Effect of Some Essential Oils on Phagocytosis and Complement

Article pubs.acs.org/JAFC

Effect of Some Essential Oils on Phagocytosis and Complement System Activity Renato Pérez-Rosés,† Ester Risco,†,§,⊥ Roser Vila,† Pedro Peñalver,# and Salvador Cañigueral*,† †

Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Joan XXIII s/n, E-08028 Barcelona, Spain § Phytonexus S.L., Na Jordana 11, E-46240 Carlet (València), Spain # Lidervet S.L., Plaça Garcı ́a Lorca 17 Baixos, E-43006 Tarragona, Spain S Supporting Information *

ABSTRACT: The aim of the present study was to investigate the in vitro activity of 15 essential oils, 4 essential oil fractions, and 3 pure compounds (thymol, carvacrol, and eugenol) on phagocytosis by human neutrophils and on complement system. Samples were characterized by GC and GC-MS. Most of the oils (nutmeg, clove, niaouli, tea tree, bay laurel, lemon, red thyme, ginger), nutmeg terpenes, eugenol, and carvacrol showed mild to moderate inhibition of phagocytosis (25−40% inhibition at doses ranging from 40 to 60 μg/mL); highest inhibitory activity was found for thymol (72% at 56 μg/mL), whereas the mixture of bornyl and isobornyl acetates showed a mild stimulating activity (21% at 56 μg/mL). All samples were inactive in the alternative pathway of complement system, whereas on classical pathway, clove oil, eugenol, palmarosa oil, red thyme oil, tarragon oil, and carvacrol showed the highest activity, with IC50 values ranging from 65 to 78 μg/mL. KEYWORDS: essential oils, phagocytosis, complement system, immune modulation, anticomplement effects

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INTRODUCTION Herbs and spices are popular food condiments due to their food flavor enhancement properties. Essential oils and oleoresins are usually responsible for this characteristic. However, modern society has begun to look at food not only for the basic nutrition it delivers but also for the health benefits it provides. Essential oils are reservoirs of bioactive compounds, and they have increased their appeal in society as they are aligned with current consumer preference for natural products. A host of beneficial effects have been reported for essential oils during the past decades, for instance, as antimicrobials, analgesics, antioxidants, and digestives. Essential oils and aromatic plants have also gained interest in animal feeding. Improvements in growth, feed consumption, and feed utilization and decreased mortality by the prophylactic use of antibiotics in animal nutrition are well documented. The use of antibiotics as growth promoters in animal production became virtually worldwide after the discovery of its benefits in the 1940s. However, the possibility of developing resistant populations of bacteria and the residue effects of using antibiotics as growth promoters such as allergy in farm animals have led to the European Union ban on the use of antibiotics on farm animals as feed additives. Evidence from some U.S. and European studies suggests that these resistant bacteria cause infections in humans that do not respond to commonly prescribed antibiotics. Hence, an intensive search for the replacement of antibiotics as feed additives has been taking place. New commercial additives derived from plants, including aromatic plants, their essential oils, and extracts, as well as some purified constituents, have been examined as alternative growth promoters. Such products have several advantages over commercial antibiotics because they have fewer residue © XXXX American Chemical Society

concerns and they are generally recognized as safe in the food industry.1 The antibacterial effect of essential oils is well established.2,3 For this reason they have gained much attention as alternatives to antibiotics. The antimicrobial mechanisms of essential oils are only partially known. Considering the large number of different groups of chemical compounds present in essential oils, it is most likely that their antibacterial activity might involve several different mechanisms. Lipophilic properties of the essential oil constituents allow them to penetrate bacterial membranes and reach the inner part of the cell, disturbing structures and rendering them more permeable. Moreover, the presence of phenol groups seems to play an important role in the antibacterial activity of essential oils. The strongest antibacterial properties against foodborne pathogens appear in volatile oils containing a high percentage of phenolic compounds such as carvacrol, eugenol, and thymol. In addition, synergistic effects between different constituents of the essential oils have been described.2,4 Besides those antibacterial properties that essential oils or their constituents have been shown to exhibit, other activities may contribute to the improvement of the health status of the animal when they are employed as feed additives.1 Among them, an improvement or modulation of the immune function may play a role. However, scientific literature on the immunemodulating activities of essential oils is limited and inconclusive,5 being a field open for new research. Received: October 11, 2014 Revised: January 7, 2015 Accepted: January 19, 2015

