were kept in the dark at ambient temperature for at least 24 h and then maintained at −20 ◦ C prior to analysis. Cytological analyses were evaluated using a fluorescent microscope using a combination of a blue excitation (488 nm) and a yellow-to-orange (515 nm) barrier filter (100× oil-immersion objective). The number of micronucleated retic-

reduction (%)   frequency of MN in A − frequency of MN in B × 100 = frequency of MN in A − frequency of MN in C where A is the group treated with CP (positive control), B the group treated with botryosphaeran solutions plus CP, and C is the group treated with isotonic saline (negative control). 2.8. Statistical analysis Data of median body weights, MN frequencies in bone marrow PCEs, and in peripheral blood RETs obtained for males and females in each treatment group were compared by the Student’ t-test (p < 0.05). The frequencies of micronucleated PCEs and RETs and median body weights were calculated for each group treatment. All results are expressed as the mean ± S.D. Data from damage-inducing and anticlastogenic activities, and median body weights were analyzed by one-way variance analysis (ANOVA), and significant differences (p < 0.05) between means determined by Tukey’s test [25]. 3. Results Results on the analysis of micronucleus frequency in RETs of peripheral blood and PCEs of bone marrow of mice treated with botryosphaeran for 15 days, and their respective control groups, are presented in Tables 1 and 2, respectively. The data indicated that botryosphaeran administered by gavage at the three doses examined was not capable of inducing micronucleus formation

Table 1 Frequency of micronucleated reticulocytes (MNRETs) in peripheral blood of mice treated orally with different doses of botryosphaeran, and botryosphaeran plus cyclophosphamide (with respective percentage of reduction) after sub-chronic treatment for 15 days. Values showing frequency and reduction of frequency are based on the combined averages of the values for males and females Treatment (mg EPS/kg b.w. per day)

Number of RETs analyzed

Number of RETs with MN/1000 RETs (mean ± S.D.)

Isotonic saline

9,000

Male 2.4 ± 0.70

Female 2.5 ± 0.65

Botryosphaeran 7.5 15 30

10,000 10,000 10,000

2.8 ± 0.45 2.2 ± 0.63 2.2 ± 0.73

Cyclophosphamide (50 mg/kg b.w.)

10,000

Botryosphaeran + cyclophosphamide 7.5 15 30

10,000 10,000 8,000

a, b, c and d are statistically different; * p < 0.05; ** p < 0.001.

Frequency (%) 0.24a

Reduction in frequency (%) –

2.6 ± 0.42 2.2 ± 0.55 2.2 ± 0.75

0.27 0.22 0.22

– – –

22.2 ± 1.48

22.4 ± 1.47

2.23b

–

13.6 ± 1.45 6.8 ± 1.81 5.75 ± 0.94

13.2 ± 1.42 6.8 ± 1.12 6.25 ± 0.76

1.34c,* 0.68d,** 0.60d,**

45 78 82

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Table 2 Frequency of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow of mice treated orally with different doses of botryosphaeran and botryosphaeran plus cyclophosphamide (with respective percentage of reduction) after sub-chronic treatment for 15 days Treatment (mg EPS/kg b.w. per day)

Number of PCEs analyzed

Number of PCEs with MN/2000 PCEs (mean ± S.D.)

Isotonic saline

18,000

Male 1.4 ± 0.29

Female 1.5 ± 0.27

Botryosphaeran 7.5 15 30

20,000 20,000 –

2.0 ± 0.37 1.2 ± 0.23 1.2 ± 0.33

2.2 ± 0.39 1.4 ± 0.25 1.0 ± 0.29

0.10 0.07 0.06

– – –

Cyclophosphamide (50 mg/kg b.w.)

20,000

43.6 ± 1.27

42.4 ± 1.32

2.15b

–

Botryosphaeran + cyclophosphamide 7.5 15 30

20,000 20,000 16,000

24.4 ± 0.98 13.6 ± 0.77 10.8 ± 0.46

24.2 ± 0.86 14.0 ± 0.85 11.0 ± 0.38

1.21c,* 0.69d,** 0.54d,**

Frequency (%) 0.07a

Reduction in frequency (%) –

45 71 78

a, b, c and d are statistically different; * p < 0.05); ** p < 0.001.

