molecules
Article
Essential Oil Analysis and Antimicrobial Evaluation of Three
Aromatic Plant Species Growing in Saudi Arabia
Hamdi El-Said 1 , Sami S. Ashgar 1 , Ammar Bader 2, *, Aljawharah AlQathama 2 , Majed Halwani 3 ,
Roberta Ascrizzi 4 and Guido Flamini 4, *
1
2
3
4
*
Citation: El-Said, H.; Ashgar, S.S.;
Bader, A.; AlQathama, A.; Halwani,
M.; Ascrizzi, R.; Flamini, G. Essential
Oil Analysis and Antimicrobial
Evaluation of Three Aromatic Plant
Species Growing in Saudi Arabia.
Molecules 2021, 26, 959. https://
doi.org/10.3390/molecules26040959
Academic Editor: Henryk H. Jeleń
Received: 15 December 2020
Department of Medical Microbiology, Faculty of Medicine, Umm Al-Qura University, Makkah 21955,
Saudi Arabia; hmibrahim@uqu.edu.sa (H.E.-S.); ssashgar70@hotmail.com (S.S.A.)
Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia;
aaqathama@uqu.edu.sa
King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health
Sciences, Riyadh 11481, Saudi Arabia; halawanima@ngha.med.sa
Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy; roberta.ascrizzi@gmail.com
Correspondence: ambader@uqu.edu.sa (A.B.); guido.flamini@unipi.it (G.F.)
Abstract: Arabian flora is a rich source of bioactive compounds. In this study, we investigated
three aromatic plant species with the aim of finding valuable sources of antimicrobial agents
against common pathogenic microorganisms. We focused especially on microorganisms, which
cause outbreaks of infectious disease during mass gatherings and pilgrimages season in Saudi Arabia. The essential oils of three aromatic plant species were hydrodistilled from flowering aerial
parts of Lavandula pubescens Decne. and Pulicaria incisa subsp. candolleana E.Gamal-Eldin, and
from leaves, stems, ripe and unripe fruits of Juniperus procera Hochst. Ex Endl. They were subsequently analyzed by gas chromatography-mass spectrometry (GC-MS). The main constituents of
L. pubescens were found to be carvacrol (55.7%), methyl carvacrol (13.4%), and β-bisabolene (9.1%).
P. incisa subsp. Candolleana essential oil was rich in linalool (33.0%), chrysanthenone (10.3%), eugenol
(8.9%), and cis-chrysanthenol (8.0%); the major components of J. procera essential oil were α-pinene
(31.3–62.5%) and δ-3-carene (7.3–30.3%). These essential oils were tested against thirteen American
Type Culture Collection (ATCC) strains of Gram-positive and Gram-negative bacteria using the
agar diffusion assay. The only effective essential oil was that of L. pubescens and the most sensitive
strains were Acinetobacter baumannii, Salmonella typhimurium, Shigella sonnei, Enterococcus faecalis and
Staphylococcus epidermidis. Carvacrol, the major constituent of L. pubescens, was tested on these strains
and was compared with vancomycin, amikacin, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays of L. pubescens essential oil and
carvacrol revealed that Gram-negative strains were more susceptible than the Gram-positive ones.
Accepted: 8 February 2021
Published: 11 February 2021
Keywords: carvacrol; Lavandula pubescens; Pulicaria incisa; Juniperus procera; holy sites health
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1. Introduction
Plants represent valuable sources of bioactive molecules belonging to various classes
of secondary metabolites. Among the physiological roles of secondary metabolites in
plants, is the resistance to phytopathogens, including bacteria, fungi and viruses [1–3].
The majority of these metabolites have the ability to interact with cellular enzymes or
cell structure, causing irreversible damage to the invasive microorganisms [4–6]. For this
reason, plant secondary metabolites have become an interesting target for the discovery of
new bioactive molecules with antimicrobial effects and variable modes of action, especially
after the recent emergence and growth of antibiotic resistance. According to the World
Health Organization (WHO), the current rise in antibiotic resistance is due to the misuse of
pharmaceutical antibiotics and is a major cause of the prolongation of illness with higher
risk of death [7].