A

DOI: 10.1021/jf504761m J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

(NaCl), ethylenediaminetetraacetic acid (EDTA), trypan blue, propidium iodide, Hanks’ balanced salt solution (HBSS) with Ca2+ and Mg2+ or without Ca2+ and Mg2+, Alsever’s solution, gelatin veronal buffer, ammonium chloride (NH4 Cl), potassium bicarbonate (KHCO3), ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA-Na4·2H2O), dimethyl sulfoxide (DMSO), paraformaldehyde, quercetin, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), sodium 5,5-diethylbarbiturate, lipopolysaccharides (LPS) from Escherichia coli O127:B8, and human sera were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fluorescent microspheric hydrophilic particles (FluoSpheres) were purchased from Invitrogen, Molecular Probes (Carlsbad, CA, USA). Red blood cell polyclonal antibody anti-sheep were bought at ICN ibérica SA (Barcelona, Spain). Sheep red blood cells in Alsever’s solution and rabbit red blood cells in Alsever’s solution were obtained from the animal facilities of the Faculty of Veterinary of the Autonomous University of Barcelona and the Faculty of Pharmacy of the University of Barcelona, respectively. Essential Oils and Compounds Studied. Essential oils from nutmeg (Myristica fragrans Houtt.), niaouli (Melaleuca quinquenervia (Cav.) S. T. Blake), clove (Syzygium aromaticum (L.) Merr. & L. M. Perry), tarragon (Artemisia dracunculus L.), coriander (Coriandrum sativum L.), juniper (Juniperus communis L.), tea tree (Melaleuca alternifolia (Maiden & Betche) Cheel), ginger (Zingiber officinale Roscoe), rosemary (Rosmarinus officinalis L.), bay laurel (Laurus nobilis L.), palmarosa (Cymbopogon martini (Roxb.) Will. Watson), cajuput (Melaleuca cajuputi Powell), lemon (Citrus limon (L.) Burm. f.), red thyme (Thymus zygis L.), and Spanish oregano (Coridothymus capitatus (L.) Rchb. f. = Thymbra capitata (L.) Cav.) were tested. The terpenic fractions from nutmeg, clove, and lemon were also included in our research. Finally, the pure compounds eugenol, carvacrol, and thymol, as well as a mixture of bornyl (76.8%) and isobornyl (21.7%) acetates, were also investigated. All samples except eugenol (Sigma-Aldrich) were supplied by Lidervet S.L. (Tarragona, Spain) and obtained from commercial sources (Lluch Essence S.L., Barcelona, Spain, and Ernesto Ventós S.A., Barcelona, Spain). All samples were kept in sealed airtight glass vials, protected from the light, at 4 °C. Characterization and Analysis of the Essential Oils. The essential oils and fractions were characterized by their composition and some physical constants. The analyses of the composition of the oils were carried out by gas chromatography (GC-FID) and gas chromatography coupled to mass spectrometry (GC-MS), using a fused silica Supelcowax 10 capillary column (60 m × 0.25 mm i.d.; 0.25 μm film thickness). GC-FID analyses were performed on a Hewlett-Packard 6890 instrument, equipped with a HP ChemStation data processor software. Helium was used as carrier gas, and a split ratio of 1:80 was employed. Samples were injected undiluted (0.1 μL). Chromatographic conditions were different depending on the essential oil and, whenever possible, were based on the methods stated in the European Pharmacopeia.24 Mass spectra were obtained using a Hewlett-Packard GC-MS instrument constituted by a GC HP 6890 coupled to a mass selective detector HP 5973N. The same analytical conditions employed in the GC-FID were applied in the GC-MS. Mass spectra were taken over m/z 35−400, using an ionizing voltage of 70 eV. Identification of the constituents of the oils and fractions was achieved by means of their linear retention indexes calculated in relation to a homologous series of n-alkanes (C8−C23) from the GCFID analysis and by comparison of fragmentation patterns in their mass spectra with those stored in our own library, in the GC-MS database (Wiley 6), and with literature data.25,26 Percentages of constituents were determined on the basis of their GC-FID peak areas using the normalization procedure without corrections for response factor. Relative density, optical rotation, and refractive index of the essential oils and fractions were determined according to the European Pharmacopoeia.24 Sample Preparation for Biological Testing. Samples of the essential oil, fractions, and pure compounds were prepared at 1.0% (v/ v) in HBSS supplemented with 10% (v/v) DMSO and 1.5% (v/v) E484 (glyceryl polyethylene glycol ricinoleate). All solvents were

