Intermediate reactive oxygen and nitrogen from macrophages induced by Brazilian lichens

Fitoterapia 75 (2004) 473–479

Intermediate reactive oxygen and nitrogen from macrophages induced by Brazilian lichens L.C. Santosa,*, N.K. Hondab, I.Z. Carlosc, W. Vilegasa a

´ ˆ ´ ˜ Paulo, Brazil Departamento de Quımica Organica, Instituto de Quımica, UNESP, Araraquara, Sao b ´ Departamento de Quımica, UFMS, Campo Grande, Mato Grosso do Sul, Brazil c ´ ´ ˆ ˆ Departamento de Analises Clınicas, Faculdade de Ciencias Farmaceuticas, UNESP, Araraquara, ˜ Paulo, Brazil Sao Received 3 September 2003; accepted in revised form 14 April 2004

Abstract The activity of ten compounds isolated from Brazilian lichen over the release of hydrogen peroxide and nitric oxide was evaluated in the culture of peritoneal macrophage cells from mice. Salazinic, secalonic A and fumarprotocetraric acids were the compounds that induced the greatest release of H2O2, whereas 12R-usnic and diffractaic acids induced the release of NO. These results indicate that lichen products have potential immunological modulating activities. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Lichens; Macrophages; Peroxide hydrogen; Nitric oxide

1. Introduction Similar to higher plants, lichens were used since antiquity as natural drugs w1x. These organisms produce secondary metabolites and many of them are known for presenting biological andyor pharmacological activities w2–4x. Lichens are organisms composed by a fungus and one or more algae. They are slow-growing organisms and their secondary metabolites are mainly depsides, depsidones, dibenzofurans, xanthones, antraquinones and terpene derivatives w2,3x. The secondary metabolites of lichens display a wide range of biological activities such as antibiotic, antitumor, *Corresponding author. Tel.: q55-16-201-6657; fax: q55-16-222-7932. E-mail address: [email protected] (L.C. Santos). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.04.002

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analgesic et al. w2,3x. Many human physiological activities such as stimulation of phagocytic cells, host-mediated tumor activity, and a wide range of antiinfective actions have been assigned to different compounds w5,9x. Phagocytic cells such as polymorphonuclear leukocytes and macrophages, respond to a variety of membrane stimulants by the production and extracellular release of a number of reactive oxygen reduction products. This coordinated sequence of biochemical reaction, known as ‘oxidative burst’, is initiated by an increase in oxygen uptake followed by the one-electron reduction of oxygen to superoxide anions (Oy 2 ) using NADPH or NADH as the electron donor and catalyzed by a NAD(P)H oxidase w6x. Macrophages are known to play an important role in host defense mechanisms. In the immune system, reactive oxygen intermediates (ROI) often function together with nitric oxide (NO), for example in macrophage killing of bacteria and tumors cells w7–9x. Currently, there is a strong tendency to study natural compounds that may be involved in the modulation of the immunological system. A simple, rapid and inexpensive method to measure the hydrogen peroxide (H2O2) produced by the cells in culture in vitro is based on the horseradish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 w9x. The measurement of the concentration of NO in biological systems is a challenging analytic problem w10x. The presence of NO in biologic systems usually is inferred based on one of its physiologic effects w11x, such as the relation of blood vessels, activation of guanylyl cyclase activity, increased cGMP concentration, production of citrulline, or inhibition of platelet aggregation w12x. NO has been too implicated as playing a role in many pathological conditions, including allergic airway diseases, pneumonitis, arthritis, vasculitis, acute rejection of allograft, and toxic shock syndrome w13x. Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea w14x, reciprocal regulation of mammalian nitric oxide synthase and calcineurin by plants calmodulin isoformes w15x has been reported in previous investigations. The objective of the present study was to determinate the release of NO and H2O2 in the culture of peritoneal macrophages in the presence of ten different compounds isolated from Brazilian lichens on mice peritoneal macrophage, by the liberation of H2O2 and NO. 2. Experimental 2.1. Plant Lichens species, collected in Vila Puraputanga, Mato Grosso do Sul State, Brazil, ˆ were identified by Profa. Dra Mariana Fleig from the Departamento de Botanica da Universidade Federal do Rio Grande do Sul and Prof. Dr Marcelo Marcelli from ˆ ˜ Paulo. A voucher of each species was deposited in the Instituto de Botanica de Sao the Chemical Departament of the Universidade Federal do Mato Grosso do Sul, Brasil. Parmotrema dilatatum (Vain.) Hale, P. tinctorum (Nyl,) Hale, P. cf. delicatulum (Vain.) Hale, P. cf. miranda (Hale) Hale, P. cf. flavescens (Kremplh)

