Immunostimulatory effects of the phenolic compounds from lichens on nitric oxide and hydrogen peroxide production

Efeito imunoestimulante de compostos fenólicos de líquens na produção de peróxido de hidrogênio e óxido nítrico

ARTIGO

Immunostimulatory effects of the phenolic compounds from lichens on nitric oxide and hydrogen peroxide production

Efeito imunoestimulante de compostos fenólicos de líquens na produção de peróxido de hidrogênio e óxido nítrico

Iracilda Z. CarlosI, ^*; Camila B. A. Carli^I; Danielle C. G. Maia^I; Fernanda P. Benzatti^I; Flavia C. M. Lopes^I; Fernanda M. Roese^II; Márcio Watanabe^II; Ana C. Micheletti^II; Lourdes C. dos Santos^III; Wagner Vilegas^III; Neli K. Honda^II

^IDepartament of Clinical Analysis, Faculdade de Ciências Farmacêuticas de Araraquara, São Paulo State University, Post Office Box 502, Araraquara-SP 14801-902, Brazil

^IIDepartament of Chemistry, Universidade Federal do Mato Grosso do Sul, Campo Grande-MS, Brazil

^IIIDepartment of Organic Chemistry, Instituto de Química de Araraquara, São Paulo State University, Post Office Box 355, Araraquara-SP 14801-970, Brazil

ABSTRACT

The effects of isolated compounds from Brazilian lichens and their derivatives on H₂O₂ and NO production were studied using murine macrophages as a part of an attempt to understand their possible immunomodulatory properties. The compound cytotoxicity was studied using MTT assay. Macrophage stimulation was evaluated by the determination of NO (Griess assay) and H₂O₂ (horseradish peroxidase/phenol red) in supernatants of peritoneal macrophage cultures of Swiss mice. This research demonstrated stimulatory activities of some phenolic compounds isolated from lichens and their derivatives on H₂O₂ and NO production. Structure-activity relationships suggest several synthetic directions for further improvement of immunological activity.

Keywords: lichens, hydrogen peroxide, nitric oxide, macrophages, phenolic compounds.

RESUMO

Os efeitos dos compostos isolados de líquens brasileiros e seus derivados na produção de NO e H₂O₂ foram estudados utilizando macrófagos murinos na tentativa de desvendar suas possíveis propriedades imunomodulatórias. A citotoxicidade dos compostos foi estudada utilizando o ensaio de MTT. A estimulação dos macrófagos foi avaliada através da determinação de NO (metodologia de Griess) e H₂O₂ (peroxidase de raíz forte/vermelho de fenol) no sobrenadante de culturas de macrófagos peritoneais de camundongos Swiss. Este estudo demonstrou atividade estimulante de alguns compostos fenólicos e seus derivados na produção de NO e H₂O₂. A relação estrutura atividade sugere inúmeras direções sintéticas para futuros melhoramentos da atividade imunológica.

Unitermos: líquens, peróxido de hidrogênio, óxido nítrico, macrófagos, compostos fenólicos.

INTRODUCTION

Lichens are a large group of organisms formed by the symbiotic association of Chlorophyta or Cyanobacteria and a fungus. Along with Cyanobacteria they play an important role as pioneer organisms in colonizing rocks. Similar to higher plants, lichens were used as natural drugs. The organisms produce secondary metabolites and many of them are known for presenting biological and/or pharmacological activities (Fahselt , 1994).

The innate immune system is mainly mediated by macrophages, dendritic cells and NK cells (Schiller et al., 2006). Macrophages are major components of the mononuclear phagocyte system that consists of closely related cells of bone marrow origin, including blood monocytes, and tissue macrophages. In every stage of the immune response macrophages are involved and they can release many compounds such as hydrogen peroxide (H₂O₂) and nitric oxide (NO). H₂O₂ is a lipid soluble molecule that crosses the cell membranes and it is scavenged by catalase and by glutathione peroxidase in biological systems (Schafer & Buettner, 2001). Studies have suggested that H₂O₂ plays an important role in the functions of macrophages (Ramasarma, 1990). It also has many different functions in the human body, acting as a signaling molecule and a cytotoxic agent in the defense system (Abe & Bradford, 1999). NO has cytotoxic properties and it is produced during host defense against invasive organisms and immunologic reactions. It is also involved in many physiologic processes of mammals, including neurotransmition, blood pressure control and inflammation (Costa et al., 2003; Garcia & Stein, 2006).

Many polysaccharides obtained from fungi are considered modifiers of the biological response and have been shown to modulate immune response. Compounds isolated from fungi had been shown to enhance immune response through the up-regulation of nitric oxide from peritoneal macrophages acting as an effective immunomodulator and an anti-tumoral agent. (Suzuki et al., 1985; Kim et al., 2004; Santos et al., 2004; Gao et al., 2005; Zheng et al., 2005). Since the symbiotic association of algae with fungus forms lichens and there are just few works about the pharmacological properties of lichens, in this research we decided to study pure compounds isolated from Brazilian lichens and their derivatives. The effects of phenolic compounds and their derivatives on H₂O₂ and NO production were studied using murine macrophages as a part of an attempt to understand their possible immunomodulatory properties.