The immune system is a complex biochemical process that enables efficient detection and removal of pathogens that threaten host viability. Immune processes have traditionally been divided into two broad, but interconnected, subsystems on the basis of their functions in host defense: the adaptive immune system and the innate immune system. Innate immunity is composed of those immunological effectors that provide robust, immediate, and nonspecific immune responses. Phagocytosis by neutrophils and complement system are two key factors of the innate immunity. Neutrophils are the first line of the host defense mechanism against microbial infection. They are recruited to the site of infection and are capable of phagocytosis and killing a wide range of bacteria. Phagocytosis is an actin-dependent mechanism by which cells ingest large particles that are usually >0.5 μm in diameter.6 Complement system is a major effector mechanism of the innate immune system. It is a complex network of plasma and membrane-associated serum proteins, which can elicit highly efficient and tightly regulated inflammatory and cytolytic immune responses to infectious organisms, tissue damaged by physical, chemical, or neoplasm insults, and other surfaces identified as “nonself”. The complement can be activated by a cascade mechanism of the classical pathway (CP), the alternative pathway (AP), or the mannan binding lectin (MBL) associated serine protease pathway. Inappropriate activation of the system may lead to pathologic reactions in a variety of inflammatory and degenerative diseases such as dermatological diseases, rheumatoid arthritis, and gout. Although other activities of essential oils have been deeply researched, few studies have examined the influence of essential oils over phagocytosis. From a literature review on the subject, it appears that essential oils of Houttuynia cordata, Allium sativum, Cinnamomum zeylanicum, Eucalyptus globulus, and Rheum palmatum are capable of stimulating phagocytosis in experiments developed either in vitro or in vivo.7−11 An inhibitory effect on phagocytosis was found in Ocimum basilicum essential oil,12 whereas those from Salvia officinalis, Lavandula angustifolia, Melaleuca alternifolia, and Origanum minutiflorum showed no effect at all.10,13,14 Pure compounds have also been examined. 3-Carene was found to be a phagocytosis inhibitor in vitro.15 On the other hand, limonene, citronellyl acetate, linalyl acetate, and methyl nonyl ketone clearly stimulated phagocytosis,7,16−18 whereas camphor, menthol, and eucalyptol were found to be rather inactive.19 The activity of eugenol on phagocytosis has not been tested on untreated cells. Nevertheless, it produced a recovery in the phagocytic function in nicotine-treated mouse peritoneal macrophages.20 Additionally, mixtures containing eugenol, either in endodontic sealers or mixed with formaldehyde and cresol, showed some phagocytosis inhibitory activity, which cannot be unambiguously assigned to eugenol.21,22 Literature on the influence of the essential oils on complement activity is even more scarce. In fact, only one retracted paper23 has been found on this topic. The aim of the present study was to investigate the in vitro phagocytic and complement system modulating activities of 15 well-characterized essential oils, 4 essential oil fractions, and 3 pure compounds.

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MATERIALS AND METHODS

Chemicals and Reagents. Water was freshly taken daily from a Milli-Q system (Millipore, Bedford, MA, USA). Sodium chloride B