Molecules 2021, 26, 959. https://doi.org/10.3390/molecules26040959
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 959
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Essential oils (EOs) have numerous commercial applications due to their diverse
biological properties and appealing fragrances. The global market thus supports a wide
variety of pharmaceutical products such as gels, creams, ointments, nano-emulsions, and
patches [8–11]. Besides their exceptional antimicrobial effects, they also possess a wide
range of pharmacological properties, including, for example, analgesic, anti-inflammatory,
antidiabetic, anti-parasitic, anticancer and antioxidant activity [12–16]. The use of EOs in
therapy has increased in recent times due to the rise in multidrug-resistant bacteria and the
high costs of new generation antibiotic drugs [17]. The chemical nature of essential oils
hinders the process of microbial resistance, since they are very complex mixtures of constituents with different structures, including monoterpenes [18], sesquiterpenes [19], diterpenes [20], sulfur-containing compounds [21,22], phenylpropanoids [20], alkaloids [23],
and phenols [24]. This large variety of chemical structures means that no single enzyme is
able to deactivate all of these compounds. Thus, EOs may represent an open frontier for
advances in medicine and pharmaceutical sciences.
The flora of Saudi Arabia is very rich and variable due to environmental diversity,
ranging from extreme arid desert to high mountains with high rainfall rates. These variations affect plant metabolism in terms of secondary metabolite chemistry and biological
activity. In fact, some Saudi plants have been found to exhibit substantial chemo-diversity
from the same plants grown in other countries and climates [25–27]. The aim of this study
was to investigate the chemical composition of hydrodistilled EOs from different organs
of three aromatic plant species collected in Saudi Arabia and to find valuable sources of
antimicrobial agents against common pathogenic microorganisms. These were the aerial
parts of Lavandula pubescens Decne. and Pulicaria incisa subsp. candolleana E.Gamal-Eldin,
as well as leaves, stems, ripe and unripe fruits of Juniperus procera Hochst. Ex Endl.
L. pubescens is reported in the literature as a species commonly found among the wild
flora in Middle-Eastern Asia and Mediterranean Africa; it is, indeed, mainly reported
as growing in Palestine [28], Yemen [29–31], Saudi Arabia [32–34]. For P. incisa subsp.
Candolleana, to the best of our knowledge, only one study is reported in the literature,
analyzing wild-growing specimens in Egypt [35]. However, Pulicaria spp. are reported as
widely used in Northern African folk-medicine [36]. These species have been chosen for
their good availability in the wild, which makes them an exploitable and easy to gather
biomass in their native range.
Recently, in Saudi Arabia, new outbreaks of multidrug-resistant pathogenic microorganisms have been recorded in intensive care units, including Gram-positive and
Gram-negative bacteria, out of which Acinetobacter baumannii, Pseudomonas aeruginosa,
Escherichia coli, and Klebsiella pnemoniae can cause fatal respiratory tract infections and
pneumonia, blood stream infections and urinary tract infections. Not only this, but also
the transmission and spread of infectious diseases during mass public gatherings such
as Hajj (an annual pilgrimage to the Holy Mecca, Saudi Arabia) poses an enormous challenge. The global spread of antibiotic-resistant bacteria by international travelers may occur
during pilgrimages or when visitors return to their home countries [37]. The pathogenic
microorganisms mentioned above have gradually become less susceptible to a broad spectrum of potent antibiotics such as imipenem, meropenem, ciprofloxacin, amikacin and
cefuroxime [38]. Due to the increasing presence of multidrug-resistant pathogenic microorganisms, the present study also aims to test these three EOs against three Gram-positive
and Gram-negative bacteria to assess their potential use as alternative antimicrobial agents.
2. Results and Discussion
2.1. Essential Oil Compositions
The six hydrodistilled EOs were analyzed by GC-MS; their complete compositions
and hydrodistillation yields are reported in Table 1.
The EO of L. pubescens (Figure 1a) revealed a total of 19 different compounds. Oxygenated monoterpenes were the predominant class, accounting for 70.1% of the whole oil.
Among them, carvacrol (55.7%) and methyl carvacrol (13.4%) were the main constituents.
Molecules 2021, 26, 959
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Monoterpene hydrocarbons were the second most abundant class, which included terpinolene (6.1%), (Z)-β-ocimene (4.1%) and myrcene (3.5%) among the most represented
compounds. Sesquiterpene hydrocarbons accounted for 13.1%, with β-bisabolene as the
most abundant (9.1%). The predominance of carvacrol and methyl carvacrol as the most
abundant compounds in the L. pubescens EO of this study is in accordance with published
compositions of the EOs hydrodistilled from several Yemeni specimens [29,30]. This species
is, indeed, reported as having a phenolic-type EO profile among species belonging to this
genus [39]. Several bioactivities of the EO are reported in the literature; for example, it is
antioxidant and antimicrobial towards a wide variety of bacterial and fungal strains responsible for human and animal diseases, e.g., Staphylococcus aureus, E. coli, Candida albicans,
and Microsporum canis [28,30].
