molecules

Article
Chemical Composition and Antimicrobial Activity of
Artemisia herba-alba and Origanum majorana
Essential Oils from Morocco
Ghita Amor 1,2 , Lucia Caputo 3 , Antonietta La Storia 2 , Vincenzo De Feo 3, * ,
Gianluigi Mauriello 2, * and Taoufiq Fechtali 1, *
1 Laboratory of Biosciences, Integrated and Molecular Functional Exploration, Faculty of Sciences and
Techniques Mohammedia, 146 Mohammedia 20650, Morocco; amor.ghitaa@gmail.com
2 Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055 Portici,
Italy; alastoria@unina.it
3 Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy;
lcaputo@unisa.it
* Correspondence: defeo@unisa.it (V.D.F.); giamauri@unina.it (G.M.); toufiqr@yahoo.com (T.F.)

Academic Editor: Daniela Rigano




Received: 7 October 2019; Accepted: 5 November 2019; Published: 6 November 2019

Abstract: Essential oils (EOs) are one of the most important groups of plant metabolites responsible
for their biological activities. This study was carried out to study the chemical composition
and the antimicrobial effects of Artemisia herba-alba and Origanum majorana essential oils against
some Gram-positive and Gram-negative bacteria, and a fungal strain isolated from spoiled butter.
The plants were collected in the region Azzemour of South West Morocco and the EOs, extracted by
hydrodistillation, were analyzed by GC-MS. The antimicrobial activity was determined using the
agar paper disc method. The main components of A. herba-alba EO were cis-thujone, trans-thujone
and vanillyl alcohol; in O. majorana EO terpinen-4-ol, isopulegol and β-phellandrene predominated.
Both essential oils exhibited growth inhibiting activities in a concentration-dependent manner on
several microorganism species. Our results demonstrated that O. majorana and A. herba-alba EOs
could be effective natural antibacterial agents in foods.

Keywords: essential oils; antimicrobial activity; chemical characterization; Artemisia herba-alba;

Origanum majorana

1. Introduction
Essential oils (EOs) are complex mixtures derived from various parts of plants with strong aromatic
components such as terpenes. They are used in many fields such as medicine, cosmetic, and food
industry [1,2]. The available literature reported that EOs possess, among others, significant antiseptic,
antibacterial, antiviral, antioxidant, anti-parasitic, antifungal, and insecticidal activities [3].
At the moment, Morocco is considered as one of the principal suppliers and producers of some
aromatic plants, such as Artemisia herba-alba Asso, Mentha pulegium L., Lavandula stoechas L., and
Rosmarinus officinalis L. Moreover, these plants produce very high added value products contributing
to the economic development of Morocco [4].
Artemisia herba-alba, chih in Arabic, belongs to the Asteraceae family; its essential oil is known for
its antimicrobial, antioxidant, insecticidal, and antispasmodic activities. It is also used in traditional
medicine as an antispasmodic and in treatment of diabetes mellitus [2,5].
Origanum majorana L. is a lamiaceous species, known for its antimicrobial, antioxidant, antidiabetic,
and antitumoral activities [6]. In traditional medicine, the plant is used as an antiepileptic and a
sedative drug [7].

Molecules 2019, 24, 4021; doi:10.3390/molecules24224021 www.mdpi.com/journal/molecules

Molecules 2019, 24, 4021 2 of 12

The aim of the present study was to identify the components of A. herba-alba and O. majorana
EOs from Morocco, and to evaluate their antimicrobial activity, against some Gram-positive and
Gram-negative bacteria, and their antifungal efficacy.

2. Results

2.1. Essential Oil Yields and Composition

Hydrodistillation of the aerial parts of A. herba alba and of O. majorana resulted in pale yellow
oils in 0.86% and 0.97% yield, on a dry mass basis, respectively. Tables 1 and 2 report the percent
composition of the essential oils; compounds are listed according to their elution on a HP-5MS column.
Fifty-eight compounds were identified, 14 for A. herba-alba, and 44 for O. majorana, accounting for
97.6% and 97.8% of the total oil, respectively. In the essential oil from A. herba-alba cis-thujone (25.5%),
trans-thujone (17.7%), vanillyl alcohol (11.5%), and nor-davanone (7.8%) are the main components.
In the essential oil from O. majorana, terpinen-4-ol (34.1%), α-terpinene (19.2%), and terpineol (8.9%)
are the main constituents.

Table 1. Chemical composition of Artemisia herba-alba essential oil.

