plants

Article
Pimpinella anisum L. Essential Oil a Valuable Antibacterial and
Antifungal Alternative
Eugenia Dumitrescu 1 , Florin Muselin 1 , Emil Tîrziu 1 , Mihai Folescu 1 , Carmen S. Dumitrescu 2 , Dora M. Orboi 2
and Romeo T. Cristina 1, *

1 Faculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, Calea Aradului
119, 300645 Timisoara, Romania; [email protected] (F.M.); [email protected] (E.T.);
[email protected] (M.F.)
2 Department Systems in Agriculture, Faculty of Management, University of Life Sciences “King Mihai I” from
Timisoara, 300645 Timisoara, Romania
* Correspondence: [email protected]; Tel.: +40-722-522-499

Abstract: Anise (Pimpinella anisum L.) essential oils are intensely investigated worldwide for the
beneficial properties, due to the specific bioactive compound’s structure. (1) Background: This study
characterized the structure of the Pimpinella anisum essential oil and evaluated its antimicrobial
properties. (2) Methods: An evaluation of the antibacterial and antifungal activity targeted strains
of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC
25923), Streptococcus pyogenes (ATCC 19615), and levure Candida albicans (ATCC 10231). Gas chro-
matography coupled with mass spectrometry (GC/MS) was used for structure identification, and the
optical density mass loss was applied for the analysis of different dilutions of aniseed essential oils
antimicrobial activity. (3) Results: A total of 13 compounds were identified, of which trans-anethole
was in the highest proportion (72.49%), followed by limonene (10.01%), anisole (5%), and α-pinene
(3.26%). The results obtained and statistically analyzed, utilizing one-way ANOVA with Bonfer-
roni’s multiple comparison test, indicated the antimicrobial activity (p < 0.001) of anise essential oil.
(4) Conclusion: Anise essential oil is a promising phyto-remedy with important antimicrobial activity
against both Gram-positive and Gram-negative pathogens. Inhibition high percentages were found
Citation: Dumitrescu, E.; Muselin, F.;
for the p. aeruginosa and S. aureus strains, but also excellent antifungal activity against C. albicans
Tîrziu, E.; Folescu, M.; Dumitrescu,
was ascertained.
C.S.; Orboi, D.M.; Cristina, R.T.
Pimpinella anisum L. Essential Oil a
Valuable Antibacterial and
Keywords: Pimpinella anisum; essential oils; composition; antimicrobial; Staphylococcus aureus; Es-
Antifungal Alternative. Plants 2023, cherichia coli; Pseudomonas aeruginosa; Streptomyces pyogenes; Candida albicans
12, 2428. https://doi.org/10.3390/
plants12132428

Academic Editors: Milan S. Stankovic

1. Introduction
and Christophe Hano
The history of aromatic anise seeds use begins as early as 5000 years ago, and it is
Received: 5 May 2023 very difficult to determine the first recorded use of anise. Some investigators believe that
Revised: 13 June 2023 anise originates from Ancient Egypt, while others attribute it to Greece or even Persia (the
Accepted: 19 June 2023
current territory of Iran) [1].
Published: 23 June 2023
Anise is part of the Apiaceae family and is an aromatic, herbaceous, annual, heliophilous
plant that prefers a warm climate. Cultivated since ancient times in Asia Minor, its use
later spread to Europe and other continents [2,3]. The plant has few leaves, with small and
Copyright: © 2023 by the authors.
white flowers arranged in the form of an umbrella. The fruits are small and green, ripening
Licensee MDPI, Basel, Switzerland. from the end of August to the end of September. They are small diachenes, ovoid, with
This article is an open access article hard-to-separate halves, with five slightly prominent ribs, lighter in color [2].
distributed under the terms and The fruits contain numerous substances and essential oils, namely anethole, present in
conditions of the Creative Commons large amounts, as well as anisaldehyde, anisic acid, and eugenol. Anise fruits also contain
Attribution (CC BY) license (https:// other fatty, proteinaceous, carbohydrate substances; mucilage; anethole; coumarins; toco-
creativecommons.org/licenses/by/ pherol; and polyphenol carboxylic acids. Some substances that demonstrate an estrogenic
4.0/). effect are also mentioned in relevant works [4].

