molecules

Article
Chemical Composition of Herbal Macerates
and Corresponding Commercial Essential Oils
and Their Effect on Bacteria Escherichia coli
Marietta Białoń 1, *, Teresa Krzyśko-Łupicka 2 , Agnieszka Pik 1 and Piotr P. Wieczorek 1
1 Faculty of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland; agnieszka.pik@op.pl (A.P.);
pwiecz@uni.opole.pl (P.P.W.)
2 Independent Department of Biotechnology and Molecular Biology, Faculty of Natural and Technical Science,
University of Opole, Kominka 6A, 45-035 Opole, Poland; teresak@uni.opole.pl
* Correspondence: Marietta.Bialon@uni.opole.pl

Received: 28 September 2017; Accepted: 29 October 2017; Published: 10 November 2017

Abstract: This study addresses the chemical composition of some commercial essential oils (clove,
juniper, oregano, and marjoram oils), as well as appropriate herbal extracts obtained in the process
of cold maceration and their biological activity against selected Escherichia coli strains: E. coli
ATTC 25922, E. coli ATTC 10536, and E. coli 127 isolated from poultry waste. On the basis of
the gas chromatography-mass spectrometry (GCMS) analysis, it was found that the commercial
essential oils revealed considerable differences in terms of the composition and diversity of
terpenes, terpenoids and sesquiterpenes as compared with the extracts obtained from plant material.
The commercial clove, oregano, and marjoram oils showed antibacterial properties against all
the tested strains of E. coli. However, these strains were not sensitive to essential oils obtained
from the plant material in the process of maceration. The tested strains of E. coli show a high
sensitivity, mainly against monoterpenes (α-pinene, β-pinene, α,β,γ-terpinene, limonene) and some
terpenoids (thymol, carvacrol). The commercial juniper oil contained mainly monoterpenes and
monoterpenoids, while the extracts contained lower amounts of monoterpenes and high amounts of
sesquiterpenes—the anti-microbiotic properties of the juniper herbal extract seem to be caused by the
synergistic activity of mono- and sesquiterpenes.

Keywords: essential oils; gas chromatography-mass spectrometry; Escherichia coli

1. Introduction
Oils or oily plants are used as spices, therapeutic agents, in herbal medicine and aromatherapy
as well as flavouring components of perfume or toilet water in the cosmetics industry. Due to their
disinfectant properties, some oils are used in the food industry and restaurant to disinfect potable
water and preserve food, as well as in the cultivation of plants and tending pets [1].
In recent years, due to the high survivability of microorganisms in the environment caused by
resistance to antibiotics and preservative agents, the natural anti-microbiological preparations, such as
extracts and essential oils, have become the centre of attention [2].
Depending on the chemical composition, essential oils and extracts can be divided into terpene
and non-terpene oils. Terpene oils contain mainly terpenes, most frequently mono-, sesqui-, and less
frequently, di-terpenes, and the non-terpene oils contain phenylpropane derivatives. Compounds
found in both of these oil groups are present, for example, in the form of hydrocarbons, alcohols,
ketones, aldehydes, phenols, esters, and acids. Some oils may also contain compounds containing
sulphur, nitrogen, and coumarins [3].

Molecules 2017, 22, 1887; doi:10.3390/molecules22111887 www.mdpi.com/journal/molecules

Molecules 2017, 22, 1887 2 of 16

The efficacy of antimicrobial action of oils and extracts depends to a great degree on the chemical
composition, which is connected with the type of oily plants, the growth conditions, harvesting,
and processing, as well as the manner of obtaining the extracts. Differences in the chemical composition
and biological activity were observed in garlic oils containing sulphur compounds [4–7], nettle [8–10],
and chamomile [11], which contain biogenic amines and angelica-containing coumarins [12–14].
Furthermore, what is also significant is the qualitative-quantitative composition of various chemical
compounds found in essential oils, since it is responsible for their antimicrobial efficacy.
Clove oil is an extremely important substance which inhibits the growth of the bacteria
(e.g., Escherichia coli, Bacillus subtilis), probably thanks to the presence of eugenol, eugenol acetate,
and caryophyllene [15,16], and so is oregano oil, perhaps due to the presence of carvacrol [17,18].
On the other hand, α- and β-pinene present in juniper and marjoram oil and, additionally, limonene in
marjoram oil may be responsible for the anti-viral, anti-fungal, and antibacterial properties [19–22].
The aim of the paper is to compare the chemical composition of chosen commercial oils with
extracts obtained from plant raw material (spices) in the process of cold maceration and hot extraction
in the Soxhlet apparatus, as well as to determine their biological activity against the E. coli strains.

2. Results
Regardless of the applied procedure, the extraction efficiency of the tested plant raw material was
similar (Table 1).

Table 1. The extraction efficacy of the tested plant raw material.

Extraction Efficacy (%)

Oil
Cold Extraction—Maceration Hot Extraction—Soxhlet Apparatus
Clove 20.3 20.6
Juniper 13.1 12.4
Oregano 4.0 3.3
Marjoram 12.2 12.6

The tested oils revealed a diversified qualitative-quantitative composition. In clove oil, a different
number of chemical compounds was identified: the most in the commercial oil (20 components),
and the least in cold and hot extracts, 14 and 13 components, respectively (Table 2). Monoterpenes
constituted the main components of these oils and in the commercial oil and after maceration they
amounted to 78% and, after hot extraction, 82% (Table 2). Eugenol was the most important part of this
group of compounds, which made up 62% of the whole composition while, in the remaining extracts,
it comprised approximately 55%. However, eugenol acetate was present in considerable amounts
(23.5% and 27.5%) only in extracts (Figure 1). The sesquiterpene-type compounds constituted around
20% of the commercial oil and maceration-obtained compositions, while 16% of the composition
of the extract obtained as the result of extraction in the Soxhlet apparatus. In this group of
compounds, β-caryophyllene was observed in the highest amounts (16% and 13%). On the other hand,
monoterpenes, which were mostly represented by eucalyptol, were present only in the commercial oil
(Table 2).
In the commercial juniper oil, there were 38 identified components, and in the extracted oils, 23 in
each of the extracts (Table 3). Monoterpenes constituted 88% of the commercial juniper oil composition
and in the extracts their amount was two times lower and equalled 45%, on average. The second
group of compounds in extracts, regardless of the manner of obtaining them, was sesquiterpenes in
the amount of 21%, on average (Table 3). α-Pinene was the main component to be found in the greatest
amounts in all the tested juniper oils (approximately 22%) (Figure 2). However, these oils differed in
terms of the quantitative-qualitative composition of the remaining components. Terpinolene, 3-carene,
β-phellandrene of the monoterpene group were identified only in the commercial oil, and the extracts
contained β-thujene and sesquiterpenes in considerable amounts (β-caryophyllene, and α-humulene).
Molecules 2017, 22, 1887 3 of 16

Table 2. Comparison of the composition of particular compounds in clove oils.

