Bioactivities of Achillea millefolium Essential Oil and Its Main Terpenes from Iran

Abstract

Achillea millefolium L. comprises several relevant species for the food, cosmetic, perfumery, and pharmaceutical industries. Gas chromatography/mass spectrometry analysis revealed borneol to be a major component of A. millefolium, with its contribution to the essential oil being 36.35%. Borneol exhibited significant lipid peroxidation inhibition and antimicrobial activity against all tested bacterial strains. In addition, borneol had the highest antioxidant activity in all conducted assays. The borneol had significantly greater radical scavenging activity than other component essential oil and the reference antioxidant Trolox. In addition, a correlation between antioxidant activity and the total phenolic content was found. The borneol significantly inhibited nitric oxide production in lipopolysaccharide-activated macrophages (an in vitro model of inflammation). These results clearly show the antimicrobial, antioxidant, and anti-inflammatory effects of the plant essential oils.

Keywords:

• Achillea millefolium
• Essential oil
• Antimicrobial
• Lipid peroxidation inhibition

INTRODUCTION

Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth.^[1–4].There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body, and to prevent the deterioration of fats and other constituents of foodstuffs. In both cases, there is some preference for antioxidants from natural rather than from synthetic sources.^[5,6].Achillea millefolium L., belongs to the Asteraceae family. Previous phytochemical investigations on the Achillea genus reported essential oils (EOs) that contain 1,8-cineole and camphor. Phytochemical studies carried out with Achillea millefolium have identified several components, including monoterpenes, sesquiterpenes, and phenylpropanoids.^[7] Therefore, our primary objective was to characterize the EO of A. millefolium, and compare its antimicrobial and antioxidant activity (AA) and anti-inflammatory potential.

MATERIALS AND METHODS

Plant Materials and Nano Zn Oxide Treatments

The aerial parts of Achillea millefolium were collected during June and July in 2013 and 2014. Voucher specimens were identified by Mr. Esmaeili and deposited, under the numbers A. millefolium (no. 158), in the private herbarium of Dr. F. Esmaeili. Seeds of Achillea millefolium were sown in Jefe pots in an experimental greenhouse, Iran (Elevation 1339 m, Latitude East 33.638, Longitude North 46.431). Plants at two- and four-leaf stage were sprayed with distilled water as a control, and nano Zn oxide at 2 and 4 mM. All sprays solution were sprayed to the point of run off. The experiment was arranged in completely randomized block design with three replications for each treatment. The temperature conditions were 24 ± 5°C and 15 ± 4°C, during days and nights, respectively; with relative humidity of 70%. Aerial parts of A. millefolium were harvested and air dried at ambient temperature in the shade. EOs were isolated by hydrodistillation for 3 h using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia.^[8] The obtained EOs were dried over Na₂SO₄ and stored in sealed dark vials, at 4°C.

Oil Isolation, Isolate Borneol, and Identification of Known Compounds

Composition of the EOs was determined by gas chromatography (GC) and mass spectrophotometry (GC/MS). The GC analysis was carried out on an Agilent Technologies 7890 GC equipped with a single injector and a flame ionization detector (FID). The analysis was carried out on fused silica capillary HP-5 column (30 m × 0.32 mm i.d.; film thickness 0.25 µm). The injector and detector temperatures were kept at 250 and 280°C, respectively. Nitrogen was used as carrier gas at a flow rate of 1 mL/min; oven temperature program was 60–210°C at the rate of 4°C/min and then programmed to 240°C at the rate of 20°C/min and finally held isothermally for 8.5 min; the split ratio was 1:50. GC/MS analysis was carried out by use of an Agilent gas chromatograph equipped with fused silica capillary HP-5MS column (30 m × 0.25 mm i.d.; film thickness 0.25 µm) coupled with 5975-C mass spectrometer. Helium was used as a carrier gas with ionization voltage of 70 eV. Ion source and interface temperatures were 230 and 280°C, respectively. The mass range was from 45 to 550 amu. Oven temperature program was the same as that given above for the GC. The constituents of the EOs were identified by calculation of their retention indices under temperature-programmed conditions for n-alkanes (C₈–C₂₅) and the oil on a HP-5 column under the same chromatographic conditions. Identification of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library or with authentic compounds and confirmed by comparison of their retention indices with authentic compounds or with those reported in the literature.^[9] For quantification purposes, relative area percentages obtained by FID were used without the use of correction factors. The oil of A. millefolium was subjected to silica gel column chromatography (silica gel 60, 180 g, 70–230 mesh) using a solvent mixture n-hexane-ethyl acetate (95:5, 90:10, 85:15, 80:20, 75:25) to isolate borneol.

