1. 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].
The genus
Artemisia is among the most widely distributed and largest genera of family Asteraceae, containing around 500 taxa [
19,
20,
21]. Many
Artemisia species grow in Northern Africa, North and Central America, and Eurasia [
22].
Artemisia vulgaris Linn., commonly known as mugwort, is a rhizomatous perennial medicinal plant [
23] and is widely used to treat to dyspepsia, rheumatic pains, fevers, diarrhea, worm infestations, vomiting, constipation, cramps, colic, hysteria, flatulence, menstrual problems, distention, epilepsy, to promote circulation, and as a sedative [
24,
25,
26,
27].
Several essential oils of aromatic plant are used for their antispasmodic, carminative, anti-parasitical, anti-inflammatory, antimicrobial, anti-helminthic, and insecticidal properties [
22]. Biological activities of these essential oils are mainly attributed to volatile compounds, such as α-pinene, camphor, caryophyllene, camphene, germacrene D, 1,8-cineole, and α-thujone [
22]. In this respect, various mugwort genotypes growing in different geographic regions showed varied components fraction. For example, oils from Italian mugwort was rich in camphor alone or together with myrcene, 1,8–cineole, or borneol [
28,
29]; German mugwort contained sabinene, myrcene, and 1,8-cineole [
22]; Indian mugwort was rich in camphor, α-thujone, or thujone isomer [
30]; French mugwort was rich in camphor, 1,8-cineole, and terpinen-4-ol [
31]; Moroccan mugwort had camphor, isothujone, and thujone as major components [
32]; Iranian mugwort dominated with α-pinene, menthol, β-eudesmol, and spathulenol [
33], Cuban mugwort was rich in sesquiterpene [
34]. However, there is no report on the chemical composition of Brazilian mugwort oils.
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.
2. Material and Methods
2.1. Plant Material and Botanical Identification
Aerial parts (before the onset of flowering) of A. vulgaris were collected from plants growing in botanical garden at the Federal University of Maranhão, Sao Luís, Maranhão, Brazil in August 2016. Plants were identified and authenticated by Dr. Eduardo B. de Almeida Junior. The voucher specimens (number 8.637) were kept at the Herbarium of Federal University of Maranhão, Sao Luis, Brazil.
2.2. 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.
2.3. 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.
2.4. 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.
2.5. 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% CO2.
2.6. 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.
2.7. 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.
2.8. 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.
2.9. 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.
2.10. 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.