Compound % Ki a Ki b Identification c
trans-Arbusculone 4.5 1048 1,2
cis-Thujone 25.5 1079 1102 1,2,3
trans-Thujone 17.7 1111 1114 1,2,3
Camphor 4.9 1150 1146 1,2,3
nor-Davanone 7.8 1200 1231 1,2
cis-Chrysanthenylacetate 4.7 1231 1265 1,2
Undec-10-en-1-al 1.3 1261 1296 1,2
Cyclosativene T 1342 1368 1,2
cis, threo-Davanafuran 5.8 1386 1415 1,2
Vanillyl Alcohol 11.5 1424 1447 1,2
n-Dodecanol 3.1 1445 1470 1,2
Isobornyl n-butyrate 4.9 1466 1491 1,2
<E>-Jasmolactone 3.4 1483 1491 1,2
Artedouglasia Oxide C 2.5 1496 1523 1,2
Total 97.6
Oxygenated monoterpene 56.4
Oxygenated sesquiterpenes 2.5
Other compounds 38.7
a Kovats retention index on HP-5 MS column; b Kovats retention index on HP Innovax column; c Identification:

1 = Kovats retention index, 2 = mass spectrum, 3 = co-injection with pure compound; T = traces, less than 0.05%.

Table 2. Chemical composition of Origanum majorana essential oil.

Compound % KI a KI b Identification c
α-Pinene 4.1 941 932 1,2,3
p-Cymene 2.6 950 1024 1,2,3
iso-Sylvestrene 0.6 952 1008 1,2,3
β-Pinene 0.2 975 974 1,2,3
α-Phellandrene 2.6 984 1002 1,2,3
δ-3-Carene 1.9 1008 1011 1,2
α-Terpinene 19.2 1021 1017 1,2,3
Limonene 0.1 1038 1029 1,2,3
1,8 Cineole 3.0 1047 1031 1,2,3
β-Ocimene 0.1 1061 1037 1,2,3
cis-Sabinene hydrate 1.3 1070 1070 1,2
Molecules 2019, 24, 4021 3 of 12

Table 2. Cont.

Compound % KI a KI b Identification c
Terpinen-4-ol 34.1 1096 1149 1,2,3
endo-Fenchyl-acetate 9.8 1114 1220 1,2
Pulegone 0.7 1122 1237 1,2
trans-Pinocarveol 0.3 1143 1139 1,2
Terpineol 8.9 1160 1133 1,2,3
cis-Limonene oxide T 1188 1136 1,2
dihydro-Linalool 0.1 1191 1135 1,2
cis-Verbenol T 1193 1141 1,2,3
Viridene 0.1 1199 1167 1,2
(E)-Isocitral 0.2 1205 1180 1,2
Thymol 0.2 1211 1290 1,2,3
Carvacrol 0.3 1220 1299 1,2,3
γ-Elemene 0.1 1233 1338 1,2,3
α-Terpinyl acetate 0.8 1242 1349 1,2
Eugenol T 1271 1359 1,2,3
Neryl acetate 0.2 1274 1361 1,2
α-Copaene T 1278 1376 1,2,3
Geranyl acetate 0.3 1293 1381 1,2,3
iso-Longifolene 0.1 1303 1390 1,2
(E)-Caryophillene 2.1 1314 1407 1,2,3
β-Duprezianene T 1321 1422 1,2
β-Cedrene T 1324 1420 1,2,3
β-Copaene T 1327 1432 1,2,3
α-Guaiene 0.2 1332 1439 1,2,3
Aromadendrene 0.3 1336 1441 1,2,3
allo-Aromadendrene 1.3 1370 1460 1,2
Valencene 0.2 1401 1496 1,2,3
Caryophyllene oxide T 1436 1583 1,2,3
Epiglobulol T 1445 1590 1,2
(-)-Spathulenol T 1453 1578 1,2
β-Atlanthol 1.6 1464 1608 1,2,3
Rosifoliol 0.1 1485 1600 1,2
Cubenol T 1497 1646 1,2
Total 97.8
Monoterpene hydrocarbons 33.1
Oxygenated monoterpene 57.9
Sesquiterpene hydrocarbons 5.1
Oxygenated sesquiterpenes 1.7
a Kovats retention index on HP-5 MS column; b Kovats retention index on HP Innovax column; c Identification:

1 = Kovats retention index, 2 = mass spectrum, 3 = co-injection with pure compound. T = traces, less than 0.05%.

