Anti-Inflammatory, Antioxidant, Antibiotic, and Cytotoxic Activities of Tanacetum vulgare L. Essential Oil and Its Constituents

Background: Tanacetum vulgare L. (Asteraceae) is a perennial herb that has been used to treat multiple ailments. Regional variability of the chemical composition of T. vulgare essential oils is well-known. Despite these regional chemotypes, most relevant studies did not analyze the complete chemical composition of the T. vulgare essential oil and its constituents in relation to their biological activities. Here, we assess the anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities of T. vulgare collected from northern Quebec (Saguenay-Lac-St-Jean), Canada. Methods: Essential oil was extracted from plants by steam distillation and analyzed using GC-FID. Biological activities of essential oil and its main constituents were evaluated in vitro. Results: We identified the major compounds as camphor, borneol, and 1,8-cineole. The oil possesses anti-inflammatory activity inhibiting NO production. It also inhibits intracellular DCFH oxidation induced by tert-butylhydroperoxide. Anti-inflammatory activity of essential oil appears driven mainly by α-humulene while antioxidant activity is provided by α-pinene and caryophyllene oxide. Essential oil from T vulgare was active against both Escherichia coli and Staphylococcus aureus with camphor and caryophyllene oxide responsible for antibacterial activity. Finally, T. vulgare essential oil was slightly cytotoxic against the human healthy cell line WS1 while α-humulene and caryophyllene oxide were moderately cytotoxic against A-549, DLD-1, and WS1. Conclusion: We report, for the first time, links between the specific compounds found in T. vulgare essential oil and anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities. T. vulgare essential oil possesses interesting biological properties.


Introduction
Tanacetum vulgare L. is a perennial herb of the Asteraceae family. This plant grows widely in Europe and Asia. Introduced into North America in the 18th century, it is well adapted to northern climates [1]. Traditionally, T. vulgare has been used as an anthelmintic, tonic, emmenagogue, antihypertensive, carminative, antispasmodic, antidiabetic, diuretic, and an anti-inflammatory compound [2,3]. Most alcohol extracts of T. vulgare are reported to have biological effects including anti-inflammatory, antioxidant, and antibacterial properties. Indeed, studies on rats and mice [4,5] have found T. vulgare extracts to have anti-inflammatory properties. Flavonoids present in the plant could be, in part, responsible for these biological effects [5,6]. Shinella et al. demonstrated that parthenolide was the main in vivo anti-inflammatory substance in T. vulgare [5]. Methanol extracts from T. vulgare showed antioxidant activity, probably due to the presence of phenolic compounds [7]. The antibacterial properties of ethanolic extracts have also been reported in studies using Escherichia coli and Staphylococcus aureus [8,9].
In our study, the essential oil of T. vulgare, collected from an area in northern Quebec (Saguenay-Lac-St-Jean, Chicoutimi, QC, Canada), was extracted by steam distillation and analyzed using GC-FID (Agilent Technologies, Santa Clara, CA, USA). We assessed the essential oils and its constituents for anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities.

Plant Material and Extraction of Essential Oil
The aerial portions of T. vulgare were collected in July 2014 in Chicoutimi, Quebec, QC, Canada. Voucher specimen no. QFA0610840 has been filed at the Louis-Marie herbarium of Laval University, Quebec, QC, Canada. Essential oil was obtained from freshly harvested aerial parts by industrial steam distillation over a 3-h period (Groupe BoréaRessources, Ferland-Boileau, QC, Canada). The essential oil extraction yields depended on the total amount of the raw material. The extracted essential oil was then stored in the dark at a temperature of 4 • C.

GC-MS Analysis
All chromatographic analyses were run on an Agilent 6890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a non-polar DB-5 column (Agilent Technologies, Santa Clara, CA, USA) and a polar SolGel-Wax column (30 m × 0.25 mm × 0.25 mm) (Agilent Technologies, Santa Clara, CA, USA) as well as two FID detectors (Agilent Technologies, Santa Clara, CA, USA). The oils were injected in an undiluted (0.1 µL injection volume, split 1:235) and undried state. The temperature program was 40 • C for 2 min, 2 • C·min −1 up to 210 • C, and then 210 • C for 13 min. Samples were also injected on an Agilent 7890A GC (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 5975C InertXL EI/CI mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), equipped with a DB-5MS column (Agilent Technologies, Santa Clara, CA, USA) using the same temperature program as above and a split of 1:1000. Compounds were identified from their retention indexes as calculated from even-numbered C8 to C36 alkane standards and/or from MS databases NIST08, HPCH 2205 and custom libraries built from pure compounds (Laboratoire LASEVE, UQAC, QC, Canada). Quantification was derived from the FID detector response on the DB-5 column without any correction factor. All standards were co-injected to validate the identification.

