Effect of the essential oil of Achillea millefolium L. in the production of hydrogen peroxide and tumor necrosis factor-alpha in murine macrophages

Efeito do óleo essencial de Achillea millefollium L. na produção de peróxido de hidrogênio e fator de necrose tumoral-alfa por macrófagos murinos

Abstracts

Macrophages release more than one hundred compounds into the extracellular environment. Among these, there are cytokines and intermediate oxygen compounds, such as TNF-alpha and H2O2. We evaluated the effect of the crude essential oil of Achillea millefolium L. (Asteraceae) by determining hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) release in cultures of peritoneal macrophages cells from Swiss mice. Commercial azulene was also tested for comparison with the essential oil. The macrophages viability in the presence of the oil was analyzed and the dilutions of 1:100 and 1:200 showed the best results. A mild production of H2O2 and a moderate liberation of TNF-alpha were observed. It was also noticed that H2O2 and TNF-alpha production using commercial azulene was higher than that produced by the oil. The essential oil of A. millefolium was able to stimulate peritoneal macrophages to produce H2O2 and TNF-alpha without causing an overproduction of these compounds. It is suggested that the essential oil can modulate macrophages activation.

Achillea millefolium; Macrophages; Hydrogen peroxide; Tumor necrosis factor-alpha; Essential oil

O efeito do óleo essencial bruto de Achillea millefolium L. (Asteraceae) foi determinado através da liberação de peróxido de hidrogênio (H2O2) e do fator de necrose tumoral-alfa (TNF-alfa) por cultura de macrófagos peritoneais de camundongos Swiss. O azuleno comercial também foi testado e comparado com o óleo essencial. Macrófagos liberam mais de cem compostos biologicamente ativos. Entre esses, citocinas e compostos intermediários do oxigênio como o TNF-alfa e o H2O2. A viabilidade dos macrófagos na presença do óleo foi analisada e as diluições de 1:100 e 1:200 do óleo essencial mostraram os melhores resultados. Pequena liberação de H2O2 e moderada produção de TNF-alfa também foram observadas. Contudo, maior liberação de H2O2 e TNF-alfa foi observada utilizando o azuleno comercial. Desse modo, observou-se que o óleo essencial de A. millefolium foi capaz de estimular os macrófagos peritoneais a produzir H2O2 e TNF-alfa sem causar superprodução desses componentes. Sugere-se que o óleo essencial pode modular a ativação de macrófagos.

Achillea millefolium; Macrófagos; Peróxido de hidrogênio; Fator de necrose tumoral-alfa; Óleo essencial

ORIGINAL PAPERS

Effect of the essential oil of Achillea millefolium L. in the production of hydrogen peroxide and tumor necrosis factor-a in murine macrophages

Efeito do óleo essencial de Achillea millefollium L. na produção de peróxido de hidrogênio e fator de necrose tumoral-a por macrófagos murinos

Flávia Cristine Mascia Lopes^I; Fernanda Paulin Benzatti^I; Cleso Mendonça Jordão Junior^II; Raquel Regina Duarte Moreira^III; Iracilda Zeppone Carlos^I,*

^IDepartamento de Análises Clínicas

^IIDepartamento de Ciências Biológicas

^IIIDepartamento de Princípios Ativos Naturais e Toxicologia, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara

ABSTRACT

Macrophages release more than one hundred compounds into the extracellular environment. Among these, there are cytokines and intermediate oxygen compounds, such as TNF-a and H₂O₂. We evaluated the effect of the crude essential oil of Achillea millefolium L. (Asteraceae) by determining hydrogen peroxide (H₂O₂) and tumor necrosis factor-a (TNF-a) release in cultures of peritoneal macrophages cells from Swiss mice. Commercial azulene was also tested for comparison with the essential oil. The macrophages viability in the presence of the oil was analyzed and the dilutions of 1:100 and 1:200 showed the best results. A mild production of H₂O₂ and a moderate liberation of TNF-a were observed. It was also noticed that H₂O₂ and TNF-a production using commercial azulene was higher than that produced by the oil. The essential oil of A. millefolium was able to stimulate peritoneal macrophages to produce H₂O₂ and TNF-a without causing an overproduction of these compounds. It is suggested that the essential oil can modulate macrophages activation.

Uniterms: Achillea millefolium. Macrophages. Hydrogen peroxide. Tumor necrosis factor-a. Essential oil.