Plants 2023, 12, 2428. https://doi.org/10.3390/plants12132428 https://www.mdpi.com/journal/plants

Plants 2023, 12, 2428 2 of 13

The seeds contain at most 6% essential oil, of which up to 90% is anethole (an organic
compound commonly used as a natural flavor and main component of essential oil extracted
from plants) and gives the plant its typical aroma. Coumarins, flavonoids, and sterols can
be found in the composition of the seeds [5].
The presence of eugenol, trans-anethole, methyl-chavicol, estragole, coumarin, anisalde-
hyde, estrols, terpene hydrocarbons, polyenes, scopoletin, and polyacetylenes as the main
compounds of aniseed essential oil were illustrated in numerous studies [6,7].
The GC and GC-MS analysis of P. anisum fruits essential oils revealed the presence
of trans-anethole (93.9%) and estragole (2.4%). In concentrations greater than 0.06%,
α-cuparene, β-bisabolene (E)-methyl-eugenol, α-himachalene, p-anisaldehyde, and cis-
anethole were ascertained [1,8,9].
The chemical components of the anise extract examined by GC-MS after supercritical
extraction using CO2 revealed that anethole (~90%), γ-hymacalene (2–4%), p-anisaldehyde
(<1%), methyl-chavicol (0.9–1.5%), cis-pseudoisoeugenyl, 2-methyl-butyrate, and trans-
pseudoisoeugenyl 2-methyl-butyrate (~1.3%) were the main compounds identified [7,10–12].
One of the phenolic glycosides of the Umbelliferae family (4-(p-d-glucopyranosyloxy) ben-
zoic acid) was also found in anise [13,14].
This plant has several pharmacological properties, including the following:
a. Antibacterial, antifungal action
Authors studied the antibacterial activity of Pimpinella anisum extracts made in methanol,
acetone, petroleum ether, and water. These extracts were tested on four pathogenic bacteria
(e.g., Staphylococcus pyogenes, Streptococcus aureus, Klebsiella pneumoniae, and Escherichia coli)
using the disc diffusimetric method [15]. Only the methanolic and aqueous extracts showed
relevant antibacterial activity against all tested bacteria and that the aqueous extract ap-
peared to be more effective when compared to the methanolic extract. The acetone and
petroleum ether extracts were not shown to inhibit the growth of the tested pathogenic
bacteria [16].
The effects of aqueous and ethanolic extracts of anise were investigated on 10 bacterial
species as well as on Candida albicans by utilizing the disc diffusimetric method. In this
study, the ethanolic extract showed significant inhibitory activity against all bacteria tested
but was not found to be effective against Candida albicans. However, the antimicrobial
effect of the aqueous extract was not illustrated against Gram-negatives Escherichia coli
and Pseudomonas aeruginosa. Furthermore, Candida albicans was sensitive to the effect of
the aqueous extract [17]. Alcoholic extracts obtained from P. anisum seeds validated the
antibacterial activity against Micrococcus luteus and Mycobacterium smegmatis [18,19].
Some authors emphasized that, besides the antibacterial activity, anise essential oil
also exhibits significant antifungal activity. The authors claimed that this is due to the
anethole in the composition of the oil [20]. In their research, the authors examined the
antifungal activity of the essential oil obtained from anise seeds and of the aqueous extract
on four species of dermatophytes and seven species of yeasts via the cylinder diffusion
method and broth dilution method. The authors illustrated the fact that the aqueous extract
of anise displayed antifungal activity against Candida (species albicans, krusei, parapsilosis,
pseudotropicalis, and tropicalis), the largest inhibition being observed in C. albicans [20].
An inhibitory effect was also demonstrated on dermatophyte species (Trichophyton
rubrum, T. mentagrophytes, Microsporum canis, and M. gypseum), the most pronounced zone
of inhibition detected being for T. mentagrophytes [21].
There are also some studies that demonstrate the antibacterial effectiveness of other
essential oils, such as tea tree essential oil on E. coli, S. typhi, C. koseri, and S. aureus but also
juniper essential oil on strains of Staphylococcus aureus [22,23].
b. Anticonvulsant action
The anticonvulsant effects of the Pimpinella anisum essential oil present in the fruits
were analyzed on seizures induced using pentylenetetrazole (PTZ) and maximal elec-
troshock (MES) in male mice. The authors showed that P. anisum boosted the threshold of
Plants 2023, 12, 2428 3 of 13