Area (%) ± Standard Deviation (SD)

Compound
Commercial Maceration Soxhlet Apparatus
Monocyclic monoterpenes
β-Phellandrene 0.07 ± 0.01 - -
α-Phellandrene 0.08 ± 0.00 - -
α-Terpinene 0.02 ± 0.01 - -
o-Cymene 0.66 ± 0.02 - -
γ-Terpinene 0.36 ± 0.04 - -
Terpinolene 0.03 ± 0.01 - -
Bi and tricyclic monoterpenes
α-Pinene 0.27 ± 0.01 - -
Camphene 0.05 ± 0.01 - -
β-Pinene 0.05 ± 0.01 - -
Sum 1.56 - -
Monocyclic monoterpenoids
Menthol 0.02 ± 0.01 - -
Eugenol 61.94 ± 0.23 54.16 ± 0.61 54.69 ± 0.21
Eugenyl acetate 4.13 ± 0.13 23.49 ± 0.61 27.65 ± 0.12
Bi and tricyclic monoterpenoids
1,8-cineole 12.07 ± 0.46 - 0.05 ± 0.007
Bornyl acetate 0.08 ± 0.02 - -
Sum 78.24 77.65 82.39
Aliphatic sesquiterpenes
α -Farnesene - 0.16 ± 0.01 -
Monocyclic sesquiterpenes
α-Humulene 3.03 ± 0.03 1.85 ± 0.04 1.41 ± 0.02
α-Elemene - 0.23 ± 0.02 0.14 ± 0.01
Bi and tricyclic sesquiterpenes
α-Cubebene 0.10 ± 0.01 0.71 ± 0.04 0.56 ± 0.03
β-Cubebene - 0.34 ± 0.02 0.24 ± 0.02
β-Caryophyllene 15.86 ± 0.19 15.76 ± 0.38 13.11 ± 0.33
α-Copaene - 2.00 ± 0.31 1.24 ± 0.22
β-Copaene - -
γ-Muurolene - 0.08 ± 0.01 -
Sum 18.99 21.13 16.70
Bi and tricyclic sesquiterpenoids
Cubebol - 0.13 ± 0.02 -
Caryophyllene oxide 0.87 ± 0.02 0.40 ± 0.03 0.45 ± 0.04
Sum 0.87 0.53 0.45
Esters
Methyl salicylate 0.14 ± 0.03 - 0.03 ± 0.01
Ketones
2,3,4-Trimethoxyacetophenone - 0.49 ± 0.15 0.18 ± 0.01
Phenols
Chavikol 0.17 ± 0.01 0.18 ± 0.04 0.21 ± 0.03
Sum 0.31 0.67 0.42
Molecules 2017, 22, 1887 4 of 16
Molecules 2017, 22, 1887 4 of 16

Clove oil
70

Area of the main components (%)

60
Eugenol
50
β-Caryophyllene
40
1,8-cineole
30 Eugenyl acetate
20 α-Humulene

10 α-Copaene

0
Commercial Cold extraction Hot extraction

Figure 1. The composition of the main components in the clove commercial oil and extracts obtained
Figure 1. The composition
in the process of the
of cold and hot main components in the clove commercial oil and extracts obtained
extraction.
in the process of cold and hot extraction.

Table
Table3.
3.Comparison
Comparisonof
ofthe
thecomposition
composition of
of particular
particular compounds
compounds in
in juniper
juniper oils.
oils.

Area (%) ± SD
Compound Area (%) ± SD
Compound Commercial Maceration Soxhlet Apparatus
Commercial Maceration Soxhlet Apparatus
Monocyclic monoterpenes
Monocyclic monoterpenes
β-Phellandrene 12.71 ± 0.23 - -
β-Phellandrene
α-Phellandrene 12.71 ± ±0.23
0.48 0.06 - - - -
α-Phellandrene 0.48 ± 0.06 - -
o-Cymene 2.46 ± 0.18 - -
o-Cymene 2.46 ± 0.18 - -
γ-Terpinene
γ-Terpinene 3.153.15 ± 0.25
± 0.25 - - - -
Terpinolene
Terpinolene 15.39 ± 0.12
15.39 ± 0.12 - - - -
p-Cymenep-Cymene - - 0.18 ± 0.01
0.18 ± 0.01 - -
LimoneneLimonene - - 3.98 3.98 ± 0.12
± 0.12 2.75
2.75 ± 0.01 ± 0.01
Bi and tricyclic
Bi and tricyclic monoterpenes
monoterpenes
α-Pineneα-Pinene 21.79 ± 0.49
21.79 ± 0.49 27.1827.18 ± 0.82
± 0.82 26.51 ± 0.09
26.51 ± 0.09
CampheneCamphene 4.49 ± 0.18
4.49 ± 0.18 - - - -
β-Pinene 4.35 ± 0.19 2.49 ± 0.10 2.32 ± 0.12
β-Pinene 4.35 ± 0.19 2.49 ± 0.10 2.32 ± 0.12
α-Thujene 1.42 ± 0.09
α-Thujene
β-Thujene 1.42 ± 0.09
2.00 ± 0.14 11.01 ± 0.30 7.37 ± 0.05
4-Careneβ-Thujene 2.00 ±
0.28 ± 0.020.14 11.01 ± 0.30
- 7.37 ± 0.05 -
3-Carene4-Carene 0.28
14.23 ± ±0.19
0.02 - - - -
2-Carene3-Carene 2.41 ± 0.18
14.23 ± 0.19 - - - -
Sabinene2-Carene 2.78 ± 0.19
2.41 ± 0.18 3.59
- ± 0.13 - 2.61 ± 0.07
Sum 87.94 48.43 41.56
Sabinene 2.78 ± 0.19 3.59 ± 0.13 2.61 ± 0.07
Monocyclic
Summonoterpenoids 87.94 48.43 41.56
Terpinen-4-ol 2.34 ± 0.07
Monocyclic monoterpenoids 0.49 ± 0.01 0.54 ± 0.01
α-Terpineol 0.45 ± 0.03 - -
Terpinen-4-ol 2.34 ± 0.07 0.49 ± 0.01 0.54 ± 0.01
Thymol methyl ether 0.28 ± 0.08 - -
α-Terpineol
α-Terpineol acetate 0.520.45 ± 0.03
± 0.02 - - - -
Thymol methyl ether 0.28 ± 0.08 - -
Bi and tricyclic monoterpenoids
α-Terpineol acetate
Bornyl acetete 0.52
4.04 ± 0.02
± 0.04 - ± 0.02
0.33 - 0.38 ± 0.01
Borneol Bi and tricyclic monoterpenoids
0.28 ± 0.03 - -
ThujoneBornyl acetete 4.04
0.11 ± 0.04
± 0.02 0.33 ± 0.02- 0.38 ± 0.01 -
Fenchyl Borneol
acetate 0.13 ± 0.02
0.28 ± 0.03 - - - -
Sum Thujone 8.15± 0.02
0.11 - 0.82 - 0.92
Fenchyl acetate 0.13 ± 0.02 - -
Sum 8.15 0.82 0.92
Monocyclic sesquiterpenes
Molecules 2017, 22, 1887 5 of 16