Figure 1. (a) Lavandula pubescens flowering aerial part; (b) Pulicaria incisa ssp. candolleana flowering aerial part; (c) Juniperus procera.
In the EOs of Pulicaria incisa ssp. candolleana (Figure 1b), 36 compounds were detected.
Its EO was rich in oxygenated monoterpenes, accounting for 64.2% of the whole oil,
with linalool (33.0%), chrysanthenone (10.3%), and cis-chrysanthenol (8.0%) as the main
constituents. Phenylpropanoids represented the second most abundant class, with eugenol
(8.9%) as the sole representative compound. The class of non-terpene derivatives accounted
for 8.7%, including mainly (Z)-jasmone (4.7%) and isopentyl 2-methylbutanoate (2.6%).
Only one previous study reported the composition of the EOs of P. incisa ssp. candolleana
collected in Egypt, with carvotanacetone (66.01–50.87%) and chrysanthenone (13.26–24.3%)
as the most representative components, of which the latter was also present in a significant
percentage (10.3%) in our sample. EOs hydrodistilled from this Egyptian sample showed
antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against
some fungi, among which Streptococcus pneumonia, E. coli and Syncephalastrum racemosum
were the most sensitive strains [35].
All EOs distilled from the different parts of J. procera (Figure 1c) were compared,
revealing minor chemical differences. EO compositions showed some common compounds in all the investigated parts, such as α-pinene (31.3–62.5%), δ-3-carene (7.3–30.3%),
α-humulene (1.5–6.9%), β-caryophyllene (1.6–6.4%), and β-pinene (3.3–4.6%). Ripe and
unripe fruits showed the least differences, while the stems EO contained higher percentages
of β-bisabolene (9.1%), which was completely absent in leaf and fruit EOs. A previous
study on the fruit EO of J. procera reported the presence of eugenol as the main constituent
(78.4%); although this phenylpropanoid was completely absent in our sample, these two
compositions shared the presence of α-pinene and β-caryophyllene, even in different
percentages [40]. The cited study, however, does not specify the ripeness stage of the hy-
Molecules 2021, 26, 959
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drodistilled material. The EOs of the leaves reported in this study shared some constituents
with Ethiopian samples, whose literature-reported EOs compositions showed α-pinene
(28.1%), δ-3-carene (29.6%), β-pinene (4.35%), elemol (1.8%) and terpinolene (4.1%) [41,42].
A published study by Burits et al. (2001) on J. Procera leaf EO reported its antioxidant
capacity [43].
Table 1. Complete chemical composition of all the hydrodistilled essential oils.
Constituents.
santolina triene
tricyclene
α-thujene
α-pinene *
α-fenchene
camphene *
thuja-2,4 (10)-diene
sabinene *
β-pinene *
2,3-dehydro-1,8-cineole
myrcene *
cis-dehydroxylinalool oxide
α-phellandrene *
δ-3-carene *
α-terpinene *
p-cymene *
limonene *
(Z)-β-ocimene *
(E)-β-ocimene *
γ-terpinene *
cis-linalool oxide (furanoid) *
terpinolene *
trans-linalool oxide (furanoid) *
p-cymenene
linalool *
isopentyl-2-methylbutanoate *
α-cyclocitral
α-isophorone *
chrysanthenone
α-campholenal
trans-pinocarveol *
camphor *
trans-pinocamphone
cis-chrysanthenol
pinocarvone
borneol *
4-terpineol *
p-cymen-8-ol *
α-terpineol *
myrtenal *
myrtenol *
verbenone *
8,9-dehydrothymol
methylcarvacrol *
cis-chrysanthenyl acetate
isopiperitenone
bornyl acetate *
p-menth-1-en-9-ol
carvacrol *
eugenol *
(E)-β-damascenone
β-bourbonene
β-elemene
(E)-jasmone
(Z)-jasmone *
β-caryophyllene *
dimethoxy-p-cymene
α-humulene *
l.r.i.
a
910
928
933
941
954
955
959
977
982
992
993
1002
1006
1013
1020
1028
1032
1042
1052
1063
1076
1089
1090
1091
1101
1102
1117
1120
1126
1127
1141
1145
1162
1163
1164
1168
1179
1185
1191
1194
1195
1207
1221
1244
1264
1271
1287
1294
1298
1358
1382
1385
1392
1393
1395
1419
1424
1455
l.r.i.