Article

Journal of Agricultural and Food Chemistry previously assayed to rule out any possible intrinsic activity. Samples were emulsified through exhaustive vortexing (10 min) and sonication (5 min, in ice). In the phagocytosis assay, nine dilutions that ranged from 5.0 × 10−3 to 7.8 × 10−7 % (v/v) in HBSS with Ca2+ and Mg2+ were tested. For the complement system assays, eight successive 2-fold dilutions (ranging from 6.67 × 10−2 to 5.21 × 10−4 % (v/v)) were prepared in barbital gelatin buffer with EGTA (for the AP essay) or barbital gelatin buffer with Ca2+ and Mg2+ (for the CP essay). Each dilution was thoroughly vortexed (10 min) immediately before use. Isolation of Human Leukocytes. Leukocytes were isolated through a controlled hemolytic shock with an ammonium chloride solution from buffy coats obtained from blood of healthy donors at the Blood and Tissue Bank of Catalonia. The pellet was suspended in HBSS without Ca2+ and Mg2+. Phagocytosis Evaluation by Flow Cytometry. The phagocytosis assay was carried out following a modification of the method described by Risco et al.27 A suspension (5.5 × 107 particles/mL) of fluorescent microspheric hydrophilic particles (2 μm in diameter) of modified carboxylate with a yellow-green fluorescence (505/515 nm), and opsonized using human serum (30 min at 37 °C), were used for the phagocytic test. In a microtiter plate, 200 μL of a suspension of human leukocytes (approximately 106 cells) was incubated with 20 μL of different treatments for 10 min at 37 °C. Treatments included a positive control of LPS (1 mg/mL). Fluorescent particles (20 μL) were added to all wells. To manage nonspecific adhesion, 50 μL of paraformaldehyde (1% w/v) was added to wells designated as nonspecific adhesion controls. Microtiter plates were again incubated for 45 min in a stirred thermally controlled chamber at 37 °C. Fifty microliters of stop solution (0.9% w/v NaCl and 0.02% w/v of disodium EDTA) at 0 °C were added to all wells. The EDTA captures cations necessary for the phagocytic process. With the purpose of discriminating viable cells, 2 μL of a propidium iodide solution (10 μg/mL) was added. Finally, 20 μL of quenching solution (trypan blue solution, 2.25 mg/mL) and 50 μL of paraformaldehyde (1% w/v) were added to all wells. A flow cytometer Cytomics FC 500 MPL system (Beckman Coulter, Inc., Brea, CA, USA) and Summit software v4.2 were employed for the analysis. Cellular viability was well over 95% in all experiments. The gate for neutrophils was established on the basis of the size and granularity. The acquisition process was stopped when 20000 viable neutrophils (negative to propidium iodide) were acquired and the green fluorescence histogram (515−548 nm) was obtained. Activity on phagocytosis (stimulation or inhibition) is calculated as percentage from phagocytic capacity (percentage of cells that ingested one or more particles), which is determined from the fluorescence distributions. Phagocytic capacity was corrected by taking in account the fluorescence due to nonspecific adhesion (particles adhered to the cell surface but not phagocyted). Examples of histograms are given as Supporting Information. Hemolytic Assay for Human Complement Activity. A hemolytic assay was used to determine the inhibition of the alternative pathway (AP) and classical pathway (CP) of complement activation as described by Klerx et al.,28 in human pooled serum. Assays were carried out in a microtiter plate, using barbital gelatin buffer with Ca2+ and Mg2+ (CP) or barbital gelatin buffer with EGTA (AP). Sensitized sheep erythrocytes (CP) or nonsensitized rabbit erythrocytes (AP) were incubated with the test samples for 30 min at 37 °C. Human pooled serum was added and incubated for 30 min (AP) or 60 min (CP) at 37 °C. The reaction was stopped by adding barbital buffer containing 10 mM EDTA. Erythrocytes were removed by centrifugation at 700g for 5 min, and the supernatant was transferred to a new flat-bottom microtiter plate. Absorbance was measured at 405 nm using a Benchmark Plus spectrophotometer (Bio-Rad, Hercules, CA, USA). Positive controls used were quercetin (CP) and an aqueous extract from Azadirachta indica (AP). Inhibition (%) was calculated from absorbance values of treated samples in relation to untreated controls. Statistical Analysis. The results were expressed as the mean ± standard deviation (SD) of at least four independent experiments. One-way analysis of variance (ANOVA) followed by a Dunnett’s test

was used to compare means of untreated controls to each treatment. In all cases, a difference was considered significant when p < 0.05. Fifty percent inhibitory concentration (IC50) was calculated by interpolation in a concentration/effect curve when possible. For data analysis, software Graph Pad Prism version 5.0 for Windows (San Diego, CA, USA) was used.