Table 3. Cont.

Area (%) ± SD
Compound
Molecules 2017, 22, 1887 Commercial Maceration Soxhlet Apparatus
5 of 16

Monocyclic sesquiterpenes
α-Humulene 0.63 ± 0.03 4.13 ± 0.14 4.76 ± 0.05
α-Humulene 0.63 ± 0.03 4.13 ± 0.14 4.76 ± 0.05
α-Elemene 0.08 ± 0.01 - -
α-Elemene 0.08 ± 0.01 - -
β-Elemene β-Elemene - - 0.65 ±0.65
0.03± 0.03 0.80 ± 0.06 0.80 ± 0.06
Elixene Elixene 0.06 ± 0.02
0.06 ± 0.02 - - - -
Germacrene DGermacrene D 0.09 ± 0.01
0.09 ± 0.01 3.57 ±3.57
0.54± 0.54 3.85 ± 0.13 3.85 ± 0.13
Bi and tricyclic sesquiterpenes
Bi and tricyclic sesquiterpenes
α-Cubebene - 1.43 ± 0.04 1.54 ± 0.05
α-Cubebene - 1.43 ± 0.04 1.54 ± 0.05
β-Caryophyllene 1.06 ± 0.01 5.06 ± 0.17 5.24 ± 0.02
β-Caryophyllene 1.06 ± 0.01 5.06 ± 0.17 5.24 ± 0.02
α-Copaene 0.05 ± 0.01 1.00 ± 0.05 1.23 ± 0.01
α-Copaene 0.05 ± 0.01 1.00 ± 0.05 1.23 ± 0.01
α-Muurolene 0.10 ± 0.01 0.61 ± 0.06 0.59 ± 0.01
α-Muurolene 0.10 ± 0.01 0.61 ± 0.06 0.59 ± 0.01
γ-Muurolene γ-Muurolene 0.090.09 ± 0.01
± 0.01 0.45 ±0.45
0.05± 0.05 0.49 ± 0.01 0.49 ± 0.01
Alloaromadendrene
Alloaromadendrene - - 0.46 ±0.46
0.02± 0.02 0.44 ± 0.01 0.44 ± 0.01
α-Cadinene α-Cadinene 0.16 ± 0.01
0.16 ± 0.01 - - - -
δ-Cadinene δ-Cadinene 0.57 ±
0.57 ± 0.01 0.01 1.38 ±1.38
0.03± 0.03 1.80 ± 0.03 1.80 ± 0.03
α-Cedrene α-Cedrene 0.080.08 ± 0.01
± 0.01 - - - -
EpizonareneEpizonarene - - 0.61 ±0.61
0.01± 0.01 0.96 ± 0.05 0.96 ± 0.05
β-Maaliene β-Maaliene - - 0.64 ±0.64
0.04± 0.04 - -
α-Selinene α-Selinene - - - - 0.73 ± 0.03 0.73 ± 0.03
LongifoleneLongifolene ± 0.01
0.170.17 ± 0.01 - - - -
Sum Sum 3.143.14 19.99 19.99 22.43 22.43
Bi and
Bi and tricyclic tricyclic sesquiterpenoids
sesquiterpenoids
Cubebol Cubebol - - 1.83 ±1.83
0.16± 0.16 1.83 ± 0.05 1.83 ± 0.05
Caryophyllene
Caryophyllene oxide oxide 0.19 ± 0.02
0.19 ± 0.02 3.82 ±3.82
0.87± 0.87 4.57 ± 0.08 4.57 ± 0.08
SpathulenolSpathulenol - - - - 1.40 ± 0.10 1.40 ± 0.10
τ-Cadinol τ-Cadinol 0.060.06 ± 0.01
± 0.01 - - - -
Sum Sum 0.250.25 5.65 5.65 7.80 7.80
Ethers
Ethers
Estragole Estragole 0.160.16 ± 0.02
± 0.02 - - - -
Hydrocarbon derivatives
Hydrocarbon derivatives
Cuparene 0.36 ± 0.01 - -
Cuparene Unseparated 0.36 ± 0.01
- 25.11 ± 0.28
- 27.29 ± 0.21
-
Unseparated - 25.11 ± 0.28 27.29 ± 0.21
Sum 0.52 25.11 27.29
Sum 0.52 25.11 27.29

Juniper oil
30
α-Pinene
Area of the main components (%)

25 Terpinolene

3-Carene
20
β-Phellandrene
15 Camphene

α-Thujene
10
β-Caryophyllene
5 α-Humulene

Limonene
0
Commercial Cold Hot extraction Caryophyllene oxide
extraction

Figure 2. The composition of the main components in the juniper commercial oil and extracts obtained
Figure 2. The composition of the main components in the juniper commercial oil and extracts
in the process of cold and hot extraction.
obtained in the process of cold and hot extraction.
Molecules 2017, 22, 1887 6 of 16

Eighteen compounds were identified in the commercial oregano oil, and more in extracts (21 and
23 components) (Table 4). The commercial oregano oil contained monoterpenes in comparable
amounts (51%) and their oxygen derivatives (49%) (Table 4). Compositions of oils obtained by
the extraction methods were differentiated and different from the composition of the commercial
oregano oil—monoterpenes and their derivatives constituted 36% each, and sesquiterpenes and
sesquiterpenoids constituted 40–50% (Table 4). p-Thymol and thymol of the monoterpenoid group
constituted the main components of the commercial oil (total of 43.9%) (Figure 3). Their presence in
extracts obtained in the cold and hot manner was significantly lower and equalled 2.5% and 3.6%,
respectively (Table 4). However, the main elements of extracts included the following: caryophyllene,
triacontyl acetate, and β-phellandrene (Figure 3), which were not identified in the commercial oil.
On the other hand, carvacrol, a compound revealing biocidal properties, was identified only in the
cold extract, in the amount of 1.8% (Table 4), although it is enumerated as one of the main components
of commercial oils. The lack of this compound can be caused by genetic changes in the plants, as well
as the environmental conditions in oregano plantations.