clonic seizures caused by the i.v. introduction of PTZ and also blocked tonic convulsions
that appear upon the i.p. injection by PTZ. Furthermore, P. anisum possessed anticonvulsant
activity against tonic seizures induced by maximal electroshocks [24].
c. Action on the digestive system
The effect of aqueous anise extract on gastric ulceration in rats was studied, the acute
gastric ulceration being induced by various harmful chemicals and the administration of
indomethacin. The results of the study showed that anise significantly inhibited the damage
to the gastric mucosa induced by indomethacin and necrotizing agents. The antiulcer effect
was corroborated both by the authors and histologically [25].
d. Analgesic and anti-inflammatory action
There are authors who demonstrated that the essential oil of Pimpinella anisum ad-
ministered to mice presented a significant analgesic effect similar to that of morphine and
aspirin [26]. Research has shown that anise essential oil has an anti-inflammatory effect
as strong as indomethacin and has an analgesic effect similar to that of 10 mg/kg.bw. of
morphine and 100 mg/kg.bw. of aspirin after 30 min [27,28].
e. Antioxidant action
In a study, the antioxidant properties of ethanolic and aqueous extracts of anise were
studied. The antioxidant properties were assessed employing different antioxidant assays
and were contrasted with synthetic antioxidants, for instance, butylated hydroxytoluene
(BHT), butylated hydroxyanisole (BHA), and tocopherol. Both studied extracts demon-
strated strong antioxidant activity by scavenging DPPH radicals and superoxide anions,
absorption of hydrogen peroxides, as well as capabilities to perform metal chelation com-
pared to BHT, BHA, and α-tocopherol. The authors concluded that, of the two extracts
studied, it seemed that the aqueous extract had a higher antioxidant capacity when com-
pared to the ethanolic extract [17].
f. Acaricidal activity
The authors also studied the acaricidal activity of p-anisaldehyde derived from aniseed
essential oil against house dust mites Dermatophagoides pteronyssinus and Dermatophagoides
farina. The results of this study showed that benzyl-benzoate and p-anisaldehyde were
the compounds that had the most significant effect on these dermatophagoides. Therefore,
p-anisaldehyde can be used for the selective control of house dust mites [29].
g. Antitumor activity
Researchers investigated the cytotoxic effect of both the extract and essential oil of
P. anisum on the AGS (gastric cancer) cell line. The results illustrated the cytotoxic effect
of alcoholic extracts and essential oils on gastric cancer cells at concentrations of 15 to
480 µg/mL but did not demonstrate any significant effect on fibroblast cells as normal
cells. The inhibitory effect on the growth of AGS cells was much greater in the case of the
methanolic extract compared to the ethanolic extract [30].
Therefore, P. anisum may present a natural source of compounds with antiproliferative
properties. In tumor growth, blood vessel formation (angiogenesis) is a necessary stage,
supplying oxygen and nutrients to tumoral cells. This process can contribute to tumor
progression, invasion, and metastasis. It is commonly used as an indicator of tumor
prognosis. Therefore, tumor angiogenesis has been determined to be of high clinical
relevance. The authors of this study evaluated the effects of extracts and essential oil on the
process of angiogenesis [30].
h. Neural activity
Few studies indicate the possible effects of Pimpinella anisum essential oil on neural
activity. The plant and, especially, the essential oils have been used in the treatment of
certain neurological diseases, including epilepsy and seizures.
Plants 2023, 12, 2428 4 of 13

There is research attempting to evaluate the possible antidepressant and anxiolytic

effects of anise oil and its ability to attenuate in vivo, Pb-induced cognitive deficits in rats
exposed to lead during gestation and lactation. The result demonstrated that exposure
to lead during intrauterine development causes problems with body development and
brain weight development and increases the level of anxiety, depression, and locomotor
hyperactivity in rats exposed to lead compared to the control rats. The authors claimed
that administration of Pimpinella anisum essential oil to lead-exposed rats led to a reduction
in anxiety, depression, and locomotor hyperactivity. The results of the study demonstrated
that lead exposure during intrauterine development induced a significant disruption of
emotional reactivity that can be improved by treatment with Pimpinella anisum essential
oil [31].
Anise essential oil is used as an expectorant, carminative, and in cough mixtures,
especially in pediatrics, and important phenylpropanoids such as trans-anethole and
estragole have a stabilizing influence on the autonomic nervous system. The aim of the
present research was to evaluate the chemical composition and antimicrobial activity of a
commercially procured anise essential oil.