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RESULTS AND DISCUSSION

The in vitro activity of 15 essential oils, 4 essential oil fractions, and 3 pure compounds (thymol, carvacrol, and eugenol) on phagocytosis by human neutrophils and complement system was investigated. As will be shown in the present section, tested essential oils were mainly phagocytosis inhibitors, whereas some of them showed a moderate but significant complement inhibitory activity in the classical pathway. Those properties might be related in some measure to the anti-inflammatory activity attributed to essential oils.29 Because inflammation is a pathogenic factor in the evolution and origin of many diseases, essential oil consumption linked to culinary herbs and spices may prevent or at least alleviate the onset of various pathologies. Characterization of the Essential Oils and Fractions. The composition of the oils and fractions analyzed is shown in Table 1. Eugenol, carvacrol, and thymol had a purity that was well over 99.0%. The mixture of bornyl acetate and isobornyl acetate contained 76.8% of the former and 21.7% of the latter, together with 1.0% of α-terpineol. The oils were also characterized by physical data, such as relative density, optical rotation, and refractive index (see the Supporting Information). Activity on Phagocytosis by Human Neutrophils. Results on the activity of essential oils, fractions, and compounds tested on phagocytosis by human neutrophils are shown in Table 2. Most of the volatile oils tested inhibited phagocytosis, although the mixture of bornyl and isobornyl acetates showed a mild stimulating activity (21.3%) at the highest concentration tested (55.90 μg/mL). Some authors have also found stimulatory effects on phagocytosis for a few essential oils, such as those of Rheum palmatum L. and Allium sativum L., and for some pure constituents, such as citronellyl acetate and linalyl acetate.8,11,18 Essential oils from cajuput, tarragon, and coriander showed no activity whatsoever on phagocytosis at the concentrations tested. At the highest concentration tested, essential oils of nutmeg, clove, niaouli, tea tree, bay laurel, lemon, red thyme, and ginger, as well as nutmeg terpenes, eugenol, and thymol, met or exceeded the 25% inhibition of phagocytosis. Thymol was the most active inhibitor of phagocytosis, giving an IC50 of 1.5 μg/ mL (Figure 1). In an early study, thymol appeared as a moderate promoter of phagocytosis, but results were produced through an in vivo experiment.30 Red thyme essential oil, with thymol as its main constituent, caused an inhibition of phagocytosis greater than 25% (p < 0.05) at the highest evaluated concentration (47.15 μg/mL). Carvacrol and eugenol, the other two phenols tested in the present study, showed weak and mild inhibitory activity (Figure 1). Eugenol inhibited phagocytosis (29% at 57.6 μg/mL, p < 0.05) with a concentration−activity profile very similar to that of clove oil (Figure 2), suggesting that it is the major compound responsible for the activity of the oil. Literature reports on this compound have shown dissimilar results. In vitro evaluations of eugenol by different authors resulted in a reduction of phagocytosis.21,22 In other in vitro experiments, it was found that eugenol was able to recover macrophage C


14.9

0.8

D

0.6

0.7

2.8 0.6

0.7

1.0

1.0

3.1

1.7

9.1

83.0

1.6 5.7 74.0

0.3

1.8 0.5

2.2

1.6

3.7

67.8

65.6

2.1

0.7

5.7

0.6 1.1

0.5

1.4

19.0

0.4

0.4

1.1 0.5

0.4

0.7

0.6

7.9

7.3 0.4 1.0

0.9 0.4

2.2

GI

5.1

CO

1.4

CLT

4.4 7.5

CL

1.8

5.6

α-pinene α-thujene camphene 6-methyl-5-hepten-2-one β-pinene sabinene Δ3-carene myrcene α-phellandrene α-terpinene limonene β-phellandrene 1,8-cineole cis-β-ocimene γ-terpinene trans-β-ocimene p-cymene terpinolene α-cubebene trans-sabinene hydrate γ-elemene linalool oxide α-copaene camphor linalool cis-sabinene hydrate β-elemene bornyl acetate β-caryophyllene isosativene terpinen-4-ol myrtenal sativene trans-carveol trans-pinocarveol trans-verbenol β-farnesene aromadendrene ar-curcumene estragole δ-terpineol α-humulene