Table 4. Comparison of the composition of particular compounds in oregano oils.

Area (%) ± SD
Compound
Commercial Maceration Soxhlet Apparatus
Aliphatic monoterpenes
β-Ocimene - 4.01 ± 0.25 4.03 ± 0.08
α-Ocimene - 0.94 ± 0.11 1.14 ± 0.07
Monocyclic monoterpenes
β-Phellandrene - 13.69 ± 0.52 13.08 ± 0.39
α-Phellandrene 0.13 ± 0.01 - -
o-Cymene 23.74 ± 0.30 5.98 ± 0.28 5.45 ± 0.11
γ-Terpinene 0.67 ± 0.02 2.04 ± 0.22 2.49 ± 0.12
Terpinolene 1.62 ± 0.05 - -
Limonene 15.47 ± 0.62 - -
Bi and tricyclic monoterpenes
α-Pinene 5.90 ± 0.04 - -
Camphene 0.80 ± 0.04 - -
β-Pinene 0.09 ± 0.01 - -
2-Carene 1.70 ± 0.08 - -
Bornylene 1.05 ± 0.04 - -
Sum 51.17 26.66 26.19
Aliphatic monoterpenoids
Linalool 1.57 ± 0.01 - -
Monocyclic monoterpenoids
Terpinen-4-ol 0.09 ± 0.01 - -
α-Terpineol 2.28 ± 0.07 - -
β-Terpineol 0.36 ± 0.02 1.34 ± 0.06 1.49 ± 0.08
Thymol 11.17 ± 0.54 2.53 ± 0.15 2.13 ± 0.09
p-Thymol 32.69 ± 0.43 - 1.52 ± 0.04
Thymol methyl ether - 0.63 ± 0.05 0.52 ± 0.02
Carvacrol - 1.80 ± 0.42 -
Isothymol methyl ether - - 0.69 ± 0.01
Bi and tricyclic monoterpenoids
1,8-cineole - - 1.41 ± 0.08
endo-Borneol 0.51 ± 0.02 - -
cis-Sabinene hydrate - 3.07 ± 0.17 2.74 ± 0.08
Sum 48.67 9.37 10.50
Molecules 2017, 22, 1887 7 of 16

Molecules 2017, 22, 1887 Table 4. Cont. 7 of 16

α-Humulene - Area
1.23 (%) ± SD
± 0.07 1.05 ± 0.05
Compound
α-Bisabolene Commercial - 1.69 ± 0.06
Maceration -
Soxhlet Apparatus
Bi andsesquiterpenes
Monocyclic tricyclic sesquiterpenes
β-Cubebene
α-Humulene - - 8.811.23 ± 0.07
± 0.24 0.01± 0.05
7.55 ± 1.05
α-Bisabolene
β-Caryophyllene - ± 0.01
0.16 1.69
10.51 ± 0.06
± 0.16 8.49 ± 0.21 -
Bi and tricyclic sesquiterpenes
β-Copaene - 0.85 ± 0.03 -
β-Cubebene
γ-Muurolene - - 1.368.81 ± 0.24
± 0.07 0.06± 0.01
1.20 ± 7.55
β-Caryophyllene 0.16 ± 0.01 10.51 ± 0.16 8.49 ± 0.21
β-Burbonene
β-Copaene -
- 3.360.85
± 0.10
± 0.03 3.14 ± 0.12 -
Alloaromadendrene
γ-Muurolene - - 1.661.36
± 0.06
± 0.07 1.45 ± 1.20
0.10± 0.06
δ-Cadinene
β-Burbonene - - 1.09 ± 0.10
± 0.07
3.36 0.03± 0.12
0.97 ± 3.14
Alloaromadendrene
β-Gurjunene - - - ± 0.06
1.66 0.81 ± 0.09± 0.10
1.45
δ-Cadinene - 1.09 ± 0.07 0.97 ± 0.03
Sum 0.16 30.56 24.66
β-Gurjunene - - 0.81 ± 0.09
Sum Bi and tricyclic sesquiterpenoids
0.16 30.56 24.66
Caryophyllene oxide
Bi and tricyclic sesquiterpenoids
- 19.41 ± 0.15 16.15 ± 0.06
Sum
Caryophyllene oxide - - 19.41± 0.15
19.41 16.15
16.15 ± 0.06
Sum Esters - 19.41 16.15
Triacontyl
Esters acetate - 14.51 ± 0.52 16.61 ± 0.25
Triacontyl
Acids acetate - 14.51 ± 0.52 16.61 ± 0.25
AcidsLinolenic acid - - 5.89 ± 0.25
Linolenic
Sumacid - - 14.51- 5.89 ± 0.25
22.50
Sum - 14.51 22.50

Oregano oil
35
p-Thymol
Area of the main components (%)

30
o-Cymene
25 Limonene

20 Thymol

α-Pinene
15
Caryophyllene oxide
10 Triacontyl acetate

5 β-Phellandrene

β-Cubebene
0
Commercial Cold Hot extraction β-Caryophyllene
extraction

Figure 3. The composition of the main components in the oregano commercial oil and extracts obtained
Figure 3. The composition of the main components in the oregano commercial oil and extracts
in the process of cold and hot extraction.
obtained in the process of cold and hot extraction.

Asfor
As for marjoram
marjoramoils, oils, the
the greatest
greatest number
numberof of compounds
compoundswas wasidentified
identifiedinin the
the commercial
commercialoiloil
(21components),
(21 components),while whileininthe
theextract
extractthere
therewere
were15 15 components
componentsfor forcold
coldand
and1212components
componentsfor forhot
hot
extraction (Table 5). The chemical analysis of these oils revealed that they are mainly
extraction (Table 5). The chemical analysis of these oils revealed that they are mainly composed of composed of
monoterpenes and monoterpenoids. In the commercial oil their composition
monoterpenes and monoterpenoids. In the commercial oil their composition was similar, and was similar, and equalled
40%. In the
equalled oilsInobtained
40%. the oils by extraction
obtained methods, the
by extraction percentage
methods, of monoterpenes
the percentage was 26–27%,was
of monoterpenes and26–
the
amount of monoterpenoids was two times larger and equalled 55% (Table 5). Limonene
27%, and the amount of monoterpenoids was two times larger and equalled 55% (Table 5). Limonene constituted the
most abundant component of the commercial oil (at least 23%) (Figure 4), and β-terpineol
constituted the most abundant component of the commercial oil (at least 23%) (Figure 4), and β- stereoisomers
terpineol stereoisomers were present in the extracts (approximately 40% of the composition), which
were present in the commercial oil in the amount of 1% (Figure 4).
Molecules 2017, 22, 1887 8 of 16

were present in the extracts (approximately 40% of the composition), which were present in the
commercial oil in the amount of 1% (Figure 4).

