Chemical Profile and Biological Activities of Essential Oil from Artemisia vulgaris L. Cultivated in Brazil

Essential oil from the leaves of Artemisia vulgaris L. (Compositae) cultivated in Brazil was investigated for its chemical composition and biological activities including antibacterial, antifungal, and anthelmintic. The constituents of essential oils isolated by hydro-distillation were examined by GC-MS and a total of 18 components were identified. The essential oil was dominated by oxygenated sesquiterpenes (44.4%), sesquiterpene hydrocarbons (33.3%), and oxygenated monoterpenes (16.6%). Caryophyllene (37.45%), germacrene D (16.17%), and humulene (13.66%) were the major components. The essential oils from A. vulgaris showed bactericidal and fungicidal properties against Staphylococcus aureus and Candida albicans, respectively. Anthelmintic activity against Haemonchus contortus was absent in this essential oil. Altogether above results indicate that essential oils from A. vulgaris can be used for various medicinal purposes.


Introduction
Increasing resistance and other side effects caused by the repeated use of similar antibiotics or anti-parasitics enforced the researchers to find suitable natural compounds as alternatives. A large number of plant derived compounds play an important role in the natural defence system against all living microorganisms [1]. In vitro investigations of antimicrobial and antifungal activities have been carried out for several medicinal crops including Baccharis trimera [2], Zingiber officinale [3,4], Mikania glomerata [5,6], Mentha piperita [3,7], Syzygium aromaticum [8], Cymbopogon citratus [9,10], Allium sativum [11,12], and Psidium guajava [6,11,13]. There is a huge interest in medicinal crops to extract oils and bioactive compounds to use them as food additives to delay or prevent growth and development of microorganisms [14][15][16][17][18].
Knowing the exact chemical composition of Brazilian mugwort oil is critical to identify its biological properties (e.g., antimicrobial, anti-parasitical, and insecticidal). Among all the pathogens, Staphylococcus aureus [7], Escherichia coli [35], and Candida albicans are of great importance to public health [36]. S. aureus, a Gram-positive and round-shaped bacterium [7] is known to cause skin and severe bloodstream infections in patients using catheters and has also been frequently associated with pneumonias associated with mechanical ventilation [24]. C. albicans is the main etiological agent of candidiasis, the most common pathogen in humans which colonizes the genitourinary tracts, gastrointestinal tracts, teeth, mouth, as well as skin of more than 70% of the healthy population [37,38]. E. coli is a bacterium commonly found in the digestive tract of humans and warm-blooded animals. Some strains of E. coli can cause serious food-borne diseases. Haemonchus contortus, a parasitic gastrointestinal nematode, is a serious threat to small ruminants' health. Diarrhea, low packed cell volume (PCV), anemia, peripheral, internal fluid accumulation, and dehydration are common signs of this nematode [39]. To ascertain the antimicrobial and anti-parasitical activity of Brazilian mugwort oils, it is necessary to test the efficacy of Brazilian mugwort oils against above pathogens.
Considering aforementioned literature, the main purpose of this study was to assess the chemical composition of essential oil extracted from the leaves of A. vulgaris grown in Brazil, and to identify its biological properties.

Extraction of Essential Oils
To extract the essential oils, leaves of A. vulgaris were dried at 40 • C in an oven with circulating air. These were then sliced into small pieces and subjected to extraction with water by hydro-distillation for 3 h using a Clevenger-type apparatus. The essential oils (1.3 mL) thus obtained was dried over anhydrous sodium sulphate and stored at 4 • C until use.

Identification of Compounds Using Gas Chromatography-Mass Spectrometry (GC-MS)
The analyses of essential oils were performed using GC-2010 plus gas chromatograph (Shimadzu, Japan) coupled to a GCMS-QP2010 SE mass spectrometry detector (Shimadzu, Japan) and equipped with an AOC20i auto-injector (Shimadzu, Japan). A capillary Rtx-5MS column (30 m × 0.25 mm i.d. × 0.25 µm film thickness, Restek, USA) was used for separation. Helium (at a flow rate of 1.0 mL/min) was used as carrier gas. Temperature was kept at 60 • C for 5 min and programmed to reach 240 • C at the rate of 3 • C per min. The samples were injected at the injected temperature of 250 • C. The injection volume was 1.0 µL in 1:30 split ratio. The mass spectra were obtained with electron impact ionization (70 eV) at full scan mode (40 to 500 m/z), using an ion source at 200 • C.

Identification of Compounds
The compounds were identified by comparing retention indices (RI) and mass spectra with data from the NIST 11.0 MS library and the literature.

Microorganisms and Growth Conditions
Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 90028, already maintained in laboratory, were used for the experiments. The strains were pre-cultured for 24 h on brain heart infusion (BHI; Difco, BD, USA) at 37 • C in the presence of 5% CO 2 .