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´ Hale, P. sphaerospora (Nyl.) Hale, Usnea Hale, Pseudoparmelia hypomiltha (Fee) meridionalis Zahlbr., U. subcavata Motyka, and Heterodermia microphylla (Kurok.) Scorepa. 2.2. Extraction and isolation of compounds Each species were Soxhlet extracted with chloroform and acetone. The extracts were concentrated in vacuo. Concentrated chloroform extracts were treated with ethanol several times to remove pigments. Then, the chloroform extract of each lichen was Si-gel CC eluting with hexane-chloroform mixtures in crescent polarity to give 12R-usnic acid (2) (U. meridionalis), atranorin (4) (P. tinctorum), diffractaic acid (3) (U. subcavata Motyka.) and secalonic acid A (9) (P. hypomiltha). The acetonic extracts yielded salazinic acid (1) (R. cetrata), lecanoric acid (5), ethyl orsellinate (8) (P. tinctorum), protocetraric acid (6) (P. dilatatum), hypostictic acid (7) (P. sphaerospora), fumarprotocetraric acid (10) (C. verticillaris Raddi.) (Fig. 1). Purity of samples (95%) was checked by TLC, HPLC, UV, IR, MS, NMR. 2.3. Animals Six-week-old male Swiss mice weighing 18–25 g were supplied by the Animal’s ˆ ˆ House of the Faculdade de Ciencias Farmaceuticas, Universidade Estadual Paulista˜ Paulo-Brasil. The animals were maintained in standard UNESP, Araraquara-Sao environmental conditions with water and food ‘ad libitum’. 2.4. Macrophage cell and determination of hydrogen peroxide (H2O2) Using the culture method in vitro and natural products, we determined the reactive compounds of oxygen w7,16x. The method consists in the determination of the liberation of H2O2, in the culture of mice peritoneal macrophages. Peritoneal thioglycollate-elicited macrophages from mice were obtained as reported previously w17x. Briefly, the peritoneal cavity was washed with 10 ml of cold PBS (buffered salting phosphate solution) and the resulting suspension was pelleted at 4 8C by centrifugation for 5 min at 300 rev.ymin, and the supernatant was removed. The cells were resuspended at a concentration of 2=106 cellsyml in a solution of phenol red containing 140 mM NaCl, 10 mM potassium phosphate pH 7.0, 5.5 mM dextrose; 0.56 mM phenol red and type II horseradish peroxidase 0.01 mgyml (Sigma). Aliquots of 0.1 ml were transferred to culture plates, flat bottomed containing 96 wells (Corning). 100 mg of test compound (50 ml of 2 mgyml solution) or 50 ml of dimethyl sulfoxide (DMSO) were added. Zymosan (250 mgy well) was used as standard. The samples were incubated for one hour at 37 8C in a humid atmosphere. The reaction was interrupted by the addition of 10 ml of 4 N NaOH. Experiments were done in quadruplicate. Absorbance was determined in an ELISA automatic photometer, at 620 nm. The results were expressed in nanomoles

L.C. Santos et al. / Fitoterapia 75 (2004) 473–479

Fig. 1. Compounds isolated from Brazilian lichens.

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of H2O2 y2=105 peritoneal cells, from a standard curve established in each test, constituted of known molar concentrations of H2O2 in buffered phenol red. 2.5. Nitric oxide (NO) measurement Thioglycolate-elicited PEC were harvested from Swiss mice using sterile PBS, pH 7.4. The cells were washed twice by centrifugation at 200 rev.ymin for 5 min at 4 8C and re-suspended in complete RPMI-1640 culture medium (Sigma) containing 100 Uyml penicillin, 100 mgyml streptomycin, 5=10y2 M mercaptoethanol and 5% inactivated fetal calf serum (Gibco, USA) to give 5=106 cellsyml, 100 ml of this suspension being added to each well of a 96 well tissue culture dish along with 500 mg of each test compound (100 ml of 5 mgyml solution) andyor 1 mgywell of Escherichia coli O26:B6 lipopolysaccharide (LPS) (100 ml of a 10 mgy ml solution). After which 50 ml aliquots of culture supernatant were mixed with 50

Fig. 2. Effects of compounds 1–10 on the production of peritoneal macrophages. The macrophages were cultured for 1 h in the presence of control DMSO (a), zymosan (250 mgywell) (b) and isolated compounds (100 mgywell). Each bar represents the mean"S.D. of four animals. Representative results of one experiment repeated four times are given. The results were statistically significant when compared to the control group (P-0.05).