CA

BLa

constituent

Table 1. Composition of the Essential Oils and Fractions Investigated JU

0.4

0.4

0.3

0.5

4.1

29.2 0.5 0.5 7.6 0.2

0.4

54.8

0.3

1.6 0.3

6.8

70.3 0.4

1.6

12.0 2.1

1.9 0.4

LE

0.1

4.3

1.4

69.1 0.3

1.7

16.6 2.7

3.0 0.6 0.1

LET

2.4

2.4 0.9

1.2

58.3

7.1

2.7

10.9

NI

4.4

1.3 1.5

3.4

2.1 4.1 2.6

11.4 40.8 1.1 3.3

14.3 2.0

NU

percentages in the essential oils and fractionsa

0.7 0.7

0.7

0.5

17.2 22.1

50.9 3.7 0.7

NUT

2.0

3.1

1.2

0.4

0.2

PA

1.6

1.2

4.5

0.2

23.2

4.6

0.6

1.2 0.6

1.8

0.4

1.2 0.6 0.8

RT

1.0

0.7

1.3 3.9

9.6 0.8

1.7

0.4

49.7

1.4

1.3 2.1

3.3

0.7

1.2 0.4

0.2

8.1

5.4

0.3 0.8

1.9

0.8 1.4

SO

9.7

9.9 0.4 4.3

RO

87.8

3.2

4.8

2.5

0.8

TA

1.7

0.5

44.5

9.3 2.9

16.5

4.5

5.9 1.1

0.8 0.7

2.5 1.2

TT

Journal of Agricultural and Food Chemistry Article


neral geranyl formate α-terpenyl acetate α-terpineol borneol ledene selinene germacrene D geranial zingiberene valencene β-bisabolene bicyclogermacrene E,E-α-farnesene δ-cadinene geranyl acetate γ-cadinene mirtenol β-sesquiphellandrene cis-α-bisabolene nerol cis-calemenene geraniol safrole geranyl butyrate geranyl valerate caryophyllene oxide β-caryophyllene oxide guaiol nerolidol viridiflorol α-cedrol eugenol elemol thymol elemicin carvacrol α-eudesmol β-eudesmol myristicin eugenyl acetate farnesol

constituent

Table 1. continued

1.3 6.4

BLa

E

0.4 0.5

0.6

0.8

11.6

CA

1.3

81.5

0.5

CL

2.7

0.4

0.8

CLT

1.5

3.2

0.5

CO

0.4

0.6

12.2

2.9

3.7

9.2

22.3

0.6

0.5 0.9

GI

0.5

0.3

JU

0.2

0.3 0.4

0.8

0.3

0.3

LE

LET

2.3 4.9

1.2 6.0

NI

4.3

2.3

1.1

NU


0.6

NUT

1.1

0.7

0.4

0.2 0.2

80.3

0.2

8.1

0.9

0.2 0.2

PA

4.3

50.0

0.4

0.4

2.0

RT

1.7 1.5

RO

72.7

0.4

0.7

0.3

SO

TA

0.6

1.5

1.4

0.6

3.3

TT

Journal of Agricultural and Food Chemistry Article


Article

99.5 99.1 100.0 100.0 99.6 99.5 97.8 100.0 100.0 99.9 100.0 99.7 95.4 100.0 98.4 99.9 99.3 100.0 identified (%)

Essential oils and fractions: BL, bay laurel; CA, cajuput; CL, clove; CLT, clove terpenes; CO, coriander; GI, ginger; JU, juniper; LE, lemon; LET, lemon terpenes; NI, niaouli; NU, nutmeg; NUT, nutmeg terpenes; PA, palmarosa; RT, red thyme; RO, rosemary; SO, Spanish oregano; TA, tarragon; TT, tea tree.