Measurement of Anti-Inflammatory Activity
We evaluated the inhibition NO production by T. vulgare essential oil and compounds as described by Legault et al. [23]. Control L-NAME was used as positive control. Murine macrophage RAW 264.7 cells were incubated with essential oil or dissolved compounds in DMSO and then stimulated with 100 ng·mL −1 LPS and incubated at 37 • C. After 24 h, we collected the cell-free supernatants and immediately determined the NO concentration using the Griess reaction. We read the absorbance at 540 nm using an automated Varioskan Ascent plate reader (Labsystems, Milford, MA, USA), and quantified the presence of nitrite by comparing with a NaNO 2 standard curve.

Evaluation of Antioxidant Activity Using Cell-Based Assays
Antioxidant activity was evaluated using the DCFH-DA assay as described by Girard-Lalancette et al. [24]. To assess antioxidant activity, we incubated the human skin fibroblasts WS1 for 1 h with a growing concentration of essential oil or dissolved compounds in DMSO. We then added 100 µL of 200 µM tert-butylhydroperoxide and immediately measured fluorescence as well as again after 90 min later. Measurements were performed on an automated plate reader (Fluoroskan Ascent FL, Labsystems, Milford, MA, USA) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Antioxidant activity was expressed as the concentration of extract inhibiting 50% (IC 50 ) of DCFH oxidation.

Evaluation of Antibacterial Activity
We tested the antibacterial activities of T. vulgare essential oil and compounds against gram-negative Escherichia coli (ATCC bacteria 25922) and gram-positive Staphylococcus aureus (ATCC bacteria 25923) using the antibacterial hydrophobic assay as described by Côté et al. [25]. This new method was developed and used in this work as classical methods including disc diffusion and microdilution assays are sometimes inefficient for testing a matrix or compounds of a hydrophobic nature. Briefly, after microorganisms passed 16-18 h at 37 • C in a nutrient broth base (Difco), we transferred 20 µL methanol containing growing concentrations of essential oil and compounds (3.1 to 200 µg·mL −1 ) onto nutrient agar of 96-well plates. We then added bacterial strains having a concentration of 5 × 10 3 colony forming units (CFU) per mL of nutrient broth. Bacterial suspension without treatment was used as negative control and bacterial suspension plus solvent was tested in parallel to demonstrate the absence of solvent toxicity. The blank consisted of culture medium only and was subtracted from all subsequent measurements of every other well. The 96-well plates were then incubated at 37 • C for 5 h to foster bacterial growth. We then added 100 µL of resazurin sodium salt solution having a concentration of 50 µg·mL −1 (Sigma R-2127, St-Louis, MO, USA) to each well. Fluorescence was read on an automated Fluoroskan Ascent FL TM plate reader (Labsystems, Milford, MA, USA) after two hours for S. aureus and three hours for E. coli. The IC 50 was determined as the lowest concentration resulting in 50% inhibition of bacterial growth.

Cytotoxic Assays
We plated in 96-well microplates-each containing a 100 µL culture medium-exponentially growing cells of human skin fibroblasts WS1, human HaCaT keratinocytes, human lung carcinoma A-549, and human colon adenocarcinoma DLD-1 at a density of 5 × 10 3 cells per well (Costar, Corning Inc., Lowell, MA, USA). The cells were allowed to adhere for 16 h before treatment. We then treated cells with T. vulgare essential oil or dissolved compounds in DMSO. The final concentration of DMSO in the culture medium was 0.5% (v/v) to avoid solvent toxicity. After 48 h, we assessed the cytotoxicity using the resazurin reduction test [26]. Fluorescence was measured on an automated Fluoroskan Ascent FL TM plate reader (Labsystems, Milford, MA, USA) using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Cytotoxicity was expressed as the concentration that inhibited cell growth by 50% (IC 50 ).