2.2. Antimicrobial Activity

The antimicrobial activity of A. herba-alba and O. majorana essential oils was tested, at different
concentrations, against 20 microorganisms, both Gram-positive and Gram-negative strains. Figure 1
shows a representative image of the antimicrobial activity. The EO of A. herba-alba showed inhibitory
effects against 15 bacterial strains, the most sensitive being Brochothrix thermosphacta 7R1, Bacillus
clausii 2226 and Salmonella Typhimurium; five strains resulted resistant to this EO: Hafnia alvei 53M,
Carnobacterium maltaromaticum F1201, Carnobacterium maltaromaticum D1203, Enterococcus faecalis 226
and Enterococcus faecalis ES1 (Table 3). O. majorana essential oil showed a wider spectrum of activity as
it was active against all microbial strains tested (Table 4).
Molecules 2019, 24, 4021 4 of 12

Figure 1. Representative antimicrobial activity of (A) Origanum majorana essential oil against Brochothrix
thermosphacta D274 at the dose of 50, 40, 20, and 15 µL (from 1 to 4, respectively) and (B) Artemisia
herba-alba essential oil against Bacillus clausii 2226 at the concentrations of 20, 15, 10, and 5 µL (from 1 to
4, respectively).

Table 3. Antibacterial activity of the essential oil of A. herba-alba.

Strain Control Essential Oil

Gentamicin Tetracyclin 5 µL 10 µL 15 µL 20 µL
B. clausii 2226 11.0 ± 1.0 16.3 ± 1.5 10.3 ± 0.6 14.7 ± 0.6 19.7 ± 1.5 a,D 24.0 ± 1.0 a,A
Br. thermosphacta
18.3 ± 1.5 19.3 ± 1.2 na 6.0 ± 0.0 6.0 ± 0.1 12.3 ± 0.6
7R1
Br. thermosphacta
6.0 ± 0.0 8.7 ± 1.2 6.0 ± 0.0 11.7 ± 0.6 a,C 14.7 ± 0.6 a,A 17.7 ± 0.6 a,A
D274
C. maltaromaticum
6.0 ± 0.0 24.3 ± 1.2 6.0 ± 0.0 9.0 ± 1.0 b 11.7 ± 0.6 a 12.3 ± 0.6 a
9P
C. maltaromaticum
6.0 ± 0.0 22.3 ± 0.6 na na na na
D1203
C. maltaromaticum
6.0 ± 0.0 23.3 ± 1.5 na na na na
F1201
C. maltaromaticum
10.0 ± 0.0 na na na na 6.0 ± 0.0 A
H1201
E. coli 32 14.7 ± 0.6 18.7 ± 1.2 na na 6.0 ± 0.0 6.0 ± 0.0
Ent. faecalis 226 6.0 ± 0.0 9.0 ± 1.0 na na na na
Ent. faecalis E21 6.0 ± 0.0 14.7 ± 0.6 na na na na
H. alvei 53M 11.7 ± 1.5 9.7 ± 0.6 na na na na
L. innocua 1770 25.3 ± 0.6 20.3 ± 1.5 na na na 6.0 ± 0.0
P. fragi 6P2 14.7 ± 0.6 17.0 ± 1.0 na 6.0 ± 0.0 9.3 ± 0.6 13.3 ± 2.1
Staph. aureus 6.0 ± 0.0 15.3 ± 0.6 na na na 6.0 ± 0.0
S. Typhimurium 9.7 ± 0.6 12.7 ± 1.2 na 6.0 ± 0.0 14.0 ± 1.7 b 17.7 ± 0.6 a,B
Serr.
12.3 ± 0.6 24.3 ± 1.2 na 6.0 ± 0.0 7.7 ± 0.6 10.3 ± 0.6
proteamaculans 20P
Str. salivarius 6.0 ± 0.0 18.7 ± 1.2 na 6.0 ± 0.0 14.0 ± 1.0 a 18.3 ± 1.5 a
Staph.
24.0 ± 1.0 29.0 ± 3.6 na na 6.0 ± 0.0 6.0 ± 0.0
saprophyticus 3S
Staph. sp.ES1 19.3 ± 1.2 29.3 ± 1.2 na 6.0 ± 0.0 8.3 ± 0.6 10.3 ± 0.6
Staph.sp.GB1 21.3 ± 1.2 27.7 ± 2.5 6 ± 0.0 11.3 ± 1.2 14.7 ± 0.6 17 ± 1.0
Data represent the diameter inhibition (in mm). Results are the mean of three repetitions ± standard deviation (SD)
of the inhibition zone. na = not active. Dunnett’s test vs. Gentamicin (a,b,c,d ) or Tetracycline (A,B,C,D ): a,A p < 0.0001;
b,B p < 0.001; c,C p < 0.01; d,D p < 0.05. B.: Bacillus; Br.: Brochothrix; C.: Carnobacterium; E.: Enterococcus; Staph.:

Staphylococcus; L.: Listeria; E.: Escherichia; H.: Hafnia; P.: Pseudomonas; S.: Salmonella; Serr.: Serratia; Str.: Streptococcus.
Molecules 2019, 24, 4021 5 of 12

Table 4. Activity of the essential oil of O. majorana.

Strain Control Essential Oil

Gentamicin Tetracyclin 5 µL 10 µL 15 µL 20 µL 40 µL 50 µL
B. clausii 2226 11.0 ± 1.0 16.3 ± 1.5 na 6.0 ± 0.0 15.3 ± 0.6b 23.3 ± 1.5 a,B 24.7 ± 0.6 a,B 28.3 ± 1.5 a,B
Br. thermosphacta
6.0 ± 0.0 8.7 ± 1.2 10.6 ± 0.6d 13.3 ± 1.2 a 18.3 ± 0.6 a,B 23.0 ± 1.7 a,B 24.3 ± 1.2 a,B 26.3 ± 1.2 a,B
D274
Br. thermosphacta
18.3 ± 1.5 19.3 ± 1.2 11.3 ± 1.2 15.3 ± 0.6 18.0 ± 0.0 20.3 ± 0.6* 20.0 ± 0.0 21.3 ± 0.6c,D
7R1
C. maltaromaticum
6.0 ± 0.0 24.3 ± 1.2 na na na 6.0 ± 0.0 6.0 ± 0.0 9.3 ± 0.6a
9P
C.maltaromaticum
10.0 ± 0.0 na 6.0 ± 0.0A 6.0 ± 0.0 A 8.7 ± 1.2 A 9.7 ± 0.6 A 9.3 ± 0.6 A 9.7 ± 0.6 A
H1 201
C. maltaromaticum
6.0 ± 0.0 22.3 ± 0.6 na na na 6.0 ± 0.0 9.3 ± 0.6 a 13.3 ± 1.5 a
D1 203
C. maltaromaticum
6.0 ± 0.0 23.3 ± 1.5 na na na 6.0 ± 0.0 6.0 ± 0.0 6.0 ± 0.0
F1 201
E. coli 32 14.7 ± 0.6 18.7 ± 1.2 9.7 ± 0.6 10.7 ± 1.2 17.7 ± 0.6 20.0 ± 0.0 a 24.3 ± 1.2 a,B 26.7 ± 0.6 a,B
Ent. faecalis 226 6.0 ± 0.0 9.0 ± 1.0 na na na 6.0 ± 0.0 6.0 ± 0.1 9.7 ± 0.7
Ent. faecalis E21 6.0 ± 0.0 14.7 ± 0.6 na na na 6.0 ± 0.0 6.0 ± 0.0 6.0 ± 0.0
H. alvei 53M 11.7 ± 1.5 9.7 ± 0.6 8.3 ± 0.6 10.3 ± 0.6 11.7 ± 0.6 C 12.7 ± 0.6 A 15.0 ± 0.0 b,A 20.3 ± 0.6 a,B
L. innocua 1770 25.3 ± 0.6 20.3 ± 1.5 na 6.0 ± 0.0 9.3 ± 0.6 11.7 ± 0.6 12.3 ± 0.6 13.7 ± 1.5
P. fragi 6P2 14.7 ± 0.6 17.0 ± 1.0 na na 6.0 ± 0.0 6.0 ± 0.0 6.0 ± 0.0 9.3 ± 0.6
Staph. aureus 6.0 ± 0.0 15.3 ± 0.6 10.7 ± 0.6 d 11.7 ± 1.5 c 16.3 ± 1.5 a 24.3 ± 2.1 a,B 27.7 ± 0.6 a,B 32.3 ± 2.5 a,B
S. Typhimurium 9.7 ± 0.6 12.7 ± 1.2 7.7 ± 2.1 11.7 ± 0.6 14.0 ± 1.7 d 17.3 ± 1.2 c,D 23.3 ± 2.9 a,B 29.7 ± 0.6 a,B
Serr.proteamaculans
12.3 ± 0.6 24.3 ± 1.2 na na na 6.0 ± 0.0 16.3 ± 1.5a 19.3 ± 1.2a
20P
Str. salivarius 6.0 ± 0.0 18.7 ± 1.2 9.7 ± 0.6 c 11.3 ± 0.6 a 13.0 ± 1.0 a 19.3 ± 1.2 a 20.7 ± 1.2 a 24.3 ± 1.2 a,B
Staph.
24.0 ± 1.0 29.0 ± 3.6 9.9 ± 1.0 11.7 ± 0.6 19.3 ± 1.2 20.3 ± 0.6 21.0 ± 1.0 24.3 ± 1.2
saprophyticus 3S
Staph.sp. ES1 19.3 ± 1.2 29.3 ± 1.2 6.0 ± 0.0 6.0 ± 0.0 14.7 ± 7.6 18.7 ± 1.2 20.0 ± 0.0 21.0 ± 1.0
Staph.sp.GB1 21.3 ± 1.2 27.7 ± 2.5 na na 5.7 ± 0.6 10 ± 0.0 14.7 ± 0.6 19.3 ± 1.2
Data represent the diameter inhibition(in mm). Results are the mean of three repetitions ± standard deviation (SD)
of the inhibition zone. na = not active. Dunnett’s test vs. Gentamicin (a,b,c,d ) or Tetracycline (A,B,C,D ): a,A p < 0.0001;
b,B p < 0.001; c,C p < 0.01; d,D p < 0.05. B.: Bacillus; Br.: Brochothrix; C.: Carnobacterium; E.: Enterococcus; Staph.:

Staphylococcus; L.: Listeria; E.: Escherichia; H.: Hafnia; P.: Pseudomonas; S.: Salmonella; Serr.: Serratia; Str.: Streptococcus.

Data analysis showed for the EO of A. herba-alba the same antimicrobial activity of tetracycline
against Streptococcus salivarius, but higher than gentamicin, and exhibited stronger antimicrobial
activity than both antibiotics against Br. thermosphacta D274, B. clausii 2226, and S. Typhimurium and
lower antimicrobial activity than that of both antibiotics against Staphylococcus sp. GB1, Staphylococcus
saprophyticus 3S, Escherichia coli 32, Br. thermosphacta 7R1, Staphylococcus sp. ES1, and Serratia
proteamaculans 20P. On the other hand, the antimicrobial activity of O. majorana essential oil was
stronger than both antibiotics against Str. salivarius, E. coli 32, Br. thermosphacta 7R1, H. alvei 53M,
Salmonella sp. ES1, Br. thermosphacta D274, B. clausii 2226, Ente. faecalis 226, S. Typhimurium, and
Staphylococcus aureus. The same antimicrobial activity as gentamicin was recorded against Staph.
saprophyticus 3S, C. maltaromaticum H1201, C. maltaromaticum F1201, Ent. faecalis E21 and lower
antimicrobial activity than both antibiotics against Staph. sp GB1 and Listeria innocua 1770.

2.3. Antifungal Activity

Table 5 reports the inhibition halos in (mm) at the dose of 20 µL of the two essential oils against
Aspergillus niger isolated from the spoiled butter. This fungal strain was sensitive to both essential
oils; the highest inhibitory activity was showed by A. herba-alba essential oil against A. niger. Figure 2
shows the antifungal activity of both EOs as determined in the same agar dish. However, diameters of
inhibition halos were measured on two separated agar dishes.
Molecules 2019, 24, 4021 6 of 12

Table 5. Antifungal activity of A. herba-alba and O. majorana essential oils.

Aspergillus niger
Artemisia herba-alba 23.6 ± 1.5
Origanum majorana 14.0 ± 1.0
Data represent the diameter inhibition (in mm). Results are the mean of three repetitions ± standard deviation (SD)
of the inhibition zone.

Figure 2. Antifungal activity of A. herba-alba (1) and O. majorana (2) essential oils against Aspergillus
niger at the dose of 20 µL.