2. Results
2.1. Phytochemical Compounds Identification
The identification of the compounds from the anise essential oil was carried out using
the Agilent gas chromatograph (Agilent Scientific, Santa Clara CA, USA) paired with the
mass spectrometer, revealing 13 different compounds.
As the most important compounds among those identified, α-pinene (3.263%), anisole
(5.003%), limonene (10.011%), and propenyl-anisole (72.499%) peaks were ascertained for
the sampled oils, representing 90.776% from the total compound structure.
Table 1 presents the compound structure identified in the studied essential oil.

Table 1. Compounds identified in anise essential oil.

Compound R. Time %
α-pinene 6.382 3.263
3-carene 9.847 1.327
α-phellandrene 10.309 1.822
Limonene 11.292 10.011
β-terpinen 11.548 2.511
1-pentanone, 1-(4-methylphenyl) 13.259 0.179
Linalool 20.840 1.962
Anisole 23.697 5.003
Propenyl-anisole 27.360 72.499
Anisaldehyde 31.552 0.326
p-(pentyloxy) acetophenone 34.046 0.089
Thymol 34.583 0.580
1-(3-methyl-2-butenoxy)-4-(1-propenyl) benzene 36.192 0.429

The chromatogram peaks are presented in the attached Supplementary Figure S1.

2.2. Antimicrobial Activity

The mean values of the three determinations for the Control and the four dilutions
tested for bacterial strains and levure and the comparative optical densities (OD) for anisum
essential oil tested for the inhibition of S. pyogenes, P. aeruginosa, S. aureus, E. coli, and C.
albicans are presented in Figures 1–5 and Table 2.
 tested for bacterial strains and levure and the comparative optical densities (OD)
sum essential oil tested for the inhibition of S. pyogenes, P. aeruginosa, S. aureus, E. c
C. albicans are presented in figures 1–5 and Table 2.
Plants 2023, 12, x FOR PEER REVIEW
Plants 2023, 12, 2428 5 of 13
2.2.1. Streptococcus Pyogenes
All anise essential oils at dilutions of 2, 4, 8, and 10 μL hindered the develop
Streptococcus pyogenes (ATCC 19615) to different degrees. The evaluation of the ant
bial activity of anise essential oils, performed using optical density mass loss re
Figure 1 the optical density (OD) of the anise essential oil and the Control.
Increasing the volume of anise oil to 10 μL led to a decline in bacterial cell
from 0.786 ± 0.005 (Control) to 0.465 ±0.003 (the 10 μL dilution), a notable valu
compared to the Control sample, also covered statistically (p < 0.001). In the Control
the optical activity was thought to present a potency of 100% and, in the case of
currently tested, inhibition of the development of Streptococcus pyogenes strains of
was observed for the 10 μL dilution.

2.2.2. Pseudomonas Aeruginosa

Figure 2 shows the optical density (OD) of the anise essential oil and the Con
Figure 1. Streptococcus pyogenes—mean values of the three determinations for the Control and the
Figure
the 1. Streptococcus pyogenes—mean values
strain.of the three determinations for the Control
four Pseudomonas aeruginosa
dilutions tested (where bacteria
*** means p < 0.001).
four dilutions tested (where *** means p < 0.001).