Table 5. Comparison of the composition of particular compounds in marjoram oils.

Area (%) ± SD
Compound
Commercial Maceration Soxhlet Apparatus
Monocyclic monoterpenes
β-Phellandrene - 1.26 ± 0.11 -
α-Phellandrene 0.23 ± 0.01 - -
γ-Terpinene 4.49 ± 0.09 1.16 ± 0.20 -
Terpinolene 1.10 ± 0.01 - -
p-Cymene 2.40 ± 0.09 0.42 ± 0.04 -
Limonene 23.47 ± 0.18 - -
Bi and tricyclic monoterpenes
α-Pinene 0.71 ± 0.01 - -
β-Pinene 0.66 ± 0.01 - -
β-Thujene 2.07 ± 0.08 - -
2-Carene 2.67 ± 0.05 - -
Tricyclene - 24.36 ± 0.15 26.37 ± 0.23
Sum 37.80 27.20 26.37
Aliphatic monoterpenoids
Linalool 17.25 ± 0.08 - -
Monocyclic monoterpenoids
Eugenol 1.55 ± 0.03 - -
p-Menth-2-en-1-ol 0.51 ± 0.03 1.06 ± 0.04 0.69 ± 0.01
Terpinen-4-ol 8.93 ± 0.11 7.06 ± 0.09 6.94 ± 0.09
α-Terpineol 9.09 ± 0.11 4.89 ± 0.04 5.63 ± 0.11
Stereoisomers of - 6.08 ± 0.09 4.91 ± 0.04
β-Terpineol 1.11 ± 0.07 35.70 ± 0.17 35.01 ± 0.06
trans-Piperitol - 0.56 ± 0.01 0.44 ± 0.01
Bi and tricyclic monoterpenoids
Camphor 0.33 ± 0.01 - -
Bornyl acetate 0.20 ± 0.01 - -
Sum 38.97 55.35 53.62
Monocyclic sesquiterpenes
Elixene 0.16 ± 0.01 - -
Bi and tricyclic sesquiterpenes
β-Caryophyllene 0.64 ± 0.02 3.89 ± 0.11 3.99 ± 0.05
Bicyclogermacrene - 0.89 ± 0.03 0.63 ± 0.02
Sum 0.80 4.78 4.62
Bi and tricyclic sesquiterpenoids
Spathulenol - 2.19 ± 0.08 2.64 ± 0.08
Sum - 2.19 2.64
Diterpenes
4-epi-Dehydroabietol - 3.31 ± 0.12 4.60 ± 0.08
Sum - 3.31 4.60
Esters
Linalyl anthranilate 16.63 ± 0.05 - -
Ethers
Estragole 5.77 ± 0.04 - -
Hydrocarbons
Eicosane - 7.29 ± 0.15 8.15 ± 0.10
Sum 22.40 7.29 8.15
Molecules2017,
Molecules 22,1887
2017,22, 1887 99of
of16
16

Marjoram oil
45
Limonene
40

Area of the main components (%)

Linalool
35
30 Linalyl anthranilate

25 α-Terpineol

20 Terpinen-4-ol
15
Stereoisomers of β-
10 Terpineol
Tricyclene
5
0 Eicosane
Commercial Cold extraction Hot extraction

Figure 4. The composition of the main components in the marjoram commercial oil and extracts
Figure 4. The composition of the main components in the marjoram commercial oil and extracts
obtained in the process of cold and hot extraction.
obtained in the process of cold and hot extraction.

The biological
The biological activity
activity of
of commercial
commercial oils oils and
and extracts
extractsobtained
obtainedin inthe
the process
process ofof cold
cold and
and hot
hot
maceration against
maceration against three
three strains
strains of E. coli
of E. coli was
was differentiated
differentiated (Table 6, Figures 5–8).
The degree
The degreeofofgrowth
growthinhibition of theoftested
inhibition the bacterial strains depended
tested bacterial both on the both
strains depended concentration
on the
and the chemical
concentration andcomposition
the chemicalrelated to the methods
composition related toofthe
obtaining
methodsoils (Table 6). oils (Table 6).
of obtaining

Table 6.
Table Thezones
6. The zonesof
ofinhibition
inhibition of E. coli
of E. coli strains
strains in
in the
the presence
presence of
of essential
essential oils.
oils.