3. Discussion
In our A. herba-alba essential oil oxygenated monoterpenes (57.3%) predominated, with cis-thujone
(25.5%) and trans-thujone (17.7%) as the main constituents. Vanillyl alcohol (11.5%) and nor-davanone
(7.8%) were in appreciable amounts.
These results agree with literature on the essential oil from A. herba-alba from different countries
that evidenced cis- and/or trans-thujone as the principal constituents [8–10]. On the other hand, other
studies showed eucalyptol (32.8%) as the main constituent of the A. herba-alba EO from Iran, and
caryophyllene acetate (10.75%) for a Jordanian EO [11,12]. These compounds are totally absent in our
essential oil. Camphor is reported as principal component in essential oil from Algeria and Tunisia
(ranging between 19.6% and 50.5%) [13,14], but it is present in a low percentage in our sample (4.9%).
Moreover, other studies evidenced that davanone is one of the main constituents, with a percent
greater than 10% [15,16]. In our EO, davanone and its derivative, cis, threo-davanafuran, accounted for
13.6% of the oil. Instead, this is the first report on the presence of vanillyl alcohol as one of the main
constituents of this EO. Other studies reported camphor as the major component of the essential oil
(17.8%–50.3%) [13,15,17–19] that, instead, is absent in our sample or chrysanthenone, present in our
EO with its derivative, iso-chrysanthenyl acetate [20,21].
Monoterpenes predominated (91.1%) in the oil of O. majorana, both hydrocarbons and oxygenated
compounds; sesquiterpenes accounted for 6.8%. The main components are terpinen-4-ol (34.1%),
α-terpinene (19.2%), and terpineol (8.9%). Our results are in agreement with many studies that reported
terpinen-4-ol among the principal constituents of the essential oil of O. majorana [22–26]. Moreover,
α-terpinene was present in similar percentage (ranging from 11.08% to 12.72%) also in essential oils
from Tunisia and Morocco [22,27]. Instead, in the EO from the Venuezelan Andes α-terpinene is
reported in lesser percentages (3.6%) [26]. trans-Sabinene hydrate was reported as one of the principal
components in other studies [28,29], in our sample its isomer was present in a low quantity (1.3%).
Linalool, absent in our essential oil, is the main compound in the EO of O. majorana from Turkey with a
percent of 88.01% [30]. Moreover, 4-terpinene and γ-terpinene were identified as the main components
in O. majorana from Taiwan and Morocco, respectively [27,31].
Molecules 2019, 24, 4021 7 of 12

Most microorganisms used in this study were sensitive to both essential oils, with the dose of
20 µL of EO sufficient to stop the growth of almost all tested Gram-positive and Gram-negative strains.
In particular, O. majorana EO resulted more active, showing a wide spectrum of activity. On the other
hand, the EO of A. herba-alba showed inhibitory effects against 15 bacterial strains.
The available literature reports the antimicrobial activity of A. herba-alba essential oil against
Staph. aureus, E. coli, and B. cereus [23,32,33]. Moreover, several studies showed a great potential
of A. herba-alba EO oil as an antibacterial agent against Klebsiella pneumoniae, Listeria monocytogenes,
Vibrio colerae, and S. Typhimurium [34–36]. Our results showed variable antimicrobial and antifungal
activity of the essential oil, being the inhibition zones in the range of 10–24 mm. Gram-positive
bacteria resulted more sensitive to this EO. The Gram-positive B. clausii 2226 was the most sensitive
tested strain, with the strongest inhibition zone (24.00 ± 1 mm). B. clausii was used as a model of
spore-forming aerobic microorganism and our findings showed that our A. herba-alba EO is suitable to
control the growth of this microorganism. It is well known that spore forming bacteria (also called
thermoduric) are the main problem in pasteurized foods, both from the point of view of food spoilage
and human intoxication. Gram-negative strains also displayed variable degree of susceptibility to this
EO. The maximum activity was showed against the pathogen strain S. Typhimurium (17.7 ± 0.6), but
C. maltaromaticum F1201, C. maltaromaticum D1203, H. alvei 53M, Ent. faecalis E21, and Ent. faecalis 226
resulted resistant, since no inhibition zone was observed. Due to the involvement of S. Typhimurium
in the majority of food intoxication across the world, the antimicrobial capability of this EO could be of
pivotal importance in the control of this microorganism in foods.
The antimicrobial activity of O. majorana essential oil appears to be similarly effective against both
Gram-positive and Gram-negative microorganisms. These results agree with literature data [21,34,35].
Data of previous research showed that O. majorana essential oil was active against a large spectrum of
different bacteria strains: E. coli, Str. agalactiae, Shigella dysenteriae, Salmonella Enteritidis, Staph. aureus,
Ent. faecalis, E. coli, and Klebsiella pneumoniae [26,37].
In our study, all tested strains were sensitive to this essential oil, with the Gram-positive S. aureus
the most sensitive with the greatest inhibition zone (32.2 ± 2.5 mm); the more sensitive Gram-negative
was S. Typhimurium, with an inhibition zone of 29.7 ± 0.6 mm.
The antimicrobial activity of both essential oils could be related to their content in oxygenated
monoterpenes, which constitute about 57.3% and 53.0% of the EOs of A. herba alba and O. majorana,
respectively. Similar findings have been already previously reported [38,39].
The major components of O. majorana EO, e.g., terpinen-4-ol, α-terpinol and α-pinene, have been
reported for their antimicrobial and antifungal properties [21]. Additionally, the main constituents
of the EO of A. herba-alba, cis- and trans-thujone and vanillyl alcohol, have been reported for their
antimicrobial, anti-inflammatory, and antioxidant activities [40,41]. Oxygenated monoterpenes exhibit
high antimicrobial activity on whole cell and possess antifungal effects. These compounds diffuse into
and damage cell membrane structures [42].
Our results showed high antifungal activity for both essential oils, with the highest inhibitory
activity shown by the EO of A. herba-alba against Aspergillus niger (inhibition zone 23.6 ± 1.5 mm).
These results are consistent with data previously reported [29,43].