b
908
926
931
939
951
953
957
976
980
991
991
999
1005
1011
1018
1027
1031
1040
1050
1062
1074
1088
1088
1089
1098
1099
1116
1118
1123
1125
1139
1144
1160
1162
1162
1165
1177
1183
1190
1193
1194
1204
1221
1244
1262
1272
1285
1291
1298
1356
1380
1384
1391
1390
1394
1418
1423
1454
Relative Abundance (%)
L. pubescens
P. incisa
J. procera
J. procera
Aerial Parts
Aerial Parts
Leaves
Stems
J. procera
J. procera
-c
3.50.2
0.3
0.2
0.3
0.2
4.1
0.4
6.1
0.7
0.1
13.4
0.2
55.7
3.8
0.1
0.1
1
0.2
2.3
0.2
33.9
1.6
0.6
4.6
3.7
-
0.4
0.3
62.5
0.5
0.7
0.3
0.1
3.4
3.3
-
0.1
0.1
31.4
1.2
0.3
0.2
3.6
4.1
-
31.3
1.1
0.5
3.3
4.2
-
-
30.3
7.3
26.8
25.8
0.5
0.2
0.2
1.6
1.0
33.0
2.6
0.4
0.3
10.3
8.0
0.9
0.7
0.3
0.2
1.3
1.8
8.9
0.3
0.4
5.7
1.1
0.2
-
0.5
2.8
3.8
0.3
0.5
0.5
2.0
0.4
2.9
3.2
0.4
2.3
1.6
0.7
0.4
0.6
1.1
0.2
0.4
0.1
0.3
0.2
1.2
0.5
0.3
0.2
0.7
1.6
1.5
0.2
2.1
0.1
5.8
0.1
0.3
0.7
0.2
5.9
6.7
0.2
2.4
0.3
4.9
0.2
0.2
0.1
0.2
6.4
6.9
Unripe Fruits Ripe Fruits
Molecules 2021, 26, 959
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Table 1. Cont.
Constituents.
(E)-β-farnesene
γ-muurolene
germacrene D
thymyl isobutyrate
neryl isobutyrate *
valencene *
viridiflorene
α-muurolene
germacrene A
α-bulnesene
β-bisabolene
trans-γ-cadinene
δ-cadinene
elemol
germacrene B
germacrene-D-4-ol
caryophyllene oxide *
cedrol *
humulene epoxide II
10-epi-γ-eudesmol
γ-eudesmol
T-cadinol
β-eudesmol
α-cadinol
α-eudesmol
Monoterpene-hydrocarbons
Oxygenated-monoterpenes
Sesquiterpene-hydrocarbons
Oxygenated-sesquiterpenes
Phenylpropanoids
Apocarotenes
Non-terpene-derivatives
Total-identified (%)
Extraction yield (% w/w)
a
c
l.r.i.
a
1459
1478
1482
1490
1492
1493
1495
1499
1505
1507
1508
1514
1524
1550
1557
1574
1582
1601
1607
1619
1632
1641
1650
1652
1653
l.r.i.
b
1458
1477
1480
1489
1491
1491
1493
1499
1503
1505
1509
1513
1524
1549
1556
1574
1581
1599
1606
1619
1630
1640
1649
1653
1652
Relative Abundance (%)
L. pubescens
P. incisa
J. procera
J. procera
Aerial Parts
Aerial Parts
Leaves
Stems
9.1
0.1
1.1
-
2.1
0.3
2
0.7
1.9
1.4
1.1
0.3
-
1.6
0.2
3.0
0.5
0.4
0.5
-
0.5
0.4
0.2
0.1
9.1
0.4
0.1
0.6
1.7
0.5
-
6.3
0.2
0.3
1.2
0.1
0.1
0.1
0.1
0.4
0.1
5.8
0.1
0.1
0.2
0.2
0.4
1.3
0.3
0.2
0.3
0.3
0.2
0.3
0.3
0.3
1.2
15.3
70.1
13.1
1.2
99.6
1.09
2.2
64.2
3.8
4.7
8.9
1.0
8.7
93.5
1.14
81.8
1.3
10.3
4.4
97.8
0.51
83.8
6.0
4.7
2.9
97.4
<0.1
76.0
1.1
19.6
2.0
98.7
2.7
74.0
0.5
20.7
4.4
99.6
2.39
Linear retentions index on a HP5-MS capillary column;
not detected.