Zones
ZonesofofInhibition
Inhibition (mm) ± SD
(mm) ± SD
Oil Concentration (%) E. coli ATTC 25922 E. coli ATTC 10536 E. coli 127
Oil Concentration (%) E. coli ATTC 25922 E. coli ATTC 10536 E. coli 127
Commercial Maceration Commercial Maceration Commercial Maceration
Commercial Maceration Commercial Maceration Commercial Maceration
Clove oil
0.25 0 0 Clove oil 0 0 0 0
0.5
0.25 12.5 ±00.7 00 9.5 ±00.7 00 00 00
0.5
1.0 12.5±±
12.5 0.7
0.7 00 9.5 ±±0.0
11.0 0.7 00 00 00
1.0
1.5 12.5±±
14.0 0.7
1.4 00 11.0±±1.4
12.0 0.0 00 9.5 ±0 0.7 00
1.5 14.0 ± 1.4 0 12.0 ± 1.4 0 9.5 ± 0.7 0
2.0
2.0
15.0 ± 0.0
15.0 ± 0.0
00 13.5 ± 0.7
13.5 ± 0.7
00 11.0 ± 0.0
11.0 ± 0.0
00
Juniper oil
Juniper oil
0.25 0 15.0 ± 0.0 0 0 0 0
0.25
0.5 0 0 15.0 ± 0.0
17.5 ± 0.7 00 00 00 00
0.5 0 17.5 ± 0.7 0 0 0 0
1.0 0 16.5 ± 0.7 0 0 0 0
1.0 0 16.5 ± 0.7 0 0 0 0
1.5
1.5 00 14.5
14.5±±0.7
0.7 00 00 00 00
2.0
2.0 00 13.5
13.5±±0.7
0.7 00 00 00 00
Oregano
Oregano oil
oil
0.25 0 0 12.5 ± 0.7 0 0 0
0.25 0 0 12.5 ± 0.7 0 0 0
0.5
0.5 14.0
14.0±± 1.4
1.4 00 20.0
20.0±±0.00.0 00 17.5
17.5 ±±0.7
0.7 00
1.0
1.0 13.5
13.5±± 0.7
0.7 00 19.0
19.0±±1.41.4 00 16.0
16.0 ±±0.0
0.0 00
1.5
1.5 13.0±±
13.0 0.0
0.0 00 16.5±±2.1
16.5 2.1 00 17.0 ±±1.4
17.0 1.4 00
2.0
2.0 14.0±±
14.0 0.0
0.0 00 14.5±±0.7
14.5 0.7 00 18.0 ±±0.0
18.0 0.0 00
Marjoram
Marjoram oiloil
0.25
0.25 00 00 00 00 7.5
7.5 ±±10.6
10.6 00
0.5
0.5 00 00 00 00 12.0 ±±0.0
12.0 0.0 00
1.0
1.0 6.5±±
6.5 9.2
9.2 00 00 00 12.5 ±±0.7
12.5 0.7 00
1.5
1.5 13.0 ±
13.0 ± 0.0 0.0 0 0 13.0 ±
13.0 ± 1.4 1.4 00 12.0 ± 0.0
12.0 ± 0.0 00
2.0 13.5 ± 0.7 0 12.5 ± 0.7 0 13.0 ± 0.0 0
2.0 13.5 ± 0.7 0 12.5 ± 0.7 0 13.0 ± 0.0 0

The commercial clove oil with the concentration of 0.5%–2% inhibited the growth of the stains in
the collection (E. coli ATTC 25922 and E. coli ATTC 10536). The E. coli ATTC 25922 strain proved to be
more sensitive, and the growth inhibition zones were 12.5 to 15 mm. On the other hand, the
Molecules 2017, 22, 1887 10 of 16
Activity of clove oils
environmental isolate25E. coli 127 revealed sensitivity only to higher concentrations of this oil (1.5%–2%).

(mm) of inhibition (mm)

However, no 22,
Molecules 2017, biocidal effect was observed of clove extracts against the tested bacterial strains (Figure
1887 20 10 5).
of 16
15
10
5 Activity of clove oils
000000 000000 0000 0000 000 000
250

Zones of inhibitionZones
control 0.25 0.5 1.0 1.5 2.0
20
Oil concentration (%)
15
10
1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127
5 000000 000000 0000 0000 000 000
20 + E. coli ATTC 25922 2 + E. coli ATTC 10536 2 + E. coli ATTC 127
control 0.25 0.5 1.0 1.5 2.0
Oilstrains
Figure 5. Zones of inhibition of the tested E. coli concentration
against(%)
the clove oils. 1: commercial oil; 2: cold
extract.
1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127

The commercial 2juniper

+ E. coli oil
ATTCdid not reveal
25922 anyATTC
2 + E. coli antibacterial
10536 2 +properties
E. coli ATTCagainst
127 any of the tested
bacterial strains, and the extract obtained in the process of maceration only inhibited the growth of
of5.E.Zones
Figure
the strain of inhibition
coli 25922 at all usedof the tested E. coli strains
concentrations of thisagainst
extractthe clove oils.
(Figure 6). 1: commercial oil; 2:
Figure 5. Zones of inhibition of the tested E. coli strains against the clove oils. 1: commercial oil; 2: cold
cold extract.
extract.

Activity of juniper oils

The commercial juniper oil did not reveal any antibacterial properties against any of the tested
bacterial strains, and25 the extract obtained in the process of maceration only inhibited the growth of
(mm) of inhibition (mm)

the strain of E. coli 25922

20 at all used concentrations of this extract (Figure 6).
15
10
Activity of juniper oils
5
000000 000 00 000 00 000 00 000 00 000 00
250
Zones of inhibitionZones

20 control 0.25 0.5 1.0 1.5 2.0

15 Oil concentration (%)
10
1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127
5
000000 000 00 000 00 000 00 000 00 000 00
20 + E. coli ATTC 25922 2 + E. coli ATTC 10536 2 + E. coli ATTC 127
control 0.25 0.5 1.0 1.5 2.0
Figure
Molecules 6.
2017,6. Zones
22,Zones of inhibition of the tested E. coli strains against the juniper oils. 1: commercial oil;11
1887 of 2:
Figure inhibition of the tested E.
Oilcoli (%) the juniper oils. 1: commercial oil; of 16
strains against
concentration
cold extract.
2: cold extract.
1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127
The commercial 2oregano
+ E. coli ATTC
Activity
oil inhibited
25922 2the
of oregano
+ E.growth
coli ATTCof10536oils
all the2 tested
+ E. colistrains of bacteria E. coli in the
ATTC 127
concentrations ranging 25 from 0.5% to 2%, yet, the maximum zone of inhibition bacteria growth was
Zones of inhibition (mm)

present at concentrations of 0.5% and 1%. The E. coli ATTC 10536 strain proved to be the most
20inhibition
Figure 6. Zones of of the tested E. coli strains against the juniper oils. 1: commercial oil;
sensitive to the oil activity. However, no biocidal activity was observed of clove macerate against the
2: cold extract. 15
tested bacterial strains
10 (Figure 7).
The commercial oregano
5 0 0 0 0oil inhibited the growth
00 0 0000 000
of all0 the
00
tested0 0strains
0
of0 0bacteria
0
E. coli in the
concentrations ranging 0 from 0.5% to 2%, yet, the maximum zone of inhibition bacteria growth was
present at concentrations control
of 0.5% and0.251%. The0.5E. coli ATTC
1.0 1.5 strain 2.0
10536 proved to be the most
sensitive to the oil activity. However, no biocidal activity was
Oil concentration (%)observed of clove macerate against the
tested bacterial strains (Figure 7).
1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127

2 + E. coli ATTC 25922 2 + E. coli ATTC 10536 2 + E. coli ATTC 127

Figure 7. Zones of inhibition of the tested E. coli strains against the oregano oils. 1: commercial oil; 2:
Figure 7. Zones of inhibition of the tested E. coli strains against the oregano oils. 1: commercial oil; 2:
cold extract.
cold extract.