4. Materials and Methods

4.1. Plant Material

The aerial parts of A. herba-alba and O. majorana were collected in the Azzemour region, South
West Morocco, in June 2018, in flowering stage, and dried in the shade. The plants were identified by
Prof. V. De Feo. A voucher specimen of each plant is stored in Department of Agricultural Sciences,
University of Naples Federico II.
Molecules 2019, 24, 4021 8 of 12

4.2. Essential Oil Extraction

One kilogram of A. herba-alba and O. majorana aerial parts was subjected to hydrodistillation for
3 h, according to the standard procedure described in the European Pharmacopoeia [44]. The oils were
solubilized in n-hexane, filtered over anhydrous sodium sulphate and stored under N2 at +4 ◦ C in the
dark, until tested and analyzed.

4.3. GC-FID Analysis

Analytical gas chromatography was carried out on a Perkin-Elmer Sigma-115 gas-chromatograph
(Perkin Elmer, Waltham, MA, USA) equipped with an FID and a data handling processor. The separation
was achieved using a HP-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film
thickness). Column temperature: 40 ◦ C, with 5 min initial hold, and then to 270 ◦ C at 2 ◦ C/min,
270 ◦ C (20 min); injection mode splitless (1 µL of a 1:1000 n-hexane solution). Injector and detector
temperatures were 250 ◦ C and 290 ◦ C, respectively. Analysis was also run by using a fused silica HP
Innowax polyethylene glycol capillary column (50 m × 0.20 mm i.d., 0.25 µm film thickness). In both
cases, helium was used as carrier gas (1.0 mL/min).

4.4. GC/MS Analysis

Analysis was performed on an Agilent 6850 Ser. II apparatus (Agilent, Roma, Italy), fitted with a
fused silica DB-5 capillary column (30 m × 0.25 mm i.d., 0.33 µm film thickness), coupled to an Agilent
Mass Selective Detector MSD 5973 (Agilent); ionization energy voltage 70 eV; electron multiplier
voltage energy 2000 V. Mass spectra were scanned in the range 40–500 amu, scan time 5 scans/s. Gas
chromatographic conditions were as reported in the previous paragraph; transfer line temperature,
295 ◦ C.

4.5. Identification of the Essential Oil Components

Most constituents were identified by comparison of their Kovats retention indices (Ri) (determined
relative to the tR of n-alkanes (C10–C35), with either those of the literature [45–47] and mass spectra
on both columns or those of authentic compounds available in our laboratories by means of NIST
02 and Wiley 275 libraries [48]. The components relative concentrations were obtained by peak
area normalization.