MATERIAL E MÉTODOS

Lichens

The lichen Parmotrema dilatatum (Vain.) Hale Parmeliaceae was collected in Mato Grosso do Sul, state, Brazil; Ramalina sp. Ramalinaceae and Parmotrema lichexanthonicum Eliasaro & Adler Parmeliaceae were obtained from the shops of decoration products. The identification was conduced by Profa. Dra. Mariana Fleig from UFRGS and Prof. Dr. Marcelo P. Marcelli from the Instituto de Botânica de São Paulo. A voucher specimen of each species is kept in our laboratory for future reference.

Extraction and isolation of compounds

P. dilatatum, P. lichexanthonicum and Ramalina sp. were extracted with chloroform and acetone exaustively. The extracts were concentrated in vacuo. The extract obtained from each lichen was fractionated on Si-gel CC and eluted with hexane-chloroform mixtures in crescent polarity to give lichexanthone [1] (P. lichexanthonicum). From the acetone extract of the lichens P. dilatatum and Ramalina sp. were isolated protocetraric acid [2] and norstitic acid [3], respectively. The purification of these compounds was conduced by treatment with small volume of acetone in an ice bath. The degree of purity for all lichen compounds was > 95% as determined by TLC, HPLC and NMR analysis. The compounds [1], [2] and [3] and the derivatives obtained from compounds [2] and [3] were studied in this research.

Derivatives

Compounds I-IX and X-XI were obtained through reactions with alcohols with the protocetraric acid [2] and norstitic acid [3], respectively. The purification of these compounds was conduced by sucessive treatments with acetone in an ice bath. Methyl protocetrarate (XII): protocetraric acid [2] was treated with CH₃I and K₂CO₃ in DMF (Garner & Park, 1992). Physodalic acid (XIII); protocetraric acid [2] was solubilized in DMF and acetylated by glacial acetic acid (Elix & Engkaninan, 1975). Conprotocetraric acid (XIV): Protocetraric acid [2] was reduced by sodium borohydride (NaBH₄) (Wolfrom & Thomson, 1963). The structures were confirmed by 1D and 2D NMR spectra and checked with literature data (Huneck & Yoshimura, 1996).

Animals

Swiss mice (six-eight weeks old, weighting 18 to 25 g) were maintained in a polycarbonate box (at 23±1 ºC, 55±5% humidity, 10-18 circulations/h and a 12 h light/dark cycle), with water and food available ad libitum. At least five animals were used for each experiment.

Peritoneal exsudate cells

Thioglycollate-elicited peritoneal exsudate cells (PEC) were harvested from Swiss mice using 5.0 mL of sterile phosphate-buffered saline (PBS), pH 7.4. The cells were washed twice by centrifugation at 200 xg for 5 min at 4 °C and re-suspended in appropriate medium for each test.

MTT assay

PEC (5x 10⁶ cells/mL) were resuspended in RPMI-1640 containing 5% heat inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin and 50 mM 2-mercaptoethanol. The suspension (100 μL) and the test material (100 μL) were added to each well of a ninety six-well tissue culture plate and the plates were incubated for 24 h. MTT solution was added (100 μL) and the plate was incubated for 3 h at 37 oC with 5% CO₂. Absorbance was read in a UV/Vis spectrophotometer at a wave length of 540 nm, with a reference filter of 620 nm. The culture medium and cells alone were used as control, representing 100% viability of the macrophages (Mosmann, 1983).

H₂O₂ production

PEC (2x 10⁶ cells/mL) were cultivated as described above and suspended in a solution containing 140 mmol NaCl, 10 mmol potassium phosphate buffer, pH 7.0, 5.5 mmol dextrose, 0.56 mmol phenol red, and 0.01 mg/mL type II horseradish peroxidase. Next, 100 μl of this suspension was added to each of the wells of a ninety six-well flat-bottom tissue culture plate and exposed to 50 μL of the compounds. Phorbol myristate acetate (PMA) solution in phosphate buffer was used as a positive control. The cells were incubated for 1 h at 37 °C in a mixture of 95% air and 5% CO₂. The reaction was stopped with 10 μL of 4 N NaOH and the samples were read at 620 nm with a Multiskan Ascent ELISA reader against a blank containing phenol red solution and 4 N NaOH. The results were expressed as nanomoles of H₂O₂/2x 10⁶ peritoneal cells, from a standard curve established in each test consisting of known molar concentrations of H₂O₂ in buffered phenol red. At least four experiments were used for the test (Pick & Keisari, 1980; Pick & Mizel, 1981).