Total Phenolic Determination

Total phenolic contents in aerial parts of A. millefolium were determined by Folin–Ciocalteu method.^[10] The total phenolic content was expressed as gallic acid equivalents (GAE; mg g^–1).

Total Flavonoid Determination

Total flavonoid contents in aerial parts of A. millefolium were measured as described previously.^[11] The total flavonoid content was calculated as rutin equivalents (mg g^–1).

Microorganisms

Gram-positive bacteria

Bacillus cereus (ATCC 10876), Enterococcus faecalis (ATCC 49452), Staphylococcus aureus (ATCC 25923).

Gram-negative bacteria

Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 35659), Salmonella typhimurium (ATCC 13311), Citrobacter freundii (ATCC 8090).

Fungal strains

Candida albicans (ATCC 10231) and Aspergillus fumigatus (ATCC 46645). The bacteria species were maintained in Mueller Hinton Agarand Tryptic Soy Agar. Strains of Candida spp. and Aspergillus spp. were maintained on Sabourand Dextrose Agar.

Antimicrobial Activity

Minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) concentrations were determined by microdilution method in 96-well microtitre plates, described by Douk et al.,^[12] and EUCAST.^[13] Briefly, fresh overnight cultures of bacteria were adjusted with sterile saline to a concentration of 1.0 × 10⁵ CFU per well, and 1.0 × 10⁴ CFU per well for fungi. EOs were added in tryptic soy broth (TSB) medium for bacteria, and sabouraud dextrose broth (SDB) medium for fungi. The microplates were incubated for 24 h at 37°C for bacteria, and 48 h at 37°C for fungi. The MIC was defined as the lowest concentration of EO inhibiting the visible growth of the test strain. However, the MIC/MBC values for bacteria and fungi were detected following the addition of 40 µL of piodonitrotetrazoliumviolet (INT) 0.2 mg/mL and incubation at 37°C for 30 min.^[14] The MBCs/MFCs were determined by serial subcultivations of 10 µL into microtiter plates containing 100 µL of broth per well and further incubation for 24 h at 37°C. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. Following positive controls were used in both experiments: antibiotics (Streptomycin) and mycotic (Fluconazole). Each test was carried out in triplicates and repeated three times.

AA

The efficacy of the EOs to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was evaluated using a spectrophotometry method.^[15,16] On basis of bleaching of the bluish-red or purple color of DPPH solution as a reagent. Briefly, a 50 µL volume of various dilutions of each sample was mixed with 5 mL of 0.004% methanol solutions of DPPH followed by 30 min incubation at ambient temperature. Thereafter, the sample absorbance was recorded against control at 517 nm. The inhibition percentages were measured using Eq. (1). The antioxidants activity of the test samples in concentration providing 50% inhibition, were considered as IC₅₀ (µg/mL).

(1)

Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls. All experiments were repeated three times and the average results and standard deviations calculated.