phagocytic capacity previously diminished by treatment with nicotine.20 Other volatile compounds and essential oils, such as 3-carene and basil oil, have been dubbed in the literature as phagocytic inhibitors.12,15 In in vivo and in vitro experiments, limonene (the main constituent of lemon oil and lemon terpenes) showed a stimulating effect over phagocytosis or the recuperation of the macrophages phagocytic capacity previously reduced.16−18 Those results differ from ours, although it is important to highlight that employed cells and methodologies were different. Essential oils from juniper, rosemary, palmarosa, and Spanish oregano, lemon terpenes, and carvacrol showed a significant inhibition of phagocytosis, but it was lower than 25% at the highest tested concentration. The measured activity of carvacrol was lower than that of its isomer, thymol (Figure 1). In some situations, a decreased phagocytic activity caused by essential oils could be a good strategy to reduce viral and bacterial infection. There is evidence that certain microbes subvert neutrophil protective functions and that neutrophils could, in fact, promote susceptibility to infection. This is due to the fact that intracellular invasion achieved with the phagocytic process can allow, in certain infections, pathogen proliferation and protection. This could be the case with leishmania parasites, Chlamydia pneumoniae or Mycobacterium tuberculosi, Epstein−Barr, and cytomegaloviruses.31 It has been shown that a selective depletion, in vivo, of neutrophils by monoclonal antibody administration did provide a protective effect against Chlamydia pneumoniae in infected mice.32 Moreover, antibiotics are also known for their modulating effects on many components of the human immune system, in addition to their direct antimicrobial effects. For example, macrolide antibiotics cause a significant reduction in the chemotactic response of neutrophils to chemokines, resulting in a markedly decreased airway neutrophilia in patients with various inflammatory pulmonary diseases.33 More than four decades earlier Forsgren and Schmeling34 had reported a similar inhibition of neutrophil chemotaxis by rifampin, tetracyclines, and chloramphenicol. Certainly, a body of evidence has emerged indicating that both antibiotics and essential oils possess modes of action which are independent of their antimicrobial activity when facing an infection. In addition, neutrophils express Fc receptors, which are responsible for phagocytosis and intracellular killing of opsonized microbial pathogens, and it has been reported that this response can be blocked by inhibitors of ROS and by inhibitors of the H2O2−myeloperoxidase−chloride system.35 Both activities have been reported in many of these essential oils in preliminary studies of our group,36 showing consistency of the relationship between ROS inhibition and phagocytosis reduction. Activity on Human Complement System. Results of the hemolytic assay for human complement activity are shown in Table 2. Different degrees of inhibitory activity were evident on the classical pathway of the complement system, whereas all essential oils were found inactive in the alternative pathway experiment. No other results on essential oils activity on complement system have been found in the literature. Nevertheless, a similar pattern of activity has also been described for other extracts or substances of terpenoid nature.37 A closer look at individual results of the classical pathway shows that eugenol and clove oil had similar moderate IC50 values (78.3 and 74.9 μg/mL, respectively). This, together with the fact that clove terpenes showed almost no activity, points to

a

NU NI LET LE constituent

Table 1. continued

BLa

CA

CL

CLT

CO

GI

JU


NUT

PA

RT

RO

SO

TA

TT


F


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Journal of Agricultural and Food Chemistry Table 2. Activity of Essential Oils, Their Fractions, and Constituents on Phagocytosis by Human Neutrophils and on Complement System phagocytosis

complement system inhibition IC50 (μg/mL)

essential oils, fractions, and constituents

concentration (μg/mL)

activity (% ± SD)

classical pathwayc

alternative pathwayd

bay laurel bornyl acetate + isobornyl acetate cajuput clove clove terpenes coriander eugenol ginger juniper lemon lemon terpenes niaouli nutmeg nutmeg terpenes palmarosa red thyme thymol rosemary Spanish oregano carvacrol tarragon tea tree

45.75 55.90 46.75 50.40 45.35 45.55 57.60 47.00 22.40 45.05 43.50 46.70 46.35 43.85 47.75 47.15 55.80 48.40 49.15 52.05 46.80 48.00

−38.7 ± 3.1* 21.3 ± 2.6* 4.1 ± 8.7 −36.1 ± 15.5* −14.6 ± 1.7* −12.8 ± 2.2 −29.4 ± 5.2* −28.5 ± 1.4* −24.3 ± 10.5* −41.0 ± 14.2* −9.7 ± 6.4* −39.1 ± 28.3* −33.8 ± 2.7* −38.5 ± 2.0* −22.1 ± 1.9* −38.0 ± 1.0* −72.2 ± 25.0* −11.3 ± 2.5* −16.2 ± 2.2* −14.9 ± 4.1* −12.9 ± 1.4 −35.2 ± 20.2*