The commercial marjoram oil, within the range of concentration 1.5%–2%, inhibited the growth
of the tested E. coli strains. It revealed a similar inhibitory activity against the strains of E. coli ATTC
25922 and E. coli 127. E. coli ATTC 10536 showed a higher sensitivity to this oil (Figure 8).

Activity of marjoram oils

 The commercial marjoram oil, within the range of concentration 1.5%–2%, inhibited the growth
of the tested E. coli strains. It revealed a similar inhibitory activity against the strains of E. coli ATTC
25922 and E. coli 127. E. coli ATTC 10536 showed a higher sensitivity to this oil (Figure 8).
Molecules 2017, 22, 1887 11 of 16

Activity of marjoram oils

25

Zones of inhibition (mm)

20
15
10
5 000000 00 000 00 000 0 000 000 000
0
control 0.25 0.5 1.0 1.5 2.0
Oil concentration (%)

1+ E. coli ATTC 25922 1 + E. coli ATTC 10536 1 + E. coli 127

2 + E. coli ATTC 25922 2 + E. coli ATTC 10536 2 + E. coli ATTC 127

Figure 8. Zones of inhibition of the tested E. coli strains against the marjoram oils. 1: commercial oil; 2:
Figure 8. extract.
cold Zones of inhibition of the tested E. coli strains against the marjoram oils. 1: commercial oil;
2: cold extract.
The commercial clove oil with the concentration of 0.5–2% inhibited the growth of the stains in the
3. Discussion
collection (E. coli ATTC 25922 and E. coli ATTC 10536). The E. coli ATTC 25922 strain proved to be more
sensitive, and the growth inhibition zones were 12.5 to 15 mm. On the other hand, the environmental
Essential oils show a differentiated chemical composition, which depends on various factors,
isolate E. coli 127 revealed sensitivity only to higher concentrations of this oil (1.5–2%). However, no
such biocidal
as the source of the
effect was raw material,
observed climateagainst
of clove extracts conditions of plantations,
the tested the(Figure
bacterial strains methods5). of obtaining
the oils, etc. [23–26]. The tested commercial essential oils revealed considerable differences
The commercial juniper oil did not reveal any antibacterial properties against any of the tested in terms
of thebacterial
composition
strains,and diversity
and the extract of terpenes,
obtained terpenoids,
in the and sesquiterpenes
process of maceration only inhibitedas the
compared
growth of with
the the
extracts obtained
strain of E. coli from plant
25922 at material.
all used The clove,
concentrations juniper,
of this and marjoram
extract (Figure 6). oils contained a lower
number of components
The commercial as compared
oregano with their
oil inhibited commercial
the growth counterparts,
of all the tested strains which mayE.be
of bacteria colicaused
in the by
concentrations
the loss rangingcompounds
of high-volatility from 0.5% toin 2%,theyet, the maximum
process of drying zone
of of
theinhibition bacteriaused
plant material growthas was
the raw
present at concentrations of 0.5% and 1%. The E. coli ATTC 10536 strain proved to
material for extraction [27]. On the other hand, the choice of extraction method mainly affected the be the most sensitive
to the oil
quantitative activity. However,
composition no biocidal
of the oils, activity
e.g., a higher wasofobserved
share of clovewas
sesquiterpenes macerate
observedagainst the testedwith
as compared
bacterial strains (Figure 7).
monoterpenes, which also may result from the nature of the raw material used for extraction purposes.
The commercial marjoram oil, within the range of concentration 1.5–2%, inhibited the growth
of the tested E. coli strains. It revealed a similar inhibitory activity against the strains of E. coli ATTC
25922 and E. coli 127. E. coli ATTC 10536 showed a higher sensitivity to this oil (Figure 8).

3. Discussion
Essential oils show a differentiated chemical composition, which depends on various factors,
such as the source of the raw material, climate conditions of plantations, the methods of obtaining
the oils, etc. [23–26]. The tested commercial essential oils revealed considerable differences in terms
of the composition and diversity of terpenes, terpenoids, and sesquiterpenes as compared with the
extracts obtained from plant material. The clove, juniper, and marjoram oils contained a lower
number of components as compared with their commercial counterparts, which may be caused by
the loss of high-volatility compounds in the process of drying of the plant material used as the raw
material for extraction [27]. On the other hand, the choice of extraction method mainly affected
the quantitative composition of the oils, e.g., a higher share of sesquiterpenes was observed as
compared with monoterpenes, which also may result from the nature of the raw material used for
extraction purposes.
The commercial clove, oregano, and marjoram oils in the concentrations ranging from 1.5% to 2%
showed antibacterial properties against all the tested strains of E. coli. However, these strains were not
sensitive to essential oils obtained from the plant material in the process of maceration.
Molecules 2017, 22, 1887 12 of 16

This suggests that the tested strains of E. coli show a high sensitivity mainly against monoterpenes,
such as α-pinene, β-pinene, α,β,γ-terpinene, limonene, and some terpenoids (thymol, carvacrol).
Hajlaoui et al. [28] linked the antimicrobial properties of marjoram essential oil with the high proportion
of oxygenated monoterpenes, such as terpinen-4-ol, α-terpinol, α-pinene, and p-cymene. Additionally,
the oxygenated compound, especially oxygenated monoterpenes and phenylpropanoids, might be
responsible for the antimicrobial activity of marjoram oil against Clostridium perfringens [29].
Other authors also connect the antibacterial properties of essential oils with the presence of
monoterpenes. For instance, Król et al. [30] and Dorman and Deans [31] link the activity of clove oil
with the presence of eugenol, β-pinene, and β-terpinene. Additionally, de Oliveira et al. [32] showed
in their study that eugenol is responsible for the phytotoxic activity of clove essential oil. On the other
hand, Fun and Baerheim [33] claim that the activity of marjoram oil is induced by limonene (23.5%),
α,β-pinene and γ-terpinene, and that a high concentration of monoterpene tricyclene leads to the
reduction of the antibacterial activity of this oil. The latter statement is confirmed by the results of
our own study, since the marjoram obtained in the extraction process with a high concentration of
tricyclene (24%) did not inhibit the growth of any tested strain of E. coli.
Furthermore, the high anti-microbiological activity of the oregano oil is connected with the
high concentration of terpenoids, carvacrol in particular [17,18,34]. However, the tested commercial
oregano oil did not contain carvacrol, but only thymol (11%), p-thymol (33%), and limonene (15.5%).
Most probably, it was these compounds present in high concentrations that were responsible for the
inhibition of E. coli. The oil obtained in the process of maceration, despite the fact it contained carvacrol
(1.8%) and thymol (2.5%), probably did not reveal any biological activity due to the low concentrations
of these compounds.
A different reaction of bacteria E. coli was observed against the commercial juniper oil.
These strains did not show sensitivity to the components of this oil; however, they were sensitive to the
action of the obtained juniper extracts. The bactericidal activity of juniper extracts against the strains
of E. coli is most probably connected with the presence of sesquiterpenes which were not isolated
from the commercial essential oil. The commercial juniper oil contained mainly terpenes (88%) and
terpenoids (8%), and the extracts (depending on the applied procedure) contained lower amounts of
terpenes (42–48%) and terpenoids (approximately 1%), yet high amounts of sesquiterpenes (25–30%).
The anti-microbiotic properties of the juniper oil obtained in the maceration process seem to be caused
by the synergistic activity of mono- and sesquiterpenes, which was also observed in the case of the
activity of coriander oil against yeasts Candida albicans [35]. The antioxidant activity of juniper berry
oil from Bulgaria against Saccharomyces cerevisiae depended on monoterpenes, mostly from α-pinene
as main component [36]. The biological activity of the tested essential oils not only depends on the
presence of active compounds, but also on the sensitivity of the tested strains of E. coli.