Rapid Screening for Antioxidants

For screening of antioxidant compounds in aerial parts of A. millefolium EO, the thin-layer chromatographic (TLC)-bioautography method was carried out.^[17,18] The diluted oil (1:20 in methanol) was spotted on silica gel sheets (silica gel 60 F254 TLC plates) and developed in n-hexane-ethyl acetate (9:1). Plates were sprayed with the methanolic solution of DPPH (0.2%). The active constituents were detected as yellow spots on a violet background. Only zones where their color turned from violet to yellow within the first 30 min (after spraying) were taken as positive results.

Activity Guided Fractionation of the EO for Antioxidants

For the isolation and identification of the active compounds in the EO, TLC was performed using the conditions previously described.^[18] The regions showing DPPH scavenging activity were scrapped off then, they were eluted with chloroform. All resulting constituents were analyzed by GC/MS and also tested for their antioxidant activities.

β-Carotene-Linoleic Acid Model System (β-CLAMS)

The β-CLAMS method by the peroxides generated during the oxidation of linoleic acid at elevated temperature.^[19] The AA of the extracts was evaluated in term of β-carotene blanching using the following formula: AA (%) = [(A₀ – A₁)/A₀] × 100. where A₀ is the absorbance of the control at 0 min, and A₁ is the absorbance of the sample at 120 min. The results are expressed as IC₅₀ values (µg/mL). All samples were prepared and analyzed in triplicate.

Reducing Power and Lipid Peroxidation Inhibition

The ability of the extracts to reduce Fe³⁺ was assayed by the method of Oyaizu.^[20] One milliliter of the aerial parts of A. millefolium EO and borneol were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% K₃Fe (CN)₆. After incubation at 50°C for 25 min, 2.5 mL of 10% trichloroacetic acid was added and the mixture was centrifuged at 650 g for 10 min. Finally, 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% aqueous FeCl₃. The absorbance was measured at 700 nm. The mean of absorbance values were plotted against concentration and a linear regression analysis was carried out. Increase absorbance of the reaction mixture indicated increased reducing power. EC₅₀ value (µg/mL) is the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid was used as positive control. Lipid peroxidation inhibition was determined by Shirwaikar et al.^[21] Ascorbic acid and Trolox was used for comparison.

Anti-Inflammatory Activity

To evaluate the anti-inflammatory potential of the oils, NO production in lipopolysaccharide (LPS)-stimulated macrophages was used. Exponentially growing macrophages (RAW 264.7 cells) were plated in 24-well microplates at a density of 2×10⁵ cells per well in 400 μL of culture medium and were allowed to adhere for 24 h at 37°C under 5% CO₂. Cells were then treated with increasing concentrations of EO and pure compounds dissolved in dimethyl sulfoxide (DMSO). The final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. Cells were then stimulated with 100 μg/mL LPS and incubated at 37°C under 5% CO₂. After 24 h, cell-free supernatants were collected and NO was measured using the modified method of Green et al.^[22] Griess reagent (50 μL of 1% sulphanilamide and 50 μL of 0.1% N-1-naphtylethylenediamine dihydrochloride in 2.5% H₃PO₄) was added in equal volume (100 μL) to cell supernatant and incubated at room temperature for 30 min. N(G)-nitro-L-arginine methyl ester (L-NAME) was used as a positive control. Absorbance was measured using an ELISA automatic microplate reader at 550 nm and the nitrite concentration determined from a regression analysis prepared with serial dilutions of sodium nitrite.^[23]

Statistical Analysis

The results are presented as mean ± SD and statistically analyzed by one way analysis of variance (ANOVA) followed by Duncan test.