RESUMO

O efeito do óleo essencial bruto de Achillea millefolium L. (Asteraceae) foi determinado através da liberação de peróxido de hidrogênio (H₂O₂) e do fator de necrose tumoral-a (TNF-a) por cultura de macrófagos peritoneais de camundongos Swiss. O azuleno comercial também foi testado e comparado com o óleo essencial. Macrófagos liberam mais de cem compostos biologicamente ativos. Entre esses, citocinas e compostos intermediários do oxigênio como o TNF-a e o H₂O₂. A viabilidade dos macrófagos na presença do óleo foi analisada e as diluições de 1:100 e 1:200 do óleo essencial mostraram os melhores resultados. Pequena liberação de H₂O₂ e moderada produção de TNF-a também foram observadas. Contudo, maior liberação de H₂O₂ e TNF-a foi observada utilizando o azuleno comercial. Desse modo, observou-se que o óleo essencial de A. millefolium foi capaz de estimular os macrófagos peritoneais a produzir H₂O₂ e TNF-a sem causar superprodução desses componentes. Sugere-se que o óleo essencial pode modular a ativação de macrófagos.

Unitermos:Achillea millefolium. Macrófagos. Peróxido de hidrogênio. Fator de necrose tumoral-a. Óleo essencial.

INTRODUCTION

Basic research on natural substances with immunomodulating properties is performed by assays primarily carried out on the stimulation of nonspecific immunity of the innate response, such as the efficiency of macrophages (Williams, 2001).

The first cells that participate in the immunological response are macrophages. The production of cytokines and intermediate compounds of oxygen is one of their functions (Roitt, 1999). These cells respond to a variety of membrane stimulants by the production and extracellular release of a number of reactive oxygen reduction products in a coordinate sequence of biochemical reactions known as "oxidative burst" (Pick, Mizel, 1981). Studies have suggested that H₂O₂ plays an important role in the functions of macrophages (Ramasarma, 1990).

Cytokines are low-molecular-weight regulatory proteins or glycoproteins secreted by various cells in the body and among them, tumor necrosis factor-a (TNF-a) is now accepted as a multifunctional cytokine that mediates key roles in acute and chronic inflammations, antitumor responses and infections (Palladino et al., 2003).

Achillea millefolium L.(Asteraceae) is a plant known as Yarrow, it is native from Europe, although perfectly adapted to Brazilian environment. This plant is used in Brazilian traditional medicine to treat respiratory infections, fever and rheumatic pains. Although the essential oil displays different chemical profiles from those observed from plants of other geographical origin, the major constituents remains as: azulene, cineol, borneol, pinenes and camphor (Lorenzi, Matos, 2002).

The purpose of the present work was to study the effects of the essential oil of A. millefolium in cultures of peritoneal macrophage cells from Swiss mice by determination of H₂O₂ and TNF-a. The commercial azulene was also tested to compare its effect with the essential oil obtained from the plant.

MATERIAL AND METHODS

Animals

Swiss mice (6-8 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.

Plant material

Leaves of Achillea millefolium L. (Asteracea) were collected from the Botanical Garden at Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, UNESP - Araraquara, São Paulo. The voucher specimen was deposited in the Herbarium of the Instituto de Biociências, Unesp - Rio Claro, under the number HRCB 35292.

Commercial azulene

A commercial azulene (Merck) sample was also tested. This sample was sonicated and sterilized. The dilution of 1:100 was used for MTT, H₂O₂ and TNF-a assays.

Distillation and preparation of the essential oil

The oil was obtained from the powdered leaves from Achillea millefolium L., using Clevenger-type apparatus. 200 g of the powdered leaves rendered about 0.6 mL of essential oil. It was sonicated with RPMI-1640 for 6 minutes and sterilized using a 0.45 µm membrane filter. The dilutions of 1:50, 1:100 and 1:200 in RPMI-1640 were analyzed. The chromatographic profile of the essential oil was studied through a comparative thin layer chromatography and the presence of azulene was demonstrated (data not shown).

Peritoneal Macrophages

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 g for 5 minutes at 4 ºC and resuspended in appropriate medium for each test.