2.2. Antimicrobial Activity

Plants 2023, 12, x FOR PEER REVIEW
The mean values of the three determinations for the Control and the four di
tested for bacterial strains and levure and the comparative optical densities (OD)
sum essential oil tested for the inhibition of S. pyogenes, P. aeruginosa, S. aureus, E. c
8 μL
C. (109.84%)
albicans and, respectively,
are presented in figures41–5
μL (49.59) of the
and Table 2. essential oil, compared to the C
leading to a decline in bacterial cell density from OD = 0.370 ± 0.008 (the Control)
0.186Streptococcus
2.2.1. ± 0.001 (dilution of 4 μL), and close to the value of 10 μL dilution (OD = 0.273 ±
Pyogenes
results
Allstatistically confirmed
anise essential (p < 0.001).
oils at dilutions of 2, 4, 8, and 10 μL hindered the developm
Streptococcus pyogenes (ATCC 19615) to different degrees. The evaluation of the ant
2.2.3.activity
bial Staphylococcus
of aniseAureus
essential oils, performed using optical density mass loss re
Figure 3 illustrates
Figure 1 the optical density the (OD)
opticalof density
the anise ofessential
the aniseoilessential
and theoil as well as the
Control.
Figure 2. Pseudomonas aeruginosa—mean values of the three determinations for the Control and the
for the
Figure 2.Staphylococcus
Pseudomonas
Increasing aureusof
the volume standard
aeruginosa—mean
anise oil strain.
values of μL
to 10 the led
threetodeterminations for the Control
a decline in bacterial cell d
four dilutions tested (where *** means p < 0.001).
four dilutions tested (where *** means p < 0.001).
from 0.786 ± 0.005 (Control) to 0.465 ±0.003 (the 10 μL dilution), a notable value
compared to the Control sample, also covered statistically (p < 0.001). In the Control
Anise activity
the optical oil dilutions impededtothe
was thought development
present a potency of of
the100%
Pseudomonas
and, in theaeruginosa
case of
27853) strain. Remarkably, the highest inhibition percentage was
currently tested, inhibition of the development of Streptococcus pyogenes strains obtained at dilu
of
was observed for the 10 μL dilution.

2.2.2. Pseudomonas Aeruginosa

Figure 2 shows the optical density (OD) of the anise essential oil and the Con
the Pseudomonas aeruginosa bacteria strain.

Staphylococcus
Figure 3.3.
Figure aureus—mean
Staphylococcus values of values
aureus—mean the threeof
determinations for the Control andfor
the three determinations thethe
fourControl
tested dilutions (where *** means p < 0.001).
four tested dilutions (where *** means p < 0.001).

In the case of the Staphylococcus aureus (ATCC 25923) strain, anise essential oi
ited its growth compared to the Control (OD = 0.495 ± 0.006) by a 41.18% inhibiti
at a dilution of 10 μL and (OD = 0.291 ± 0.005) (p < 0.001). Thus, we can say that the
volume also had the highest inhibition rate in the case of Staphylococcus aureus stra

2.2.4. Escherichia Coli

 volume also had the highest inhibition rate in the case of Staphylococcus aureus stra

2.2.4. Escherichia Coli

Plants 2023, 12, 2428

The optical density of anise essential oil in the case of Escherichia coli is shown
6 of 13
ure 4.

Plants 2023, 12, x FOR PEER REVIEW

2.2.5. Candida Albicans

The optical density of anise essential oil in the case of levure Candida albicans is
Figure 4. Escherichia coli—mean values of the three determinations for the Control and the four tested
in Figure
dilutions
Figure
5. *** meanscoli—mean
(where
4. Escherichia p < 0.001). values of the three determinations for the Control and
tested dilutions (where *** means p < 0.001).

In the case of Escherichia coli (ATCC 25922), the percentage of inhibition produ
anise essential oil was 35.83% at a dilution of 4 μL (OD = 0.675 ± 0.009) and 26.9
dilution of 10 μL (OD = 0.769 ± 0.008) (p < 0.001).