b
J. procera
J. procera
Unripe Fruits Ripe Fruits
values from the literature [44,45]; * comparison with authentic standards;
2.2. Antimicrobial Activity of the Essential Oils
The EOs of L. pubescens, P. incisa ssp. candolleana, and J. procera leaves were tested
against 13 different microbial strains, using diffusion assay on agar plates at the concentration of 20 µL per well (equivalent to 200 µg); vancomycin, amikacin (30 µg per disc),
and ciprofloxacin were used as positive controls (Table 2). Only the EO of L. pubescens
was significantly active, and, among all tested strains, only towards Enterococcus faecalis
ATCC 51299 (inhibition zone 12 mm), Staphylococcus epidermidis ATCC 12228 (10 mm),
Salmonella typhimurium ATCC 700720 (13 mm), A. baumannii (CRE) ATCC 1605 (15 mm),
and Shigella sonnei ATCC 25931 (11 mm). A. baumannii has been reported by Haseeb et al.
(2016) to be among the most common isolated Gram-negative pathogens, with high resistance rate to tobramycin, and E. faecalis and S. epidermidis are the most frequently reported
Gram-negative pathogens during pilgrimage season [46]. In addition, antibiotic-resistant
S. sonnei infections are commonly reported in mass gathering events [47]. Thus, finding
antimicrobial activity in the EO of L. pubescens that works against these reported resistant
pathogens will open the door to utilizing the essential oil of this plant as an alternative
natural control against the spread of resistant pathogens during mass gatherings.
Molecules 2021, 26, 959
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Table 2. Antimicrobial evaluation of essential oils (EOs) in agar diffusion assay (200 µg/well),
R = Resistant.
Microbial Strains
L. pubescens
Aerial Parts EO
P. incisa
Aerial Parts EO
J. procera
Leaves EO
Enterococcusfaecalis ATCC * 51299
12 mm
R
R
Enterococcusfaecalis(VRE) ATCC 51299
R
R
R
Staphylococcus aureus ATCC 25923
R
R
R
Staphylococcus aureus (MRSA) ATCC 43300
R
R
R
Staphylococcus epidermidis ATCC 12228
10 mm
R
R
Salmonella typhimurium ATCC 700720
13 mm
R
R
Klebsiella pneumonia (ESBL) ATCC 14028
R
R
R
Klebsiella pneumonia (CRE) ATCC 1705
R
R
R
Acinetobacter baumannii (CRE) ATCC 19605
15 mm
R
R
Shigella sonnei ATCC 25931
11 mm
R
R
Pseudomonas aeruginosa ATCC 15442
R
R
R
Proteus mirabilis ATCC 3071
R
R
R
Escherichia coli ATCC 35218
R
R
R
* ATCC: American Type Culture Collection; VRE: Vancomycin-resistant Enterococci; MRSA: Methicillin-resistant
Staphylococcus aureus; ESBL: Extended spectrum beta-lactamases; CRE: Carbapenem-resistant Enterobacteriaceae.
2.3. Antimicrobial Activity of L. Pubescens and Carvacrol
The EO of L. pubescens was tested at two concentrations (200 µg and 300 µg per
well) to obtain better information about its efficacy in comparison with its most abundant
constituent (carvacrol). The EO exerted better inhibitory results against A. baumannii (CRE)
ATCC 1605, compared to those obtained with carvacrol. This increased efficacy could
be a synergistic effect of other constituents of L. pubescens EO. When testing carvacrol,
inhibition zones were more pronounced than those of the crude oil for S. epidermidis ATCC
12228 (15 mm) and S. typhimurium ATCC 700720 (20 mm), A. baumannii (CRE) ATCC 1605
(15 mm), and S. sonnei ATCC 25931 (15 mm). Gram-positive strains were more sensitive
to the antibiotic vancomycin (30µg disc), showing more pronounced inhibition zones for
E. faecalis (18.5 mm) and S. epidermidis (28 mm).
In the case of Gram-negative strains, the antibiotic amikacin (30µg disc) showed inhibition activity against S. typhimurium (20 mm) and A. baumannii (CRE) (15 mm), while
S. sonnei was observed to be resistant. All strains were not affected by dimethyl sulfoxide
(DMSO), which was used as a negative control (Table 3). The antimicrobial activity of
carvacrol has been reported similarly by others against E. faecalis [48], S. typhimurium [49],
and A. baumannii indicating its potential effects in infectious disease control; antibacterial
and antibiofilm activity against Salmonella enterica serotype; antimicrobial activity of essential oils-derived volatile compounds against several nosocomial pathogens including
representative multidrug-resistant A. baumannii clinical isolates [9].