neither in peripheral blood RETs nor in bone marrow PCEs of the mice, because the data did not present any statistical differences compared with the negative control group. Furthermore, there was no statistical difference observed between the results obtained for males and females (Student’ t-test), demonstrating that gender differences did not modify activity of the EPS in the animals or their body weights. Variance analysis showed no significant differences among the median body weight gains of the groups of mice treated with EPS and the negative control group (p < 0.05) during experiments evaluating genotoxic activity of botryosphaeran (data not shown). The MN frequencies in peripheral blood RETs and bone marrow PCEs of mice receiving botryosphaeran plus CP, and their respective control groups, are shown in Tables 1 and 2, respectively. The results demonstrate the anticlastogenic potential of botryosphaeran at the three doses examined. Data in Table 1 confirmed the anticlastogenic potential of botryosphaeran in peripheral blood RETs at the three EPS doses presented. The statistical differences were significant for all doses of EPS in relation to the positive control group (p < 0.05) for EPS 7.5 mg/kg b.w. per day, and (p < 0.001) for EPS 15 and 30 mg/kg b.w. per day. Botryosphaeran at doses 15 and 30 mg/kg b.w. per day presented statistical differences in relation to the dose at 7.5 mg/kg b.w. per day. The smallest dose presented statistical differences among the other two doses (15 and 30 mg/kg b.w. per day). Reductions in the MN frequency of 45, 78 and 82%, respectively, were observed for botryosphaeran at doses of 7.5, 15 and 30 mg EPS/kg b.w./day (Table 1). The MNPCEs frequencies presented significant statistical differences at all dose levels examined in relation to the positive control group (p < 0.05) for 7.5 mg EPS/kg b.w. per day, and (p < 0.001) for 15 and 30 mg EPS/kg b.w. per day. The smallest dose presented statistical differences among the other two doses (15 and 30 mg/kg b.w. per day). Reductions in the MN frequency of 45, 71 and 78%, respectively, were observed for botryosphaeran doses of 7.5, 15 and 30 mg EPS/kg b.w. per day (Table 2).

Variance analysis again showed that there was no statistical difference in the median body weight gains of the groups of mice treated with EPS and the positive control group (p < 0.05) in evaluating the anticlastogenic activity of botryosphaeran (data not shown). 4. Discussion Many microbial polysaccharides have been reported to present biological activities. Some in their in situ form as in mushrooms have been used in traditional Chinese and Japanese medicines for centuries to treat human diseases [4]. The mechanism of action of these carbohydrate macromolecules, however, is not clearly understood in spite of the knowledge of their activities as biological response modifiers [26]. The protective activity of these polysaccharides is mediated through stimulation of the immune system, possibly due to immunological host-mediated mechanisms involving the action of various immuno-competent cells [4]. It is thought that ␤-glucans exert their effects by activating cells of the immune system through binding to surface receptors; for example, the C-type lectin-like Dectin-1 receptor on leukocytes recognizes ␤(1 → 3)- and ␤(1 → 6)- linked glucans [3]. Dectin-1 is expressed on phagocytic cells, including macrophages and neutrophils, and mediates the internalization (signal transduction) and cellular responses to ␤-glucans through unique mechanisms [27]. The present study demonstrated that botryosphaeran from B. rhodina MAMB-05 did not present clastogenic or aneugenic activity in vivo in mice after administration by gavage over a 15 day period. These findings were in agreement with those reported for other fungal EPS including the ␤-1,3-glucans, schizophyllan and scleroglucan [28], and polysaccharides extracted from mushrooms [29,30]. It is important to mention that most of the polysaccharides possessing biological activities reported in the literature were used as crude extracts and were not purified beforehand. Many of these polysaccharides were obtained from sources such as mushrooms that contain ␤-glucans [31–33] with similar structural features to