NO production

PEC were harvested from Swiss mice using sterile PBS, pH 7.4. The cells were washed twice by centrifugation at 200 xg for 5 min and resuspended in complete RPMI-1640 culture medium containing 100 U/mL penicillin, 100 mg/mL streptomycin, 5x 10^-2 M mercaptoethanol and 5% inactivated fetal calf serum. The medium of this composition was denoted complete RPMI-1640 (RPMI-1640-C) and was used for cell counts in a Neubauer chamber (Boeco, Germany) and the cell suspension was adjusted to a concentration of 5x 10⁶ cells/mL. A 100 μL of this suspension was added to each well of a ninety six well tissue culture plate with different tested compounds. Escherichia coli O26:B6 lipopolysaccharide (LPS) was used as a positive control. The plates were then incubated for 24 h at 37 ºC under, 7.5% CO₂. The nitrite concentrations were indirectly measured by a quantitative colorimetric assay using Griess reagent system: 50.0 μL aliquots of supernatant were added to 50.0 μL of Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine and 3% H₃PO₄), incubated at room-temperature for 10 min, and the absorbance was measured at 540 nm in an Elisa microplate reader (Multiskan Ascent Labsystems). Supernatants from quadruplicate cultures were assayed in four experiments and reported as μmols NO/5x 10⁵ cells as calibrated against solutions of known NaNO₂ concentration. (Green et al., 1982).

Statistical Analysis

Data were expressed as mean±standard deviation, and the Dunnet t-test (Microcal Origin 5.0) was used to determine the significance of the differences between the negative control and tested compounds.

RESULTS AND DISCUSSION

Nowadays, compounds isolated from botanical sources as mushrooms, algae, lichens and higher plants have attracted a great deal of attention in the biomedical area because of their broad spectrum of therapeutic properties and relatively low toxicity (Schepetkin & Quinn, 2006).

The cytotoxic effect of the compounds tested in peritoneal macrophages was assessed by the MTT assay as previously described (Table 1, Figure 1) (Mosmann, 1983). The tested concentration was show in the Table 1. It was used the concentration which the viability was higher or the same of 50%.

Initial stage of the immune response macrophages is involved and they can release many mediators such as hydrogen peroxide (H₂O₂) and nitric oxide (NO). The killing of intracellular and extracellular pathogens by phagocytes is due in part to the production of reactive oxygen intermediate (ROIs). The H₂O₂ is responsible for killing internalized bacteria in phagocytosis. The NO has central functions in innate immunity and regulation of immune functions. It has important roles in inhibiting tumor cell adhesion, arresting the growth of tumor cells and contributing to the control of microbial pathogens (Maragos et al., 1993; Kong et al., 1996; Rajan et al., 1996; Bogdan et al., 2000; Carlos et al., 2003; Chakravortty & Hensel, 2003). The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as H₂O₂ and superoxide (MacMicking et al., 1997).

Our results showed that the compounds I, V, VIII and IX induced a small production of H₂O₂ (Figure 2), however the compound VI showed a high production of this mediator when it was added to the cell culture (approximately 18 nmols/2 x 10⁵ cells). Analyzing the production of NO (Figure 3), it was observed that compounds VIII, IX and XIII induced a small production of this mediator when compared with the negative control, with IX presenting statistical difference, p < 0.05. The compounds I, II and V were more representative in the production of this mediator; all of them showed a significative liberation of NO, approximately 23, 90 and 13 μmols/5 x 10⁵ cells, respectively (p < 0.001). It is important to notice that compound V was also derivatived from compound II (Santos et al., 2004).

The compound [2] (protocetraric acid) presents three functions: alcohol, acid and aldehyde that were modified in this study and showed high production of NO. When the aldehyde function was modified for alcohol (XII) or the acid function was modified for ester (XIV), H₂O₂ and NO production was not observed. In contrast, when the alcohol function was substituted for ether, induced a small NO production, exception compound V demonstrated a higher release of this mediator. However, a small production of H₂O₂ induced by compounds V, VIII and IX and a high liberation caused by compound VI can be explained due to higher liphophylic of the molecule because of the presence of the n-hexyl radical in the position 9'. The norstitic acid [3] and their derivatives (X and XI) did not induced the production of H₂O₂ and NO by the macrophages. The lichexanthone [1] showed significative production of NO and its derivatives must be subsequently studied.

This research demonstrated stimulatory activities of some phenolic compounds isolated from lichens on H₂O₂ and NO production by murine macrophages, suggesting that these compounds are activators of macrophages. The induction of H₂O₂ and NO may contribute in the immunoprevention of cancer and infecctious diseases.

In conclusion, structure-activity relationships suggest several synthetic directions for further improvement of immunological activity. Protocetraric acid derivatives appear to be promising as immunostimulating agents.

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Publication Dates

• Publication in this collection
  19 May 2010
• Date of issue
  Dec 2009