4.6. Antibacterial Assay

The antibacterial activity was evaluated in vitro, by means of the agar diffusion test on the plate.
The activity of the essential oils was tested on the 20 microorganisms reported in Table 6. All of them
belong to the collection of the Department of Agricultural Sciences, University of Naples Federico II.
Microbial strains were previously grown in TSB tryptone soya broth for 24 h. A volume of 0.1
mL of the microbial suspensions (about 1 × 108 CFU/mL) was uniformly distributed on Nutrient agar
plates in sterile conditions. Different amounts of essential oils were spotted on the inoculated plates:
50, 40, 20, 15, 10, and 5 µL for O. majorana and 20, 15, 10, and 5 µL for A. herba-alba essential oils. After
10 min, under sterile conditions, plates were then incubated at optimal growth condition culture of
each strain. The antimicrobial activity was evidenced by measuring the diameter (in mm) of the zone
of inhibition. Ethanol was used as the negative control; tetracycline (10 µg) and gentamycin (10 µg)
were used as positive controls. Each experiment was carried out in three independent replicates and
result is the average with standard deviation.
Molecules 2019, 24, 4021 9 of 12

Table 6. Source and optimal growth conditions of microorganisms.

Gram Microorganism Source Growth Conditions

B. clausii 2226 Supplement TSB 24h at 30 ◦ C
Br. thermosphacta 7R1 Meat TSB 24h at 20 ◦ C
Br. thermosphacta D274 Meat TSB 24h at 20 ◦ C
C. maltaromaticum 9P Meat TSB 24h at 20 ◦ C
C. maltaromaticum D1203 Meat TSB 24h at 25 ◦ C

Positive C. maltaromaticum F1201 Meat TSB 24h at 25 ◦ C

C. maltaromaticum H1201 Meat TSB 24h at 25 ◦ C
Ent. faecalis 226 Milk TSB 24h at 30 ◦ C
Ent. faecalis E21 Milk TSB 24h at 30 ◦ C
Staph. aureus Meat TSB 24h at 37 ◦ C
Staph. saprophyticus 3S Fermented meat TSB 24h at 37 ◦ C
Staph. sp. ES1 Fermented meat TSB 24h at 37 ◦ C
Staph. sp. GB1 Fermented meat TSB 24h at 37 ◦ C
L. innocua 1770 Milk TSB 24h at 30 ◦ C
E. coli 32 Meat TSB 24h at 37 ◦ C
Str. salivarius Milk TSB 24h at 30 ◦ C
H.alvei 53M Meat TSB 24h at 30 ◦ C
Negative Pseud. fragi 6P2 Meat TSB 24h at 20 ◦ C
S. Typhimurium Chicken meat TSB 24h at 30 ◦ C
Serr.proteamaculans20P Meat TSB 24h at 25 ◦ C
B.: Bacillus; Br.: Brochothrix; C.: Carnobacterium; E.: Enterococcus; Staph.: Staphylococcus; L.: Listeria; E.: Escherichia; H.:
Hafnia; P.: Pseudomonas; S.: Salmonella; Serr.: Serratia; Str.: Streptococcus.

4.7. Antifungal Activity

The antifungal activity was evaluated in vitro, using the agar well diffusion method on the plates.
The activity was tested against Aspergillus niger isolated from a spoiled butter sample and identified by
phenotypic characteristics. The fungus was previously grown in TSA agar plates at 28 ◦ C until spore
formation. Then, 1 mL of a spore suspension in quarter strength Ringer solution, containing about
1 × 108 spores per mL, was uniformly distributed on Nutrient agar plates in sterile conditions, then a
hole was punched with sterile cork and 20 µL of each EO was introduced into the well; the plates were
incubated at 28 ◦ C for 4–5 days. Ethanol was used as the negative control. The antifungal activity was
evaluated by measuring diameter of the inhibition area. Each experiment was carried out in three
independent replicates and result is the average with standard deviation.

4.8. Statistical Analysis

Data of each experiment were statistically analyzed using GraphPad Prism 6.0 software (GraphPad
Software Inc., San Diego, CA, USA), followed by comparison of means (one-way ANOVA) using
Dunnett’s multiple comparisons test, at the significance level of p < 0.05.

5. Conclusions
The composition of Artemisia herba-alba and Origanum majorana essential oils growing in Morocco
was analyzed and its antibacterial and antifungal activity investigated. The results indicated an
important antimicrobial activity against different microorganisms especially from O. majorana essential
oil. Thus, they can maybe be applied in food industry as natural preservatives, due to their antibacterial
Molecules 2019, 24, 4021 10 of 12

properties. Further organoleptic features and toxicological studies are required to prove the safety of
the oils.

Author Contributions: Conceptualization, T.F., G.M., and V.D.F.; formal analysis, L.C. and G.A.; investigation,
G.A., A.L.S.; writing—original draft preparation, G.A. and L.C.; writing—review and editing, T.F., G.M. and V.D.F.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of A. herba-alba and O. majorana essential oils are available from the authors.

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