Molecules 2021, 26, 959
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Table 3. Antimicrobial evaluation of carvacrol in dimethyl sulfoxide (DMSO) 10% w/v and L. pubescens EO in DMSO 10%
v/v in agar diffusion assay. R = Resistant. Data expressed as (mean ± SD) of two replicates.
Diameter of Zone of Inhibition (mm)
Tested ATCC Strains
Carvacrol
300 µg/well
L. pubescens
EO
200 µg/well
Enterococcus faecalis
ATCC 51299
12 ± 0.00
12 ± 0.00
14 ± 0.00
-
-
18.5 ± 0.71
R
Staphylococcus epidermidis
ATCC 12228
15 ± 0.00
10 ± 0.00
15 ± 0.00
-
-
28 ± 0.00
R
Salmonella typhimurium
ATCC 700720
20 ± 0.00
13 ± 0.00
19 ± 0.00
29 ± 0.00
20 ± 0.00
-
R
Acinetobacter baumannii (CRE)
ATCC 1605
15 ± 0.00
15 ± 0.00
24 ± 0.00
9 ± 0.00
15 ± 0.00
-
R
Shigella sonnei
ATCC 25931
15 ± 0.00
11 ± 0.00
16 ± 0.00
30 ± 0.00
R
-
R
L. pubescens
EO
300 µg/well
Ciprofloxacin Amikacin
30 µg/well
30 µg (Disc)
Vancomycin
30 µg (Disc) DMSO
As shown in Table 4, Gram-negative strains exhibited a higher susceptibility to both
EO and carvacrol than the Gram-positive ones (Table 4).
Table 4. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) expressed in µg/mL of carvacrol, L. pubescens EO, ciprofloxacin and vancomycin hydrochloride; data expressed as (mean ± SD)
of two replicates.
Tested ATCC Strains
Carvacrol
L. pubescens EO
Vancomycin
Ciprofloxacin
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
Enterococcus faecalis
ATCC 51299
500 ± 0.00
1000 ± 0.00
312 ± 0.00
625 ± 0.00
-
-
0.06 ± 0.00
0.12 ± 0.00
Staphylococcus epidermidis
ATCC 12228
500 ± 0.00
1000 ± 0.00
312 ± 0.00
625 ± 0.00
-
-
0.06 ± 0.00
0.12 ± 0.00
Salmonella typhimurium
ATCC 700720
250 ± 0.00
500 ± 0.00
78 ± 0.00
156 ± 0.00
0.70 ± 0.17
1.4 ± 0.65
-
-
Acinetobacter baumannii
ATCC 1605
250 ± 0.00
500 ± 0.00
78 ± 0.00
156 ± 0.00
15 ± 0.00
30 ± 0.00
-
-
Shigella sonnei
ATCC 25931
250 ± 0.00
500 ± 0.00
78 ± 0.00
156 ± 0.00
0.70 ± 0.17
1.4 ± 0.65
-
-
As shown in Tables 3 and 4, both carvacrol and L. pubescens EO could be promising
candidates for the development of formulas to be used mainly for the treatment of intestinal
diseases caused by S. typhimurium and S. sonnei. The essential oil contact with these
pathogens results in microorganism deactivation and a formulation of carvacrol with
tetracycline hydrochloride was previous successfully used for the treatment of local mouth
bacterial infections and candidiasis [8]. Further horizons could be established by the
combinations of carvacrol or L. pubescens EO with classic antibiotics for the treatment of
enteric pathogens.
3. Materials and Methods
3.1. Plant Material
Flowering aerial parts of Lavandula pubescens and different organs (leaves, stems,
ripe and unripe fruits) of Juniperus procera were collected at Wadi Thee Ghazal, Near Taif,
Makkah Province (GPS coordinates 21◦ 05′ 56.1” N 40◦ 20’33.1” E), in June. Flowering aerial
parts of Pulicaria incisa ssp. candolleana were collected at Jabal Al-Lawz, Tabuk province
(GPS Coordinates 28◦ 51′ 18.1” N, 35◦ 23′ 22.6” E), in November. Plants were photographed
(Figure 1) and voucher specimens were deposited in the herbarium of the pharmacognosy
lab, Umm Al-Qura University (L. pubescens, LP-EOM/SA-IT; J. procera JP-EOM/SA-IT;