Figure 5. Candida albicans—mean values of the three determinations for the Control and the four
Figure 5. Candida albicans—mean values of the three determinations for the Control and
tested dilutions (where *** means p < 0.001 and * means p < 0.05).
tested dilutions (where *** means p < 0.001 and * means p < 0.05).
2.2.1. Streptococcus Pyogenes
In the case of Candida albicans (ATCC 10231), the percentage of inhibition pr
All anise essential oils at dilutions of 2, 4, 8, and 10 µL hindered the development of
by anise oilpyogenes
Streptococcus was 72.03% compared
(ATCC 19615) to the
to different Control
degrees. (OD = 0.830
The evaluation of the ± 0.007) for 2 μL d
antimicrobial
(OD
activity of anise essential oils, performed using optical density mass loss reveal in Figureof1 10 μL
= 0.232 ± 0.007) and 61.60% compared to the Control for a dilution
the optical
0.008). In density
the case(OD) of the
of the anise essential
levure Candidaoil and the we
albicans, Control.
can say that Pimpinella anisum e
Increasing the volume of anise oil to 10 µL led to a decline in bacterial cell density
oil was much more effective at the lowest dose used.
from 0.786 ± 0.005 (Control) to 0.465 ±0.003 (the 10 µL dilution), a notable value when
In Table
compared to the2, the comparative
Control optical
sample, also covered densities
statistically (p (OD) forInanisum
< 0.001). essential
the Control group, oil te
the inhibition
the optical of was
activity S. pyogenes,
thought to P.present
aeruginosa, S. aureus,
a potency of 100%E.and,
coli,inand C. albicans
the case of the oilare pres
currently tested, inhibition of the development of Streptococcus pyogenes strains of 40.86%
was observed
Table for the 10 µL dilution.
2. The comparative optical densities for anisum essential oil tested, SEM values, and
bition percentage for S. pyogenes, P. aeruginosa, S. aureus, E. coli, and C. albicans standard stra
2.2.2. Pseudomonas Aeruginosa
Concentration
Figure 2 shows the optical density (OD) of the anise essential oil and the Control for Inh
the Pseudomonas aeruginosa bacteria strain. I II III x¯ SEM
/Replica
Anise oil dilutions impeded the development of the Pseudomonas aeruginosa (ATCC
27853) strain. Remarkably, the BHI+strain
highest inhibition0.786 0.787
percentage was 0.786
obtained0.7864 0.0005
at dilutions 1
ofStreptococcus
8 µL (109.84%)pyogenes 10 4μL
and, respectively, µL (49.59)0.465 0.466 0.464
of the essential 0.465 to0.0003
oil, compared the 40
Control, leading to a decline in bacterial cell density from OD = 0.370 ± 0.008 (the Con-
8 μL 0.593 0.591 0.594 0.5928 0.0005 24
trol) to OD = 0.186 ± 0.001 (dilution of 4 µL), and close to the value of 10 µL dilution
4 μLconfirmed
(OD = 0.273 ± 0.001), results statistically 0.714 0.712 0.714 0.7132 0.0007
(p < 0.001). 9
2 μL 0.722 0.724 0.721 0.7218 0.0006 8
BHI+strain 0.368 0.370 0.371 0.3697 0.0008 1
10 μL 0.274 0.268 0.276 0.2726 0.0019 26
Plants 2023, 12, 2428 7 of 13

Table 2. The comparative optical densities for anisum essential oil tested, SEM values, and the
inhibition percentage for S. pyogenes, P. aeruginosa, S. aureus, E. coli, and C. albicans standard strains.