155.4 ± 3.5 126.1 ± 4.1 97.3 ± 4.6 74.9 ± 3.2 nc 154.4 ± 3.5 78.3 ± 3.9 nc nc nc nc 91.3 ± 4.2 nc nc 75.2 ± 3.0 67.8 ± 2.9 125.5 ± 4.4 nc 118.1 ± 7.8 78.1 ± 4.7 64.8 ± 3.4 97.0 ± 4.2

na na na na na na na na na na na na na na na na na na na na na na

a

b

Highest concentration tested. bn = 4; *, statistically significant (p < 0.05). cn = 6; nc, activity did not allaw calculation of IC50; positive control, quercetin (IC50 = 33.7 μg/mL). dn = 4; na, not active; positive control, aqueous extract from Azadirachta indica (IC50 = 226.4 μg/mL).

a

eugenol as the source of activity in clove oil (Figure 3). Other phenol-rich essential oils (red thyme, Spanish oregano, and

Figure 1. Activity of eugenol, thymol, and carvacrol on phagocytosis by human neutrophils (stimulation %, mean ± SD).

Figure 3. Activity of clove oil, clove terpenes, and eugenol on the classic pathway of the human complement system (inhibition %, mean ± SD).

tarragon) also displayed a similar mild inhibitory activity. It is interesting to highlight that carvacrol, the main constituent of the Spanish oregano oil, was more active that the essential oil, whereas thymol, the major component of red thyme oil, was less active than the essential oil. Maybe in red thyme essential oil other substances take part in its activity, whereas carvacrol remains as the main source of activity for Spanish oregano. All phenols tested, either monoterpenes (thymol and carvacrol) or phenylpropanoids (eugenol), had a similar behavior (Figure 4). In the essential oils tested from the Melaleuca genus, some activity was found, although it was moderate. This was the case

Figure 2. Activity of clove oil and eugenol on phagocytosis by human neutrophils (stimulation %, mean ± SD). G



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Article

AUTHOR INFORMATION

Corresponding Author

*(S.C.) Phone: +34 934 024531. Fax: +34 934 035982. E-mail: [email protected]. Present Address ⊥

(E.R.) Phytonexus S.L.

Funding

This work was financially supported by Lidervet S.L. (Tarragona, Spain). R.P.-R. enjoyed a grant from the Generalitat de Catalunya and the European Social Fund during the development of this research. Notes

All biological samples, either from animals or humans, were obtained under the relevant ethical approval, by the Ethical Committees of the Faculty of Pharmacy of the University of Barcelona and the Blood and Tissue Bank of Catalonia. The authors declare the following competing financial interest(s): Pedro Peñalver is shareholder of the company Lidervet S.L. (Tarragona, Spain). The work presented in this paper was financially supported by Lidervet S.L.

Figure 4. Activity of eugenol, carvacrol, and thymol on the classic pathway of the human complement system (inhibition %, mean ± SD).

of niaouli, tea tree, and cajuput oils. Tea tree oil is rich in terpinen-4-ol (44.5%), whereas in niaouli and cajuput oils 1,8cineole is the major constituent (58.3 and 67.8%, respectively). Essential oils of bay laurel (IC50 = 155.4 μg/mL) and rosemary (showing a 27% inhibition at 154.7 μg/mL, p < 0.01) were also rich in 1,8-cineole (49.7 and 65.6%, respectively), which suggests a possible role of this compound in the anticomplementary activity. Other oxygenated monoterpene-rich oils and fractions showed moderate to weak activities, with IC50 values of 75.2 μg/mL for palmarosa oil (with 80.3% of geraniol), 126.1 μg/mL for the mixture of bornyl and isobornyl acetates, and 1540.4 μg/mL for coriander oil (with 74.0% of linalool). Results confirm that oxygen-containing compounds (either phenylpropanoids or monoterpenes) confer higher inhibitory activity of the classical pathway of the complement system than monoterpene hydrocarbons. Among oxygen-containing compounds, phenols, especially eugenol and carvacrol, showed the best profile. Hemolytic effects of the essential oils tested were also examined. In vitro testing for hemolytic action has been used as one of the methods of triage for different toxic agents. Hemolysis tests are a simple tool to detect membrane toxicity and cytotoxicity. Most of the essential oils studied caused erythrocyte lysis at concentrations >200 μg/mL, showing thus a low toxicity toward membranes. The most noticeable action on erythrocyte membrane integrity was rendered by juniper oil and nutmeg terpenes, which caused erythrocyte membrane lysis at concentrations

Effect of Some Essential Oils on Phagocytosis and Complement

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