P. incisa ssp. candolleana PIC-EOM/SA-IT).
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3.2. Chemicals and Reagents
Solvents (n-hexane HPLC grade, dimethyl sulfoxide (DMSO) analytical grade and
carvacrol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Mueller Hinton Agar
was purchased from HiMedia Laboratories Pvt, Ltd. (Mumbai, India). Ciprofloxacin was
purchased from Acros (New Jersey, USA), vancomycin and amikacin paper discs were
purchased from Bioanalyse (Ankara, Turkey), vancomycin hydrochloride was kindly gifted
by Hikma (Amman, Jordan).
3.3. Essential Oil Extraction
The air-dried plant material was finely crushed and subjected to EO hydrodistillation
in a Clevenger-type apparatus for 2 h. Aliquots of the obtained EOs were diluted to 10% in
HPLC grade n-hexane prior to GC-MS injection, while the remaining parts were stored in
freezer at −18 ◦ C until antimicrobial testing.
3.4. Gas Chromatography-Mass Spectrometry Analyses and Peak Identification
Gas chromatography-electron impact mass spectrometry (GC-EIMS) analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara,
CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc., Santa Clara,
CA, USA) capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent
5977B single quadrupole mass detector (Agilent Technologies Inc., Santa Clara, CA, USA).
The oven temperature program was set to rise from 60 ◦ C to 240 ◦ C at 3 ◦ C/min. Temperatures were set as follows: injector temperature, 220 ◦ C; transfer-line temperature,
240 ◦ C. The carrier gas was He, at 1 mL/min flow. The acquisition was performed with
the following parameters: full scan, with a scan range of 35–300 m/z; scan time: 1.0 s;
threshold: 1 count. The identification of the constituents was based on the comparison of
their retention times (tR ) with those of pure reference samples and of their linear retention
indices (LRIs), which were determined relatively to the tR of a series of n-alkanes (C9–C25).
The detected mass spectra were compared with those listed in the commercial libraries
NIST 14 and ADAMS, as well as in a homemade mass-spectral library, built up from pure
substances and components of EOs of known composition and MS literature data [44,50].
3.5. Diffusion Assay on Agar Plates
As recommended by the National Committee for Clinical Laboratory Standards
(NCCLS manual), the antimicrobial activity of EOs of the investigated plants was assayed by the diffusion method. The tested bacterial strains were: E. faecalis—ATCC
51299, E. faecalis (Vancomycin-resistant Enterococci, VRE), S. epidermidis—ATCC 12228,
S. aureus —ATCC 43300, S. aureus (Methicillin-resistant Staphylococcus aureus, MRSA)
43300, S. typhimurium—ATCC 14028, A. baumannii, (Carbapenem-resistant Enterobacteriaceae, CRE)—ATCC 19605, S. sonnei–25931, K. pneumonia (Extended spectrum betalactamases, ESBL)—ATCC 700603, K. pneumonia (CRE)—ATCC 1705, P. aeruginosa,
Proteus mirabilis—ATCC 43071, and E. coli—ATCC 35218. Above acronyms are as follows:
ATTC: American Type Culture Collection; VRE: Vancomycin-resistant Enterococci; MRSA:
Methicillin-resistant Staphylococcus aureus; ESBL: Extended spectrum beta-lactamases;
CRE: Carbapenem-resistant Enterobacteriaceae. Each bacterial strain was suspended in
Mueller–Hinton Broth and adjusted to 0.5 McFarland scale turbidity. The surface of Muller–
Hinton agar plates was swabbed in three directions with standard inoculum, using sterile
cotton swabs. The plates were allowed to dry for 10 min before 3-mm wells were cut
into the Muller–Hinton agar using sterile plastic pipettes. Then, the wells were filled
with 20 µL and 30 µL of EO dissolved in DMSO at the rate of 1/10 v/v, since the EO of
Lavandula pubescens was the only active one, the test was repeated by using the pure major
constituent carvacrol at the concentration of 10 mg/mL in dimethyl sulfoxide (20 mg
of carvacrol was dissolved in 2 mL of DMSO), 30 µL per well (corresponding to 300 µg
per well), with vancomycin (30 µg disc) as a positive control for Gram-positive bacterial
strains while amikacin (30 µg disc) and ciprofloxacin dissolved in DMSO with the rate
Molecules 2021, 26, 959
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1000 µg/1000 µL (30 µg per well) were used as a positive control for Gram-negative bacterial strains; furthermore, dimethyl sulfoxide was used as a negative control. All plates
were incubated at 37 ◦ C for 24 h under aerobic conditions. After the incubation period, the
plates were examined, and the diameter of each inhibition zone was measured.