Determination of Antimicrobial and Anti-Fungal Activities in Essential Oils
The antimicrobial and anti-fungal activities in essential oils of A. artemisia were determined through MIC, the microdilution technique according to the broth dilution methodology proposed by the Clinical and Laboratory Standards Institute (CLSI, 2013). In short, sterile 96-well plates were prepared with 50 µL of BHI broth and 50 µL of the essential oil followed by serial dilutions (1:2 to 1:128) done in triplicate After dilution, 2 µL of microbial suspension (0.5 on the MacFarland scale) was added to the samples. Then, 25 µL of chloramphenicol (20 µg/mL) was added to the well as positive control and 50 µL of BHI and 2 µL of microbial suspension were used as growth control. The plate was incubated at 35 • C in an incubator for 24 h for bacteria and 48 h for fungus. After the incubation period, 5 µL of the resazurin (0.1% w/v) was added.

Parasitological Procedures
Eggs and third stage larvae (L3) were obtained from a donor sheep with a monospecific infection of Haemonchus contortus. Experimental procedures were performed in accordance with the guidelines of the Animal Ethics Committee of Maranhão, Federal University and approved under protocol number 23115018061/2011-11.

Egg Hatching Assay (EHA)
Fresh faeces were collected, and the eggs were recovered using 25 µm sieves. Recovered eggs were added to a saturated NaCl solution and centrifuged (3000 rpm) for 3 min; floating eggs were recovered using a 25 µm sieve [40]. A suspension of 100 eggs/well was placed in a plate and 100 µL of essential oils of A. vulgaris and control (2%, v/v, methanol) was added. The essential oils of A. vulgaris were diluted in 2% (v/v) methanol at concentration of 10 mg/mL. Tests were performed with four replicates. The plate was incubated at 27 • C and RH > 80% for 48 h. Larvae and unhatched eggs were counted under an inverted microscope.

Larval Exsheathment Inhibition Assay (LEIA)
This test was performed according to Bahuaud et al. [41]. The essential oil of A. vulgaris was diluted in 2% (v/v) methanol and evaluated at concentration 1.2 mg/mL. The negative control was performed with 2% (v/v) methanol and PBS (0.1 M phosphate, 0.05 M NaCl, pH 7.2). The L3 larvae were incubated in the different treatments for 3 h at 22 • C. After incubation, the larvae were washed and centrifuged (3000 rpm) three times with PBS. Approximately 1000 larvae/tube was subjected to the artificial exsheathment process by contact with sodium hypochlorite (2.0%, w/v) and sodium chloride (16.5%, w/v). Tests were performed with four replicates. The percentages of larval exsheathment process were monitored at 0, 20, 40 and 60 min intervals by observing under an inverted microscope.

Larval Migration Inhibition Assay (LMIA)
The evaluation of larval migration inhibition was performed according to Jackson and Hoste [42]. Initially, H. contortus L3 larvae were subjected to the exsheathment process with 2.0% (w/v) sodium hypochlorite (w/v). After being sieved, the larvae were centrifuged in distilled water for 5 min at 407× g and were re-suspended in distilled water and centrifuged again. Larval suspension (100 µL; approximately 100 larvae) and essential oil of A. vulgaris (1000 µL at 10 mg/mL) were added to microtubes. The suspension was incubated for 2 h at 27 • C and ≥80% RH. After that, the tubes were centrifuged at 1500× g for 10 min and the supernatant was removed, reducing the volume by approximately 300 µL. Using culture plates, 200 µL of the samples at the concentrations described above were added to the wells, and then an apparatus containing 25 µm sieves were submerged in each well. A 50 µL volume of larval suspension was added to the corresponding apparatus and incubated for 2 h at 27 • C and ≥80% RH. Each apparatus was then removed carefully and the mesh was washed. Lugol was added to each well and larvae of each well and filter were counted. The assay was performed in quadruplicate and methanol (2%, v/v) was used as a negative control.