Results and Discussion
The essential oil of T. vulgare obtained by steam distillation method was yellow with a distinct odor. The extraction yield obtained after 3 h was 0.23% (w/w). The refraction index was 1.4797, and the density measured using pycnometer was 0.92396 g·mL −1 . Table 1 presents the chemical composition of the essential oil as determined by GC-FID analysis. The oil was mainly comprised of oxygenated monoterpenes such as camphor (30.48%), borneol (14.80%), and 1,8-cineole (10.80%). The essential oil also contained camphene (7.29%), bornyl acetate (5.53%), and α-pinene (4.43%). Our results confirm the findings of Collin et al. [17] for T. vulgare essential oil from northern Quebec. We evaluated the biological properties of T. vulgare essential oil, including anti-inflammatory, antioxidant, antibiotic, and cytotoxic effects, in vitro using cellular models. Our results for anti-inflammatory activity ( Table 2), showed that the T. vulgare essential oil inhibited NO production (induced by LPS) with an IC 50 of 72 µg·mL −1 . Evaluation of the anti-inflammatory properties of the main compounds present in the oil showed that the most active compound was α-humulene with an IC 50 of 15 µg·mL −1 ( Table 2). Fernandes et al. [27] reported that α-humulene reduced rat paw edema that had been induced by carrageenan. It also inhibited COX-2 and iNOS expression. Moreover, the authors concluded that α-humulene might represent an important compound for the management and/or treatment of inflammatory diseases [27]. Other compounds, including α-terpinene, α-pinene, and β-pinene were also effective with an IC 50 ranging from 30 to 46 µg·mL −1 while caryophyllene oxide also inhibited NO production induced by LPS with an IC 50 of 183 µg·mL −1 . In a previous study, α-pinene was shown to inhibit NF-κB and JNK activation as well as the expression of iNOS [28]. The anti-inflammatory properties of β-pinene and α-terpinene have also been reported [29,30] as has caryophyllene oxide in vivo on mice models [31].
The essential oil of T. vulgare demonstrated antioxidant activity by significantly inhibiting the oxidation of DCFH induced by tert-butylhydroperoxide with an IC 50 of 51 µg·mL −1 ( Table 2). Using in vitro methods including DPPH, ABTS, and FRAP assays, Shaparov et al. [32] also reported a moderate level of antioxidant activity of T. vulgare essential oil. Unfortunately, the chemical composition of the oil was not analyzed and the compounds responsible for the activity were not identified. The antioxidant activity of essential oils is often attributed to terpenoids such as carvacrol, thymol, and eugenol [33]. However, our results for T. vulgare essential oil showed two compounds, α-pinene and caryophyllene oxide, to be particularly effective having a respective IC 50 of 3.4 and 6.2 µg·mL −1 . A moderate level of antioxidant activity for α-pinene, based on a DPPH assay, had been previously reported by Dai et al. [34]. However, to the best of our knowledge, this is the first time the antioxidant activity of caryophyllene oxide has been reported.  Numerous Tanacetum species produce essential oils that are known to have antibacterial activity including T. balsamita, T. polycephalum, T. parthenium, T. longifolium, and T. cilicicum [35][36][37]. Antibacterial activity of T. vulgare from Roumania [9] and Slovakia [38] were also reported against gram-positive bacterial strains using the disc diffusion method. Moreover, T. vulgare essential oil from Tajikistan was found to be weakly effective against E. coli and MRSA [32]. In our study, we used a new antibiotic assay developed to test hydrophobic compounds as described in the Material and Methods section [25]. The results, presented in Table 2, show that T. vulgare essential oil was effective against S. aureus and E. coli with an IC 50 of 59 µg·mL −1 and 241 µg·mL −1 , respectively. We also tested the antibacterial activity of the main constituents of T. vulgare essential oil against E. coli and S. aureus. Camphor was active against both bacterial strains having an IC 50 ranging from 22 to 26 µg·mL −1 . Moreover, caryophyllene oxide and γ-terpinene were also active against S. aureus having a respective IC 50 of 10.4 µg·mL −1 and 50 µg·mL −1 . The antibacterial activity of camphor, caryophyllene oxide, and γ-terpinene has been previously reported in the literature [39,40] although the results are sometimes contradictory. For example, Kotan et al. [41] found camphor to be ineffective as an antibacterial compound. Our use of a new method that is better suited to hydrophobic matrices and compounds [25] may explain the differences in the study outcomes.
We assessed the cytotoxic activity of T. vulgare essential oil and its main compounds against human lung carcinoma A-549, colon adenocarcinoma DLD-1, human keratinocytes HaCaT, and healthy WS1 cell lines. T. vulgare essential oil was slightly cytotoxic against A-549 and WS1 with an IC 50 higher than 200 µg·mL −1 ( Table 2). Essential oil was also slightly cytotoxic against human keratinocytes HaCaT with an IC 50 of 199 µg·mL −1 . Interestingly, DLD-1 (colon adenocarcinoma) was more sensitive to T. vulgare essential oil than other cell lines, having an IC 50 of 105 µg·mL −1 . To the best of our knowledge, no studies have previously reported the cytotoxicity of T. vulgare essential oil against cancer cell lines. The major constituents of camphor, borneol, and 1,8-cineole, were all inactive with an IC 50 > 200 µg·mL −1 . However, some minor compounds in the oil, including α-pinene, α-humulene, α-terpinene, β-caryophyllene, β-pinene, γ-terpinene, camphene, caryophyllene oxide, and limonene possessed a moderate cytotoxicity against all cell lines, including the tested cancer cell lines, with an IC 50 ranging from 28 to 112 µg·mL −1 . The cytotoxicity of these compounds against various cancer cell lines has also been previously reported [42][43][44][45][46].

Conclusions
Analysis of the chemical composition of T. vulgare essential oil from northern Quebec, QC, Canada, demonstrated that it was mainly comprised of oxygenated monoterpene such as camphor, borneol, and 1,8-cineole. The essential oil inhibited NO production (stimulated by LPS and induced by RAW 264.7). The compound α-humulene was, in part, responsible for this anti-inflammatory effect. The antioxidant activity of the essential oil was, in part, due to α-pinene and caryophyllene oxide. Caryophyllene oxide (as well as camphor) was also responsible for antibiotic activity. Finally, T. vulgare essential oil inhibited the growth of colorectal cancer cells. In conclusion, T. vulgare essential oil possesses interesting biological properties. This study reports, for the first time, the links between the compounds found in T. vulgare essential oil and anti-inflammatory, antioxidant, antibacterial, and cytotoxic activities. Additional investigations of T. vulgare should be carried out for its potential use for cosmetic or pharmaceutical purposes.