3.6. Microdilution of Broth Assay
The microdilution method, using 96-well microtiter plates according to the Clinical
and Laboratory Standards Institute (CLSI) guidelines [16], was conducted to evaluate
the antibacterial activity. Performance standards for antimicrobial susceptibility testing
were based on the 18th informational supplement of CLSI document Wayne (PA Clinical
Laboratories Standards Institute, pp. 46–52) [9].
3.7. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC)
MICs and MBCs of L. pubescens EO, carvacrol, ciprofloxacin, and vancomycin hydrochloride were determined with the broth micro-dilution method, with sterile 96-well
microtiter plates for the determination of the MIC and MBCs of the tested samples. All
samples were dissolved in DMSO, with exception of vancomycin hydrochloride which
was dissolved in water. Basically, the first column in microtiter plates contained 200 µL of
EO, carvacrol, or antibiotics and the other subsequent wells contained 100 µL of Mueller–
Hinton broth. EO, carvacrol and antibiotic were serially diluted by transferring 100 µL
to the next well to produce serial dilutions. Mueller–Hinton broth (100 µL containing
the bacteria 0.5 McFarland) was added to each well containing 100 µL of the tested EO,
carvacrol or antibiotics. Sterilized Mueller–Hinton broth alone was used as the negative
control, and bacterial broth with dimethyl sulfoxide (DMSO) was only used as control.
The microdilution plates were incubated at 37 ◦ C overnight. The MIC was determined by
selecting the lowest concentration of sample that completely inhibited the growth of the
organism and compared with the growth control (Table 4). Wells with no visible growth
in MIC were sub-cultured using 10 µL of the selected wells and placed on Muller–Hinton
agar plates. The MBC was determined by taking 10 µL of the selected column and placing
it on the Mueller–Hinton agar plates as well. All plates were incubated for 24 h at 37 ◦ C
and the colony forming units (CFUs) were counted. MIC was determined by selecting
the lowest concentration of the tested sample that completely inhibited the visible growth
of a microorganism after overnight incubation in the well. The MBC was defined as the
lowest concentration of the sample that prevents any growth of an organism after being
sub-cultured on the Mueller–Hinton agar plate [9].
4. Conclusions
This study proposed the use of aromatic wild-growing species of Saudi Arabia as
potential, natural sources of bioactive antimicrobial agents. As available and exploitable
biomass, local wild-growing species can represent a promising source of new bioactive natural compounds, especially in the light of alternatives to traditional medicinal compounds,
towards which antibiotic-resistance is a scary but growing phenomenon. Moreover, their
availability and the facility of the proposed extraction method (hydrodistillation) constitute
a remarkable cost reduction compared to existing antimicrobial agents.
L. pubescens and J. procera are abundantly distributed in the high region of Asir and
Hijaz mountains, so they could be a source for mass production of essential oils, especially
after the successful plantation in that region, while P. incisa subsp. candolleana belongs to
an important genus, rich in essential oil with potential antimicrobial properties [51,52].
Our results encourage us to continue investigation into the possible mechanism of
action of carvacrol, especially against A. baumannii, which is causing an increasing number
of deaths in vulnerable patients. In addition, the EO of L. pubescens can be further studied
for its use as an alternative natural approach to lowering the spread of infectious diseases
during large gatherings and pilgrimages, such as Hajj. Furthermore, it could be of interest
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to verify a possible synergistic effect between the pure constituents of EOs and a number
of antibiotics.
Author Contributions: Conceptualization, A.B.; methodology, A.B., G.F. and H.E.-S.; software, R.A.,
H.E.-S. and S.S.A.; validation, A.B. and G.F.; formal analysis, R.A., H.E.-S. and S.S.A.; investigation,
R.A. and A.A.; resources, M.H; data curation, R.A.; writing—original draft preparation, A.B., A.A.
and M.H.; writing—review and editing, G.F., R.A. and A.A.; supervision, A.B.; project administration, A.B.; funding acquisition, A.B. All authors have read and agreed to the published version of
the manuscript.
Funding: The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura
University for supporting this work by Grant Code: (20UQU0055DSR).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.
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