Effect of Essential Oils on the Release of TNF-α and CCL2 by LPS-Stimulated THP‑1 Cells

Plants and their constituents have been used to treat diverse ailments since time immemorial. Many plants are used in diverse external and internal formulations (infusions, alcoholic extracts, essential oils (EOs), etc.) in the treatment of inflammation-associated diseases, such as those affecting the respiratory tract or causing gastrointestinal or joint problems, among others. To support the traditional uses of plant extracts, EOs have been assessed for their alleged anti-inflammatory properties. However, the effect of EOs on the release of cytokines and chemokines has been much less reported. Considering their traditional use and commercial relevance in Portugal and Angola, this study evaluated the effect of EOs on the in vitro inhibition of the cytokine tumor necrosis factor-α (TNF-α) and the chemokine (C-C motif) ligand 2 (CCL2) by lipopolysaccharide (LPS)-stimulated human acute monocytic leukemia cells (THP-1 cells). Twenty EOs extracted from eighteen species from seven families, namely from Amaranthaceae (Dysphania ambrosioides), Apiaceae (Foeniculum vulgare), Asteraceae (Brachylaena huillensis, Solidago virgaurea), Euphorbiaceae (Spirostachys africana), Lamiaceae (Lavandula luisieri, Mentha cervina, Origanum majorana, Satureja montana, Thymbra capitata, Thymus mastichina, Thymus vulgaris, Thymus zygis subsp. zygis), Myrtaceae (Eucalyptus globulus subsp. maidenii, Eucalyptus radiata, Eucalyptus viminalis) and Pinaceae (Pinus pinaster) were assayed for the release of CCL2 and TNF-α by LPS-stimulated THP-1 cells. B. huillensis, S. africana, S. montana, Th. mastichina and Th. vulgaris EOs showed toxicity to THP-1 cells, at the lowest concentration tested (10 μg/mL), using the tetrazolium dye assay. The most active EOs in reducing TNF-α release by LPS-stimulated THP-1 cells were those of T. capitata (51% inhibition at 20 μg/mL) and L. luisieri (15–23% inhibition at 30 μg/mL and 78–83% inhibition at 90 μg/mL). L. luisieri EO induced a concentration-dependent inhibition of CCL2 release by LPS‑stimulated THP-1 cells (23%, 54% and 82% inhibition at 10, 30 and 90 μg/mL, respectively). These EOs are potentially useful in the management of inflammatory diseases mediated by CCL2 and TNF‑α, such as atherosclerosis and arthritis.


Introduction
Since ancient times, man has used the plant kingdom as a source for clothing, construction, fuel, food, spices and medicines, as well as for poisons. Nowadays, around half the pharmaceutical drugs used in developed countries, such as aspirin, are of plant origin [1]. Traditional medicine is still the main source of health care for 80% of the people in developing countries, where medicinal plants are commonly used for the treatment of several ailments, notably inflammatory diseases. Timber and oleoresin production [33] pt/en: Official two-letter codes of Portuguese and English languages, respectively. * African name adopted in Portuguese. ** African name given to the wood and adopted in Portuguese and English.
Chemokines constitute a family of chemoattractant cytokines. These are small heparinbinding proteins involved in atherosclerosis by promoting directed migration of inflammatory cells. Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), has been detected in atherosclerotic lesions [34]. CCL2 is also a potent mediator of chronic inflammation, triggering, for instance, inflammation in rheumatoid arthritis [35]. In addition, inflammatory response is characterized by increased production of tumor necrosis factor-α (TNF-α) [35]. TNF-α, interleukin (IL)-1 and IL-6, secreted by macrophages, lymphocytes, natural killer cells and vascular smooth muscle cells, are considered pro-atherogenic cytokines [36]. Despite the reported anti-inflammatory potential of several EOs (Table 2), their effect on the release of CCL2 is much less reported than the release of TNF-α. Table 2. Previously reported anti-inflammatory activity of the essential oils (EOs) from the species under study.
[51] Essential oils are gaining commercial relevance in several countries, such as Portugal or Angola, as an additional source of income in a context of a more sustainable use of the local flora. Nevertheless, despite these essential oils being traded, national or internationally, for specific markets, it is ever more important for their added value to gather scientific support for their alleged biological properties. Given the traditional and commercial use of EOs for medicinal and cosmetic purposes and the knowledge of the ability, of their monoterpene and sesquiterpene constituents, to act as anti-inflammatories [56,57], the present work evaluated twenty EOs obtained from eighteen plant species collected in Portugal and Angola (Table 1) for their effect on the release of CCL2 (MCP-1) and TNF-α by lipopolysaccharide (LPS)-stimulated THP-1 cells.