MTT assay for cell viability

PEC (5x10⁶ cells/mL) were re-suspended in RPMI-1640 containing 5% heat inactivated fetal bovine serum, 100 IU/mL penicillin, 100 µg/mL streptomycin and 50 mM 2-mercaptoethanol. The suspension (100 µL) and the samples (100 µL) were added to each well of a 96-well tissue culture plate and they were incubated for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed and absorbance measured at 540 nm with a Multiskan Ascent ELISA reader (Labsystems, Helsinki, Finland) equipped with a 620 nm reference filter. Only cells and culture medium (RPMI-1640) were used as a control that corresponds to 100% of macrophages viability.

H₂O₂ measurement

PEC (2x10⁶ 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, called complete buffer. Next 100 µL of this suspension was added to each of the wells of a 96-well flat-bottom tissue culture plate and exposed to 50 µL of different dilutions of essential oil, commercial azulene and Zymosan at 5 mg/mL (positive control). The cells were incubated for 1 h at 37 ºC in a 5% CO₂ atmosphere. The reaction was stopped with 50 µL of 5 N NAOH and measured at 620 nm. The results were expressed as nanomols of H₂O₂/2 x 10⁵ peritoneal cells (Pick, Mizel, 1981).

TNF-a production and assay

For TNF-a production, adherent PEC was stimulated with 50 mL of essential oil dilutions, commercial azulene or lipopolysaccharide (LPS) from E. coli 0111:B4 at 10 µg/mL, as positive control. After 24 h incubation, the supernatants were removed and stored at -80 ºC until assayed. L929 mouse tumor cells were used to measure TNF-a levels as described previously (Carlos et al., 1994).

Statistical analysis

Data are expressed as mean ± standard deviation, and the Student's t-test (Microcal Origin 5.0) was used to determine the significance of the differences between the control and experimental groups.

RESULTS AND DISCUSSION

The effect of the essential oil of A. millefolium on the macrophage viability was determined by MTT assay and the 1:50 dilution showed only 30% of viable cells. Viability levels higher than 70% were observed in the 1:100 and 1:200 dilutions. The viability of the cells in the presence of commercial azulene was 66.86 (Table I).

Studies have shown that H₂O₂ has important functions in intracellular and intercellular reactions of different cells. The production of H₂O₂ in all tested samples of the essential oil was not very expressive in this work (less than 25 nmols/ 2.10⁵ cells), even though a small quantity is an important result (Figure 1). Under normal physiological conditions it is generated in small quantities and rapidly used or degraded. However long exposures and high concentrations of this mediator can destroy biological structures and lead to irreversible cell damage (Ramasarma, 1990).

Nowadays it has become apparent that the regulation of immune responses is controlled in part, by cytokines produced by macrophages. According to Figure 1, all tested samples of the essential oil produced moderate quantities of TNF-a, around 80 Units. When the commercial azulene was tested, both H₂O₂ and TNF-a production were higher than the samples with plant essential oil. It is probably due to the purity of the commercial azulene.

Overproduction of pro-inflammatory cytokines and reactive oxygen species, which in adequate amounts are involved in normal and localized immune defenses, leads the development of an oxidative stress, which occurs in endotoxic shock (Víctor, De La Fuente, 2003). The production of H₂O₂ is a natural cellular process, although in high concentrations it can damage DNA, leading to cell death (Ramasarma, 1990). In this work, the production of H₂O₂ and TNF-a was small and moderate, respectively. These results suggest that the essential oil has a positive effect when it is utilized in the dilutions tested in this study.

Candal et al. (2003), studying the essential oil of A. millefolium confirmed that it possess strong anti-oxidative activity but low antimicrobial activity in vitro. Previous studies performed in our laboratory showed that essential oil of A. millefolium can induce a mild NO release (14,99±0,61 µmol/5x10⁵ cells) at a 1:100 dilution by peritoneal macrophages (Lopes et al., 2003).

Further studies are necessary to find out which Yarrow's components have a specific effect in the immune system. Nevertheless, our findings may contribute to a better understanding of beneficial effects of this folk medicine.

ACKNOWLEDGEMENTS

This work was supported by FAPESP (02/03638-1).

Recebido para publicação em 21 de dezembro de 2004.

Aceito para publicação em 22 de maio de 2005.

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Publication Dates

• Publication in this collection
  10 Mar 2006
• Date of issue
  Sept 2005

History

• Accepted
  22 May 2005
• Received
  21 Dec 2004