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botryosphaeran. Some studies have also involved isolated glucans from the cell wall of yeasts, S. cerevisiae [34] and Candida albicans [35], but few studies have reported the EPS from submerged fermentation [36,37], as is the case with botryosphaeran. Cyclophosphamide, an alkylating agent used in chemotherapy to treat cancers [38], induces chromosomal damage in bone marrow and peripheral blood cells. Consequently, CP has been used as a cytostatic agent to induce cellular damage in the micronucleus test for evaluating mutagenic and antimutagenic activities [38]. The micronuclei originate from chromosome fragments or chromosome loss events during mitotic or meiotic division. Accordingly, the evaluation of micronucleus frequencies in vivo is one of the primary genotoxicity tests [39] recommended internationally by regulatory agencies for product safety assessment The increase in the frequency of micronucleated polychromatic erythrocytes or micronucleated reticulocytes in treated mice is an indication of induced chromosome damages [39,40]. The role of microbial polysaccharides in preventing mutagenesis in CP-induced clastogenicity is thought to be due to their scavenging of the highly reactive hydroxyl groups on metabolites of CP, and this therefore represents one of the most promising approaches in antimutagenesis and anticarcinogenesis [41–43]. Botryosphaeran exercised strong potential protective action in relation to the clastogenicity of CP resulting in the reduction of frequencies of 78 and 82% of micronucleated cells in the RETs of peripheral blood, and 71 and 78% of micronucleated PCEs of bone marrow cells when administered at doses of 15 and 30 mg/kg b.w. per day, respectively. In comparing the reduction in MN frequency by botryosphaeran with literature reports of other fungal ␤-glucans derived by fermentation with similar composition and structure (e.g., at doses of 100 mg EPS/kg b.w., ␤-glucans from S. cerevisiae [34], Aspergillus niger [44] and Grifola frondosa [45] resulted in maximum reductions of between 44 and 58%), it is obvious that the results obtained with botryosphaeran are very significant, and interestingly occurred at much lower EPS doses. The antimutagenic activity of pestalotan, an extracted EPS from the fungus Pestalotia sp., has been evaluated in vivo [37]. In its native form, and where the side branching chains were chemically modified, pestalotan was not mutagenic and presented antimutagenic and antitumour activities in mice. The chemical structure of pestalotan is not dissimilar to botryosphaeran, although its degree of branching was somewhat higher (37% compared to 22% for botryosphaeran [11]). The method of administrating EPS by gavage presents advantages over other administrative routes reducing adverse effects such as formation of granulomas, microembolisation and inflammation, but also promotes a more comfortable, less-distressing situation for the animal [46]. A major obstacle to clinical utilisation of ␤-glucans is their relatively high molecular weight, and in general, poor solubility in aqueous solutions. This can lead to their inactivity when administered orally because of reduced amounts of EPS in blood circulation [34]. To overcome this drawback, ultrasonication is often necessary to solubilise EPS [36], or water-soluble derivatives of EPS are prepared by derivatisation through carboxymethylation or sulfation [36,47].

It is unlikely that botryosphaeran, through its complex structure, acts as an energy source for the mouse, as its digestive system would need to produce enzymes specifically hydrolysing the ␤(1 → 3)- and ␤(1 → 6)- glucosidic bonds to digest this polysaccharide. As far as the authors are aware, enzymes of this kind are unknown in animal systems (http://www.brenda.unikoeln.de/). A mutagenic agent, however, can weaken an animal promoting cachexia and result in weight loss. Therefore, an evaluation in body weight changes of the animals during a study for genotoxic and protective activities is an important parameter. The work reported herein demonstrated no significant statistical differences in the experimental treatments assessing chromosome damage and anticlastogenic activities in relation to the body weight of the animals, thus concluding botryosphaeran did not serve as an energy source. These results were furthermore corroborated by the findings of other studies evaluating antimutagenic activity (e.g., mushroom of Lentinula edodes [48] incorporated into diets fed mice likewise did not influence body weight gains and reduced the frequency of MNPCEs induced by N-ethyl-N-nitrosourea), and antitumour activity (e.g., ␤-glucans extracted from the cell wall of C. albicans administered to mice infected with P815 mastocytoma cells resulted in suppression of tumour growth, and enhanced host-defense response to the tumour [35]). 5. Concluding remarks We have described for the first time the biological activity exercised by the exopolysaccharide botryosphaeran from B. rhodina MAMB-05, and demonstrated this EPS was incapable of producing chromosomal damage, i.e., it was not mutagenic. Furthermore, botryosphaeran exhibited anticlastogenic activity against the in vivo DNA-damaging effect of CP leading to a reduction of cyclophosphamide-induced MN frequencies in bone marrow and peripheral blood of mice. Botryosphaeran thus possesses some promising biological activities as demonstrated by this work, and this will open new perspectives for its biological activities and applications. Studies in progress in our laboratory have indicated that botryosphaeran is effective in promoting hypoglycaemia and hypocholesterolaemia in rats (unpublished data), and when sulfated exhibited strong anticoagulant activity (unpublished data). Acknowledgements The authors are grateful to CAPES (Brazil), and Fundac¸a˜ o Arauc´aria do Paran´a (Brazil) for financial support (Project No. 5777). C.C.B.O. Miranda thanks CAPES for a Master’s scholarship, E.A.I. Fonseca and J.M. Serpeloni thank PIBIC/CNPq/UEL for scientific initiation scholarships, and R.F.H. Dekker acknowledges CNPq (Brazil) for a Fellowship as senior visiting research professor. References [1] Y.H. Shon, K.S. Nam, J. Ethnopharmacol. 77 (2001) 103–109. [2] G.D. Brown, S. Gordon, Immunity 19 (2003) 311–315.

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