Plant Material
Collective and/or individual samples, from cultivated and wild-growing medicinal and aromatic plants, were collected from mainland Portugal ( Table 3). As a rule, the plant material was collected during the local producers' harvesting season. For herbaceous species, this was usually at the flowering phase, whereas for trees, it was at landscaping time. If not immediately extracted, the plant material was stored at −20 • C until essential oil (EO) isolation. Dried aerial parts from commercially available products sold in local herbal shops were also analyzed, as well as the essential oils isolated from oleoresin, in the case of Pinus pinaster, and from the two species from Angola (Table 3). A total of twenty essential oils isolated from eighteen species from the Amaranthaceae, Apiaceae, Asteraceae, Euphorbiaceae, Lamiaceae, Myrtaceae and Pinaceae families were tested. A voucher specimen of each plant species, collected from the wild state condition, was deposited in the Herbarium of the Botanical Garden of Lisbon University, Lisbon, Portugal. For commercial plant material, a reference sample from each plant is retained at the CBV laboratory and is available upon request.

Extraction and Chemical Analysis of the Essential Oils
Essential oils were extracted by hydrodistillation for 3 h, using a Clevenger-type apparatus, according to the European Pharmacopoeia [59], and stored at −20 • C until analysis. The EOs were analyzed by gas chromatography (GC) for component quantification and gas chromatography coupled to mass spectrometry (GC-MS) for component identification.

Gas Chromatography (GC)
Gas chromatographic analyses were performed using a Perkin Elmer Clarus 400 gas chromatograph equipped with two flame ionization detectors (FIDs), a data handling system and a vaporizing injector port into which two columns of different polarities were installed: a DB-1 fused-silica column (polydimethylsiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column ((50% phenyl)-methylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific Inc.). The oven temperature was programmed from 45 to 175 • C, at 3 • C/min, and subsequently at 15 • C/min up to 300 • C, and then held isothermal for 10 min; injector and detector temperatures were 280 • C and 300 • C, respectively; the carrier gas, hydrogen, was adjusted to a linear velocity of 30 cm/s. The samples were injected using a split sampling technique, ratio 1:50. The volume of injection was 0.1 µL of n-pentane-essential oil solution (1:1). The percentage composition of the volatiles was computed, by the normalization method from the GC peak areas, and calculated as the mean values of two injections, from each sample, without using the response factors.

Gas Chromatography-Mass Spectrometry (GC-MS)
The GC-MS unit consisted of a Perkin Elmer Clarus 600 gas chromatograph, equipped with a DB-1 fused-silica column (30 m × 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific, Inc.), and interfaced with a Perkin Elmer 600T mass spectrometer (software version 5.4.2.1617, Perkin Elmer, Shelton, CT, USA). Injector and oven temperatures were as above; transfer line temperature, 280 • C; ion source temperature, 220 • C; the carrier gas, helium, was adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; scan range, 40-300 u; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to n-alkane indices and GC-MS spectra from a lab-made library, created with reference essential oils, laboratory-synthesized components, laboratory-isolated compounds and commercially available standards.