RESULTS AND DISCUSSION

Chemical Composition of A. millefolium EO

The constituents of the obtained EO of A. millefolium treated with nano Zn oxide are presented in Table 1. Twenty-four components were identified in nano Zn oxide-treated plants (Table 1). The differences were supposed to be the effects of nano Zn oxide on chemical composition of A.millefolium EO. α-Pinene, camphene, limonene, borneol and carvacrol were increased with nano Zn oxide-treatment (Table 1). The yield of the A. millefolium oil was 1.54% v/w (2 mM) and 2.01% (4 mM). Nano Zn oxide significantly increased the yield of EO (Table 1). It seems that the use of nano-particles causes increasing in pod and dry leaf weight and finally will increase total yield.^[24] Lu et al.^[25] has shown that application of nano fertilizers could increase the nitrate reductase enzyme in soybean (Glycine max L.), increase its abilities of absorbing and utilizing water and fertilizer, promote its antioxidant system, and, in fact, accelerate its germination and growth. A previous report by Afsharypour et al.^[26] indicated the major constituent of the EO of Achillea tenuifolia was caryophyllene oxide and in other studies, borneol was the second most abundant constituent of oil.^[27]

TABLE 1 Chemical compositions (%, w/w) of from the aerial parts of A. millefolium

	Treatment
Compound	Nano Zn oxide (2mM)%	Nano Zn oxide (4mM)%	RI	Identification methods
α-Pinene	6.45	7.45	932	MS, RI
Camphene	2.14	4.65	950	MS, RI
Sabinene	1.02	1.87	975	MS, RI
α-Phellandrene	0.07	0.41	1000	MS, RI
Limonene	2.36	4.53	1020	MS, RI
trans-limonene oxide	0.25	1	1140	MS, RI
Borneol	20.14	36.35	1160	MS, RI
γ-Terpinene	1.14	1.45	1195	MS, RI
Carone	1.54	1.1	1200	MS, RI
Carvone	2.35	1.45	1240	MS, RI
Bornyl acetate	2	1.74	1281	MS, RI
Thymol	10.14	10.01	1300	MS, RI
Carvacrol	8.14	10.14	1308	MS, RI
β-caryophyllene	1.25	1.74	1418	MS, RI
γ-Elemene	1.08	1.01	1430	MS, RI
γ-Muurolene	0.74	0.1	1476	MS, RI
β-Selinene	0.87	0.41	1490	MS, RI
β-Guaiene	0.7	0.98	1496	MS, RI
α-Selinene	0.35	0.11	1490	MS, RI
Calarene	0.47	0.14	1500	MS, RI
Calacorene	0.74	0.35	1529	MS, RI
Ccaryophyllene oxide	1.05	0.21	1579	MS, RI
Torreyol	1.45	0.78	1640	MS, RI
Cadalene	2.14	0.61	1670	MS, RI
Yield	1.54	2.01
Total	68.58%	88.59%

^aPercentage composition determined on column HP 5. ^b

The retention Kovats indices were determined on HP 5 capillary column in reference to n-alkanes. MS: mass spectroscopy, RI: retention index.

Extraction Yield, Total Phenolic Contents, and Total Flavonoid Contents

The AA of plant extracts has been correlated to their total phenolic content due to their property of scavenging free radicals.^[28] It is well-known that phenolic compounds contribute to quality and nutritional value in terms of modifying color, taste, aroma, and flavor and also in providing benefical health effects.^[29] They also serve in plant defense mechanisms to prevent damage by microorganisms, insects, and herbivores.^[29] Moreover, a few studies on the A. millefolium revealed that they are good dietary sources of antioxidants. Thus, we determined the total phenolic and flavonoid contents of the methanol extracts of A. millefolium wild vegetables. As shown in Table 2, the extraction yield of A. millefolium ranged from lowest 66.14 ± 24 mg g^–1 (control) to highest 100.14 ± 55 mg g^–1 (Nano Zn oxide [4mM]). Among the three A. millefolium extracts, A. millefolium treated with nano Zn oxide at 4 mM showed the highest total phenolic content (162.14 ± 57 mg g^–1) and showed the highest total flavonoid content (121.47 ± 64 mg g^–1). These results showed that the total phenolic and total flavonoid contents have an obvious variation in various concentrations.