4. Materials and Methods

4.1. Plant Material

The scientific material consisted in the commercial oils: clove (Eugenia caryophyllus) leaf oil
˛ Poland; juniper (Juniperus communis) oil from Etja, Elblag,
from Etja, Elblag, ˛ Poland; oregano
(Origanum vulgare) leaf oil from Aromatika, Kiev, Ukraine; and marjoram (Majorana hortensis) oil
from Profarm, L˛ebork, Poland. The dried clove flower bud Syzygium aromaticum from Prymat,
Jastrz˛ebie-Zdrój, Poland; juniper berries (Juniperus communis) from Kawon-Hurt, Gostyń, Poland;
dried marjoram leaves Majorana hortensis from Kawon-Hurt, Gostyń, Poland; and dried oregano leaves
(Origanum vulgare) from Dary Natury, Grodzisk, Poland were used to obtain the appropriate herbal
extracts in the process of cold and hot maceration in the Soxhlet apparatus.
Molecules 2017, 22, 1887 13 of 16

4.2. Bacterial Strains

The biological activity of all the studied essential oils was assessed against three E. coli
strains—E. coli ATTC 25922, E. coli ATTC 10536, and E. coli 127 isolated from poultry waste (strains
came from the collection of the Independent Department of Biotechnology and Molecular Biology,
University of Opole, Opole, Poland).

4.3. Study Methods

4.3.1. Physicochemical
(a) Maceration—Twenty grams of dry plant material was minced in a mortar and ground with 100 cm3
of dichloromethane. The mixture was percolated under reduced pressure. Next, anhydrous
magnesium sulphate was added to the solution which was percolated again. The solution was
placed in the weighed round-bottom flask and concentrated in the rotary evaporator Heidolph
Laborota 4000 efficient (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany).
The cooled flask was weighed again, and the extraction efficiency was calculated (%).
(b) Extraction in the Soxhlet apparatus—Fifteen grams of dry plant material was minced in a mortar
and moved to the thimble, which was placed in the Soxhlet apparatus. Dichloromethane (150 cm3 )
was poured into the round-bottom flask together with a boiling stone. The extraction set was
mounted and the process was carried out for 4 h. After cooling, anhydrous magnesium sulphate
was added to the solution, which was then percolated under reduced pressure. The solution was
placed in the weighed flask and the solvent was evaporated in the rotary evaporator. The cooled
flask was weighed again and the extraction efficiency was calculated (in %).

4.3.2. The Gas Chromatography Mass Spectrometry Analysis

The Hewlett Packard HP 6890 series GC system chromatograph (Hewlett Packard, Waldbronn,
Germany) was used for the study, which was coupled with the Hewlett Packard 5973 mass selective
detector (Hewlett Packard, Waldbronn, Germany) The chromatograph was equipped with the
non-polar, high-temperature ZB-5HT capillary column (length, 30 m; inner diameter, 0.32 mm; film
thickness, 0.25 µm, Phenomenex Inc., Torrance, California, USA). The on-column injector was used
and 1 µm of a sample was introduced. The results of carrying out the process: initial temperatures,
both of the injector and the oven were 60 ◦ C, and the temperature was increased by 10 ◦ C per minute
up to 280 ◦ C, the auxiliary temperature was 300 ◦ C. Helium was used as the carrier gas and its
flow was 2 mL/min. The components were identified by comparison of their mass spectra with
the spectrometer database of the NIST 11 Library (National Institute of Standards and Technology,
Gaithersburg, MD, USA). Each chromatographic analysis was repeated three times. The average value
of relative composition of essential oil percentage were calculated from the peak areas.

4.3.3. Biological
The assessment of biological activity of commercial oils and macerates against the growth of three
tested E. coli strains was carried out by the diffusion cylinder-plate method on the Nutrient Lab-AgarTM
medium [5]. The media were inoculated with 1 cm3 of standard bacterial suspension with the optical
density of ζ = 2 and the wavelength of 550 nm. The results were presented as a mean value of the
growth inhibition diameter (in mm). The inhibition effect was assumed to be the lack of growth around
wells, growth stimulation–intensified growth around wells and the neutral effect–growth inhibition at
the edges of the wells. The control was water with 0.05% Tween 80. Essential oils and extract were used
in the following concentrations: 0.25%, 0.5%, 1%, 1.5% and 2% (v/v). Each experiment was repeated
four times.
Molecules 2017, 22, 1887 14 of 16

5. Conclusions
Commercial essential oils and oils obtained in the process of extraction from plant material
differed in terms of the content and the quantitative ratio of terpenes, terpenoids, and sesquiterpenes,
as well as their antibacterial activity.
The commercial clove, oregano, and marjoram oils in concentrations of 1.5–2% revealed the
antibacterial activity against all tested E. coli strains.
Essential oils obtained from plant material in the process of maceration did not reveal bactericidal
activity, except for the juniper oil.
The bactericidal activity of the juniper extract against E. coli strains is probably related to presence
of sesquiterpenes which were not isolated from the commercial essential oil.

Author Contributions: M.B. conceived and designed the chemical experiments; T.K.Ł. conceived and designed
the biological experiments; A.P. performed the experiments; M.B., T.K.Ł. and P.P.W. analysed the data; M.B. and
T.K.Ł work concept; M.B. and T.K.Ł wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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