In Vitro Inhibition of TNF-α and CCL2
This assay was performed according to Campana et al. [60]. Briefly, THP-1 cells (ATCC TIB-202) were cultivated in RPMI 1640 medium supplemented with 0.05 mM 2mercaptoethanol, 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL gentamicin at 37 • C in an atmosphere containing 5% CO 2 . The medium was renewed twice a week when the cell concentration reached 1.0 × 10 6 cells/mL. The cells were transferred to a 96-well microplate at a concentration of 100,000 cells per well and incubated for 18 h with RPMI supplemented with 1% FBS to initiate serum starvation, which was kept throughout the experiment.

Family/Species and Control
Concentrations (µg/mL)  Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma), added at 200 ng/mL, was employed as the inflammatory stimulus. The plate was incubated at 37 • C overnight. After this period, the plate was centrifuged (1800 g, 5 min, 16 • C), the supernatant collected and TNF-α release measured using the cytokine-specific sandwich quantitative ELISA according to the manufacturer's instructions (TNF-α duo set, DY210, R&D Systems, Minneapolis, MN, USA). CCL2 release was measured using the cytokine-specific sandwich quantitative ELISA according to the manufacturer's instructions (Human CCL2/MCP-1 duo set, DY279, R&D Systems, Minneapolis, MN, USA). Dexamethasone was employed as positive control (0.3 µM). The statistical significance of differences was calculated employing the software GraphPad Prism, version 5.0 (GraphPad Software Inc., San Diego, CA, USA), using ordinary one-way ANOVA/Newman-Keuls multiple comparison test. All the experiments were performed in triplicate.

Chemical Composition of the Essential Oils
All essential oils were fully chemically characterized. Table 3 reports only their main constituents (≥10%), since, in some cases, duly marked in Table 3, the detailed composition was previously reported, or their composition was overall very similar to data from prior studies. In the case of EOs from Angola, the complexity of the EOs still requires additional characterization for the full identification of some minor components.
Although a few studies evaluated the EO composition from Brachylaena huillensis aerial parts, only three studies reported the essential oil composition from the wood or saw powder of this species [16,17,70]. Although no detailed composition has been reported, α-amorphene was the dominant constituent in the studies of Klein and Schmidt [70] and of Maitai et al. [16] (17% and 15%, respectively), whereas β-caryophyllene (19%) was the major constituent described by Oliva et al. [17]. In the present study, α-amorphene was the second main component, together with gleenol (both 6%), whereas β-caryophyllene was found only in trace amounts. Baarschers et al. [20] reported the isolation of diterpenes from Spirostachys africana wood, but no previous studies addressed the EO composition from the wood.