TABLE 2 Effect of nano Zn oxide on extraction yields, total phenolic contents, and total flavonoid contents of A. millefolium extracts

	Extract	Extraction yield^a	Total phenolic^b	Total flavonoid^c
1	Control	66.14 ± 24	78.14 ± 32	80.24 ± 68
2	Nano Zn oxide (2mM)	68.47 ± 21	80.14 ± 78	84.24 ± 19
3	Nano Zn oxide (4mM)	100.14 ± 55	162.14 ± 57	121.47 ± 64

The data are expressed as mean ± SD.

^aExpressed as mg of extract per gr dry material.

^bExpressed as mg of gallic acid per gr dry extract.

^cExpressed as mg of rutin per gr dry extract.

Antimicrobial Activity

The in vitro antimicrobial activities of borneol against the studied microorganisms were assessed by the MIC and MBC/MFC (Table 3). According to the results given in Table 3, borneol exhibited significant antimicrobial activity against all tested strains. Inhibition values were in the following range: MIC 2.5 ± 0.05 (Escherichia coli) to 25.0 ± 0.11µg/mL (Pseudomonas aeruginosa), and MBC 2.5 ± 0.08 µg/mL (E.coli) to 20.0 ± 0.65 µg/mL (Staphylococcus aureus) for bacteria, and MIC 3.0 ± 0.35 (Candida albicans) to 4.5 ± 0.65 µg/mL (Aspergillus fumigatus), and MFC 3.0 ± 0.21 (C.albicans) to 4.5 ± 0.84 µg/mL (A.fumigatus) for fungi. Results obtained from MIC and MBC/MFC indicated that the antimicrobial activity of the borneol against E. coli and C. albicans was greater than those of EOs. Among the individual constituents of EOs, carvacrol, isoeugenol, nerol, citral, and sabinene exhibited potent anti-H. pylori effects.^[30] The major components of thyme and oregano EOs, thymol and carvacrol, inhibited pathogenic bacterial strains, such as E. coli, Salmonella enteritidis, S.choleraesuis, and S. typhimurium.^[31] Eugenol, terpenen-4-ol, and carvacrol showed an inhibitory effect against the growth of four strains of E. coli O157:H7 and L. monocytogenes, but results obtained indicated that three isolated compounds was greater than those of monoterpenes (benzyl alchol, camphor, cinnamaldehyde, borneol, and cineole) but weaker than those of monoterpenes (thymol, carvacrol, carveol, and geraniol).^[32] Comparing the results of borneol with that of standard, streptomycin and fluconazole, it was concluded that the oils possess more potent anti-oral-pathogen activity. The borneol expressed higher antibacterial activity both antibiotics tested. In our study, most of the antimicrobial activity in EOs from A. millefolium appears to be associated with borneol (Table 3).

TABLE 3 Antimicrobial activity of the EOs and borneol (µg/mL) from A. millefolium using minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) test