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ml of Griess reagent (1% wyv sulfanylamide, 0.1% wyv naphthylethylenediamine and 3% H3PO4), incubated at room-temperature for 10 min, and the color reaction determined at 550 nm in an ELISA reader Spectra (Shell) and Rainbow (Shell) (TECAN, Austria). Supernatants from quadruplicate cultures were assayed in triplicate (five determinations) and reported as the mean NOy 2 concentration. 2.6. Statistical analysis Data are expressed as mean"S.D., and the Student’s t-test was used to determine the significance of the differences between the control and experimental groups (PF0.05) considered statistically significant. 3. Results and discussion Fractionation of lichen species afforded the known compounds 1–10 (Fig. 1). The data presented in Fig. 2 summarize the release of H2 O2 expressed as nmol after

Fig. 3. Effect of compounds 1–10 on the nitrite production of peritoneal macrophages. The macrophages were cultured for 24 h in the presence of control DMSO (a), lipopolysaccharide (1 mgywell) (b) and isolated compounds (100 mgywell). Representative results of one experiment repeated four are given. Data shown are mean"S.D. The results were statistically significant when compared to the control group (P-0.05).

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1 h of incubation of several lichen compounds. From the tested compounds, only compound 1, 9 and 10 were able to release 65.68, 20.74 and 33.08 nmol of H2O2, respectively, while zymosan, a powerful immunostimulator, was able to release 275.41 nmol. Determination of the NO concentration demonstrated that several lichen compounds were able to increase the release of NO (Fig. 3). Compounds 2 and 3 (Fig. 1) were the most active compounds when compared to the positive control B (lipopolysaccharide of E. coli). Compounds 1, 6 and 10 also presented higher activity than the positive control B. In conclusion, our results suggest that lichen compounds may contribute to the induction of immunostimulatory effects. However, further tests involving the ‘oxidative burst’ (Oy 2 ), cytokines and NO production are required. Acknowledgments We thank to FAPESP for financial aid for a fellowship to L.C. Santos and CNPq for a grant to W. Vilegas. The authors are grateful to Marisa Campos Polesi Placeres, ´ ´ ˆ ˆ of the Laboratorio de Imunologia Clınica da Faculdade de Ciencias Farmaceuticas ˜ Paulo, Brazil. of UNESP, Araraquara, Sao References w1x Vartia KO. Antibiotics in lichens. In: Ahmadjian V, Hale ME, editors. The lichens. New York: Academic Press, 1973. w2x Huneck S. Naturwissenschaften 1999;86:559. w3x Muller ¨ K. Appl Microbiol Biotechnol 2001;56:9. w4x Fahselt D. Symbiosis 1994;16:117. w5x Halliwell B, Gutteridge JMC. Biochem J 1984;219:1. w6x Pick E, Mizel D. J Immunol Methods 1981;46:211. w7x Schmidt HHHW, Walter U. Cell 1994;78:919. w8x Nathan C. Cell 1995;82:873. w9x Moreira RRD, Carlos IZ, Vilegas W. Biol Pharm Bull 2001;24:201. w10x Archer SL, Hampi V. Biochem Biophys Res Commun 1992;188:590. w11x Furchgott RF, Vanhoutte PM. FASEB J 1987;3:2007. w12x Frederick L, Kiechle MD, Malinski T. Clin Chem 1993;100:567. w13x Nathan CJ. Clin Invest 1997;100:2417. w14x Chan MMY, Fong D, Ho CT, Huangs HI. Biochem Pharm 1997;54:1281. w15x Cho MJ, Vagh PL, Kondo R, Lee SH, Davis JP, Rehl R, Heo WD, Johnson JD. Biochemistry 1998;37:15 593. w16x Pick E, Keisari Y. J Immunol Methods 1980;38:161. w17x Carlos IZ, Zini MMC, Sgarbi DBG, Angluster J, Alviano CS, Silva CL. Mycopathologia 1994;127:189.