In Vitro Inhibition of TNF-α Release by LPS-Stimulated THP-1 Cells
The potential anti-inflammatory activity of essential oils (EOs) was investigated by measuring TNF-α release by lipopolysaccharide (LPS)-stimulated THP-1 cells by employing an immunoassay. The toxicity of the EOs on THP-1 cells was accessed to determine the adequate EO working concentrations. When the cell viability of THP-1 cells was higher than 90%, samples were considered non-cytotoxic and adequate for further analysis. According to the availability of EO, at least three concentrations were checked for each essential oil (Table 4). Data in Table 4 also include information on EOs which were not assessed further due to being toxic (ND) to differentiate them from those that showed no inhibition (NI).
The potential anti-inflammatory activity of L. luisieri, F. vulgare and T. capitata EOs has been previously reported using different in vitro and in vivo models ( Table 2), but as far as we know, the anti-inflammatory potential of D. ambrosioides EO has not been addressed to date. Recently, the anti-inflammatory activity of alcoholic or hydroalcoholic extracts of D. ambrosioides was reported as showing the ability to reduce interleukin 6 (IL-6), myeloperoxidase (MPO), nitric oxide (NO) and adenosine-deaminase (ADA) activity and TNF-α and, therefore, they are potentially useful in wound healing and in the treatment of arthritic processes [13,71]. The oxygen-containing monoterpene ascaridole was identified as a constituent of D. ambrosioides ethanolic extract by Grassi et al. [13], a compound also identified in the essential oils evaluated in the present work (Table 3).
Despite carvacrol being the main compound of T. capitata EO (Table 3), this phenol-like oxygen-containing monoterpene may not be the only compound accountable for T. capitata EO activity. Indeed, other carvacrol-rich EOs, such as those of S. montana and Th. zygis (Table 3), were not able to reduce TNF-α release. The presence of antagonists in these EOs can also not be ignored. Moreover, it is relevant to highlight the important role of the minor compounds and/or some of the compounds' enantiomeric ratio in the overall activity of EOs. Often overlooked, these factors can contribute to synergistic or antagonistic actions determining differences in the EOs' activities [56,72]. These results make it difficult to predict the effect of different species' essential oils that share the same major component for TNF-α release.
Th. pulegioides and, particularly, Th. vulgaris EOs, with thymol, an isomer of carvacrol as the main component (Table 3), were toxic for THP-1 cells, even at lower concentrations (Table 4). Th. vulgaris EO has been reported to show anti-inflammatory activity, including the capacity of reducing TNF-α release, this activity being related solely to the higher carvacrol content [37,49,50,52]. On the other hand, Th. zygis and Th. vulgaris EOs, which have thymol as the main constituent, have been reported to decrease TNF-α secretion by human macrophages derived from THP-1 monocytes and activated by oxidized (ox)-LDLs [51]. Dexamethasone at 0.3 µM had > 90% inhibition.

In Vitro Inhibition of CCL2 Release by LPS-Stimulated THP-1 Cells
Inflammatory changes in arterial lesions are characterized by the recruitment and activation of monocytes/macrophages, which are regulated by CCL2. This chemoattractant cytokine has been shown to play a vital role in the initiation and progression of arteriosclerotic lesions in experimental animals [73]. The effect of the essential oils on CCL2 release by LPS-stimulated THP-1 cells was also evaluated.
Along with L. luisieri EO, the ascaridole-and iso-ascaridole-rich D. ambrosioides EO was also able to reduce CCL2 release by LPS-stimulated THP-1 cells, as observed for TNF-α, although in a weaker manner (Table 4). The absence of these compounds in the remaining non-active EOs may suggest that these volatile compounds have an important role in the suppression of some inflammatory processes in which TNF-α and CCL2 are involved. Despite the traditional application of D. ambrosioides as a vermifuge and against vomiting [14], this is the first report on the effect of its essential oil on the release of the pro-inflammatory cytokine TNF-α and the chemokine CCL2. For this reason, this EO and its main component ascaridole, and/or its isomers, should be further investigated to explore their anti-inflammatory activity.

Conclusions
Inflammatory disorders are usually treated with steroidal anti-inflammatory drugs (SAIDs) or non-SAIDs (NSAIDs). Nevertheless, because these drugs present multiple negative side effects, it is important to assess and validate the use of other potential antiinflammatory agents, namely, essential oils. Moreover, some of these essential oils are by-products from landscaping activities or other industries, thus constituting an added value to countries' local flora.
EOs' chemical complexity and variability (existence of chemotypes and/or the enantiomeric ratio of some components), their hydrophobicity and, sometimes, their scarcity, have been considered some of the limitations to their use in diverse formulations. Nevertheless, EOs are Generally Regarded as Safe (GRAS), and the knowledge on their biological properties should be further explored, in solo formulations and in combination therapies, as potential anti-inflammatory agents. This approach would contribute to the goal of decreasing the use of SAIDs and, therefore, preventing or diminishing these drugs' adverse side effects.

Convention on Biodiversity:
The authors obtained, and acknowledge, the appropriate authority to access plant samples, other than commercially available plant material, essential oils or oleoresin, used for research as required under the framework of the United Nations Convention on Biodiversity.

Conflicts of Interest:
The authors declare no conflict of interest.