	A. millefolium EOs	Borneol	Antibiotic (Streptomycin)
	MIC	MIC	MIC
Microorganisms	MBC	MBC	MBC
Bacillus cereus	7.0 ± 0.12	3.5 ± 0.27	10 ± 0.70
	7.0 ± 0.21	3.5 ± 0.41	15 ± 0.32
Enterococcus faecalis	7.5 ± 0.21	4.0 ± 0.28	10 ± 0.59
	7.0 ± 0.74	4.0 ± 0.17	10 ± 0.52
Staphylococcus aureus	20.0 ± 0.14	3.5 ± 0.01	250 ± 0.0
	20.0 ± 0.65	3.5 ± 0.05	250 ± 0.0
Escherichia coli	4.5 ± 0.31	2.5 ± 0.05	25 ± 0.5
	5.0 ± 0.52	2.5 ± 0.08	25 ± 0.5
Pseudomonas aeruginosa	25.0 ± 0.11	10 ± 0.74	100 ± 0.34
	20 ± 0.05	10 ± 0.24	150 ± 0.87
Proteus mirabilis	10.0 ± 0.54	4.5 ± 0.61	20 ± 0.54
	8.0 ± 0.12	4.0 ± 0.08	15 ± 0.5
Salmonella typhimurium	5.5 ± 0.24	4.5 ± 0.06	15 ± 0.05
	4.5 ± 0.14	4.5 ± 0.11	15 ± 0.02
Citrobacter freundii	6.5 ± 0.08	4.5 ± 0.17	15 ± 0.87
	6.0 ± 0.14	4.5 ± 0.20	20 ± 0.56
Microorganisms	A. millefolium EOs	Borneol	mycotic (Fluconazole)
	MIC	MIC	MIC
	MFC	MFC	MFC
Candida albicans	4.5 ± 0.21	3.0 ± 0.35	4.5 ± 0.00
	4.5 ± 0.24	3.0 ± 0.21	5 ± 0.00
Aspergillus fumigatus	4.5 ± 0.65	3.5 ± 0.28	5 ± 0.9
	4.5 ± 0.84	3.0 ± 0.47	3.5 ± 0.5

All tests were performed in triplicate, repeated three times twice (means ± SD). Statistically, the differences were significant at p ≤ 0.01 or at least at p ≤ 0.05 (in few cases), using Duncan test. C1: dihydro-a-cyclogeranyl hexanoate.

AA

AA is a complex process usually occurring through several mechanisms. Due to its complexity, the evaluation of the AA for pure compounds or extracts should be carried out by more than one test method.^[33] The lower IC₅₀ value indicates a stronger ability of the extract to act as a DPPH scavenger while the higher IC₅₀ value indicates a lower scavenging activity of the scavengers as more scavengers were required to achieve 50% scavenging reaction. The results presented in Table 4 revealed that EOs and borneol exhibited a remarkable activity. In particular, borneol exhibited clearly a higher activity (11.11 ± 0.60 µg/mL) followed by A. millefolium EO (12.97 ± 0.14 µg/mL; Table 4). The positive controls butylated hydroxytoluene (BHT) and ascorbic acid exhibited IC₅₀ values equal to 13.87 ± 0.22 µg/mL and 12.97 ± 0.11 µg/mL, respectively. Table 4 depicts the inhibition of β-carotene bleaching by the EOs and borneol. Because of high antioxidant and free radical-scavenging activities of EOs, further investigation was carried out to identify its active constituents. Therefore, a preliminary screening was initially carried out using the dot-blot DPPH staining method on TLC. As the borneol presented a significant AA in the assays and bioautography test, it was subjected to the TLC for isolation of the active compounds. Components identified and their AA relative percentages have been shown in Table 5. The major compound found in the active band was borneol (45.14%; Table 5). According to these results, there is a relationship between total phenolic contents and AA.

TABLE 4 Antioxidant EOs and borneol: Scavenging activity (expressed as IC₅₀ values: µg/mL), and β-carotene bleaching test. Reducing power was expressed as EC₅₀ values (µg/mL). Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls

Tested compounds	IC50 (μg/mL)
A. millefolium EO	12.97 ± 0.14 µg/mL
borneol	11.11 ± 0.60 µg/mL
A. millefolium EO (β-Carotenes IC50 µg/mL)	12.47 ± 0.28 µg/mL
A. millefolium EO (PR EC50 µg/mL)	12.97 ± 0.21 µg/mL
BHA	13.87 ± 0.22 µg/mL
AA	12.97 ± 0.11 µg/mL

Values are mean ± SD of three replications. *IC₅₀ values have been presented with their respective 95% confidence limits.

TABLE 5 Components identified and their antioxidant activity relative percentages

Compounds	%
Thymol	21.11
Borneol	45.14
Other components	36.68

Results are expressed as a percentage of antioxidant activity relative. Experiments were carried out in triplicate.