Chemical Composition of Essential Oil Extracted from Brazilian A. vulgaris Leaves
The essential oil extracted (0.5%, v/v) from the leaves of Brazilian A. vulgaris was analyzed by GC-MS chromatography ( Table 1). The obtained results indicated the presence of 18 chemical compounds in the chromatogram, constituting 100% of the total components detected. The highest compositions of the compounds were 37.45% of caryophyllene, followed by germacrene-D (16.17%) and humulene with 13.66%. Caryophyllene (β-caryophyllene) [43] is a natural bicyclic sesquiterpene usually found in various essential oils including clove, rosemary, and Cannabis [44,45]). This compound is commonly found mixed with α-humulene and isocaryophyllene in essential oils [43]. Caryophyllene is mainly used as a flavoring or fragrance enhancer in spice blends, citrus flavors, chewing gums, soap, detergents, creams or lotions, food products, and beverages. It is known to possess anaesthetic and anti-inflammatory activities [43]. Rasmann and colleagues [46] reported that sesquiterpene olefin (E)-b-caryophyllene plays an essential role in different separate pathways of induced defenses response against herbivores. Caryophyllene can act as antimicrobial agent in defense mechanisms against pathogens [47] such as Pseudomonas aeruginosa and Bacillus subtilis [48]. Caryophyllene is also reported to have allelopathic potential and shown to inhibit the seedlings development of various plant species [49,50].
Germacrenes, a group of volatile organic hydrocarbon compounds (particularly sesquiterpenes), produced in various plant species are known to act as insecticidal, antimicrobial, and insect pheromones [51,52]. This volatile organic compound has been observed in bryophytes, gymnosperms and angiosperms. Interestingly, germacrene D plays an important role as a precursor in sesquiterpenes synthesis such as selinenes and cadinenes [53,54]. Reportedly, germacrene D as predominant components has been found in the leaves of five plants extracts viz. Bursera fagaroides, B. mirandae, B. exselsa, B. copallifera, and B. ruticola [55]. Humulene, also known as αhumulene or α-caryophyllene is a naturally occurring monocyclic sesquiterpene characteristic of Humulus lupulus [56]. It is also found in Abies balsamea, Salvia officinalis, Comptonia peregrine, Cordia verbenacea, and Myrica gale [57][58][59]. Generally terpenes play a role in plants as an anti-herbivore defenses mechanism [60]. Lutz et al. determined the chemical profile of essential oils obtained from seven species of Artemisia grown in Canada and studied their antimicrobial and antioxidant activities [61].
Interestingly, monoterpenes hydrocarbons were not found in the essential oil in the present investigation, whereas one diterpene (phytol, 2.94%) was detected. Diterpenes are rare in essential oils and the presence of phytol can be considered as a standard for this species oil. According to Hussein et al. [19], the essential oil from aerial part of mugwort extracted in Egypt contained camphor and 3,5-dimethylcyclohexene as the major components (both with 13.83%), followed by germacrene-D and α-cubenene with 10.44%. However, they found phytol acetate instead of phytol, which were detected in the present work. In contrast to present findings, the monoterpene fraction dominated in the mugwort oils from European countries.

Anti-Microbial Activities Identification
The antimicrobial activity of essential oil of A. vulgaris was obtained (Table 2) from microdilution in BHI broth and expressed as Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and Minimum Fungicidal Concentration (MCF). The MIC of essential oil of A. vulgaris leaves in Escherichia coli ATCC 25922, Staphylococcus aureus (ATCC 25923), and Candida albicans ATCC 90028 were found at the 1:64, 1:16, and 1:32 dilution titre, respectively. MIC is considered as the lowest concentrations for recognizing the susceptibility of microorganisms to antimicrobial and is applied to judge the performance of all other methods of susceptibility testing. The minimum bactericidal concentration (MBC) affected by extracted essential oil of A. vulgaris leaves were reported in E. coli at the 1:8 dilution titre. MBC as the lowest concentration act as an antibacterial agent required to kill a microorganism. It could be identified from MIC experiments by sub-culturing to agar plates media that do not contain the test agent. MFC values for C. albicans were 1:8 dilution titre. The results revealed variable inhibition activity against E. coli, S. aureus, and C. albicans. The antimicrobial activity was evident with the increase in concentration of essential oil extracted from leaves of A. vulgaris. Reportedly, the essential oil of A. vulgaris presented lowest bactericidal potential for E. coli. The essential oil of A. vulgaris showed bactericidal activity for S. aureus and fungicide for C. albicans in the same concentration [24].

Anthelmintic Activity
The A. vulgaris essential oil was tested in high concentrations (10 mg/mL) against eggs (Egg hatching assay) and on two different larval behavior (10 mg/mL and 1.2 mg/mL in LMIA) of main gastrointestinal nematode from small ruminant H. contortus. The efficacy of A. vulgaris essential oil was low in inhibition of eggs hatch (7.4 ± 6.5%), either in larval exsheathment (4.1 ± 12.8%) and larval migration (1.9 ± 4.1 %). Extracts of several species of Artemisia were described as antihelmintic effect against H. contortus [62][63][64] and extract of A. vulgaris was efficient in vivo models against Trichinella spiralis [65]. However, few studies were performed using essential oils of Artemisia species against nematodes [66,67]. To our knowledge, this is the first report of A. vulgaris essential oil to test against nematode. Besides the efficacy of extracts of A. vulgaris on T. spiralis [65], the essential oil was not effective against eggs hatch (7.4 ± 6.5%), larval exsheathment (4.1 ± 12.8%), and larval migration (1.9 ± 4.1%). Essential oils of plants with (E)-caryophyllene and β-caryophyllene as main compound showed activity against eggs and adult of Schistosoma mansoni and inhibited larval migration of Strongyloides ratti, respectively [68][69][70]. β-Caryophyllene was effective for in vitro inhibition of the enzyme Glutathione S-transferase (GST), an important enzyme of detoxification in nematodes [71], however, the data are contradictory, with no efficacy of β-caryophyllene against a root-knot nematode, Meloidogyne incognita [72].

Conclusions
The essential oil from A. vulgaris cultivated in Brazil is a potential alternative source of caryophyllene, germacrene D, and humulene. The volatile compounds from this plant species possess antifungal and anti-bacterial properties. Hence, there is a potential for using this essential oil from A. vulgaris as disinfectants and preservatives against microorganisms. More studies are required to be focused on isolating the compounds from essential oils and to investigate their biological activities and mode of action in order to use these volatile compounds at commercial level.

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