Lipid Peroxidation Inhibition

According to the results obtained, EOs and borneol significantly inhibited the formation of thiobarbituric acid reactive substance (TBARS) in brain homogenates in a concentration-dependent manner (Table 6). The suppressive power on the lipid peroxidation of borneol were found to be the most potent (80.41 ± 0.24), followed by A. millefolium EO (78.54 ± 0.21 µg/mL). Ascorbic acid and Trolox showed significant suppressive power on lipid peroxidation in mice brain homogenate with IC₅₀ value of 75.61 ± 0.21 and 72.18 ± 0.35 µg/mL (Table 6). Phenolic compounds exist in most plant tissues as secondary metabolites, i.e., they are not essential for growth, development or reproduction but may play roles as antioxidants and in interactions between the plant and its biological environment. Phenolics are also important components of the human diet due to their potential AA, their capacity to diminish oxidative stress induced tissue damage resulted from chronic diseases and their potentially important properties such as anticancer activities.^[34] These results indicated that borneol displayed significant AA in inhibiting peroxidation of rat brain homogenate in vitro.

TABLE 6 Lipid peroxidation inhibition of EOs and borneol (expressed as IC₅₀ values: µg/mL). Trolox and ascorbic acid (AA) were used as positive controls

Tested compounds	IC50 (μg/mL)
A. millefolium EO	78.54 ± 0.21 µg/mL
Borneol	80.41 ± 0.24 µg/mL
Trolox	72.18 ± 0.35 µg/mL
AA	75.61 ± 0.21 µg/mL

Experiments were carried out in triplicate and the results are expressed as mean ± SD.

Anti-Inflammatory Activity

The traditional use of EOs as anti-inflammatory agents suggests that they possess potent anti-inflammatory activity. The anti-inflammatory activity of EOs and borneol was evaluated on RAW 264.7 macrophages which were stimulated to induce an overproduction of NO. As Table 7, the borneol exhibited a strong inhibitory effect on LPS-induced NO secretion with 88 ± 0.05 and inhibition observed at 45.0 μM. Comparatively, the L-NAME, used as positive control inhibited NO release by 73 ± 0.05% (Table 7). Borneol was found to be the most active compound, inhibiting NO production by 88 ± 0.05% at 45.0 μM (Table 7). Therefore, this compound may be responsible for the anti-inflammatory activity of the oil. The anti-inflammatory potential of the borneol may be directly related to its scavenging ability and/or capacity to inhibit inducible NO synthase expression, the enzyme responsible for the release of high amounts of NO, during inflammatory conditions. Indeed, inflammatory mediators, such as NO, have been reported to contribute to mutagenesis.^[35] This radical is an important regulator of physical homeostasis, whereas large amounts have been closely correlated with the pathophysiology of a variety of diseases and inflammations.^[35] EOs seem to be a good source of antioxidant and anti-inflammatory natural products.^[36–38]

TABLE 7 Effects of A. millefolium EO (45.0 μg/mL), and borneol (45.0 μM) on NO production in LPS-stimulated RAW-264.7 macrophages

Tested compounds	NO inhibition (%)
A. millefolium EO	78 ± 0.21
Borneol	88 ± 0.05
L-NAME	73 ± 0.05

Results are expressed as a percentage of nitrite production by control cells maintained in culture medium. Experiments were carried out in triplicate and the results are expressed as mean ± SD.

CONCLUSION

EOs showed variations in their chemical components and physicochemical profiles. The present study is the first report describing the anti-inflammatory activity of A. millefolium EO. In addition, a high correlation between the AA and the total phenolic content was found. Considering that Achillea millefolium showed an acceptable yield in EO, which confers an industrial potential interest, and its volatile oil has a pleasant smell and is safe in bioactive concentrations, the results presented here emphasize its potential use as an antioxidant and anti-inflammatory for the food, cosmetic, and nutraceutic industries. Also, the high content of borneol makes this species an important natural source of this compound.