Concentration
I II III x¯ SEM Inhibition(%)
/Replica
BHI+strain 0.786 0.787 0.786 0.7864 0.0005 100.00
Streptococcus pyogenes 10 µL 0.465 0.466 0.464 0.465 0.0003 40.86%
8 µL 0.593 0.591 0.594 0.5928 0.0005 24.61%
4 µL 0.714 0.712 0.714 0.7132 0.0007 9.3%
2 µL 0.722 0.724 0.721 0.7218 0.0006 8.21%
BHI+strain 0.368 0.370 0.371 0.3697 0.0008 100.00
10 µL 0.274 0.268 0.276 0.2726 0.0019 26.28%
Pseudomonas aeruginosa 8 µL 0.777 0.775 0.778 0.776 0.0008 109.84
4 µL 0.184 0.189 0.186 0.1864 0.0010 49.59
2 µL 0.331 0.332 0.334 0.3324 0.0008 10.11
BHI+strain 0.496 0.495 0.495 0.4954 0.0006 100.00
10 µL 0.292 0.289 0.291 0.2906 0.0009 41.18
Staphylococcus aureus 8 µL 0.459 0.458 0.461 0.4594 0.0006 7.26
4 µL 0.366 0.365 0.363 0.3646 0.0010 26.44
2 µL 0.362 0.360 0.366 0.3626 0.0013 26.71
BHI+strain 1.053 1.050 1.052 1.052 0.0008 100.00
10 µL 0.769 0.767 0.771 0.769 0.0008 26.90
Escherichia coli 8 µL 0.918 0.919 0.916 0.917 0.0010 12.77
4 µL 0.678 0.674 0.673 0.675 0.0009 35.80
2 µL 1.106 1.101 1.105 1.104 0.0009 4.92
BHI+strain 0.831 0.832 0.828 0.8304 0.0007 100.00
10 µL 0.319 0.317 0.320 0.3186 0.0008 61.60
Candida albicans 8 µL 0.796 0.795 0.795 0.7953 0.0007 4.23
4 µL 0.244 0.244 0.247 0.245 0.0027 70.42
2 µL 0.234 0.233 0.230 0.2323 0.0007 72.03
Where BHI = Brain Heart Infusion; x¯ = Arithmetic mean; SEM = Standard Error of the Mean.

2.2.3. Staphylococcus Aureus

Figure 3 illustrates the optical density of the anise essential oil as well as the Control
for the Staphylococcus aureus standard strain.
In the case of the Staphylococcus aureus (ATCC 25923) strain, anise essential oil inhibited
its growth compared to the Control (OD = 0.495 ± 0.006) by a 41.18% inhibition rate, at a
dilution of 10 µL and (OD = 0.291 ± 0.005) (p < 0.001). Thus, we can say that the highest
volume also had the highest inhibition rate in the case of Staphylococcus aureus strain.

2.2.4. Escherichia Coli

The optical density of anise essential oil in the case of Escherichia coli is shown
in Figure 4.
In the case of Escherichia coli (ATCC 25922), the percentage of inhibition produced by
anise essential oil was 35.83% at a dilution of 4 µL (OD = 0.675 ± 0.009) and 26.90% at a
dilution of 10 µL (OD = 0.769 ± 0.008) (p < 0.001).

2.2.5. Candida Albicans

The optical density of anise essential oil in the case of levure Candida albicans is shown
in Figure 5.
In the case of Candida albicans (ATCC 10231), the percentage of inhibition produced
by anise oil was 72.03% compared to the Control (OD = 0.830 ± 0.007) for 2 µL dilu-
tion (OD = 0.232 ± 0.007) and 61.60% compared to the Control for a dilution of 10 µL
(0.319 ± 0.008). In the case of the levure Candida albicans, we can say that Pimpinella anisum
essential oil was much more effective at the lowest dose used.
Plants 2023, 12, 2428 8 of 13

In Table 2, the comparative optical densities (OD) for anisum essential oil tested for
the inhibition of S. pyogenes, P. aeruginosa, S. aureus, E. coli, and C. albicans are presented.
Analyzing the inhibition rate percentage (IRP), we perceived that in the case of Gram-
negative strains (Pseudomonas aeruginosa and Escherichia coli in our case) and of the Candida
albicans levure, the IRP was greater in the case of the lower dilutions in all cases, with 10 µL
dilution generating inferior results.

3. Discussion
Following the chromatographic analysis of anise essential oil, 13 compounds were
identified, the major ones being propenyl-anisole (syn. trans-anethole) in proportion to
72.49%, limonene 10.01%, anisole 5%, and α-pinene (3.263%). These results are consistent
with those of other authors of the domain, who identified trans-anethole, a phytoestrogen,
as the major compound in Pimpinella anisum essential oil [32–36].
Moreover, in a study conducted by Orav et al. [7], the main component of Pimpinella
anisum L. fruits essential oil obtained from various geographical areas of Europe was trans-
anethole. Chemical characterization of essential oils is very important for understanding
their biological properties. Plants are known to produce many secondary metabolites, of
which most have distinctive pharmacological actions, including phenolic acids, flavonoids,
and tannins [37].
Pure essential oils are mixtures of over 200 components, normally mixtures of terpenes
or phenylpropane derivatives. They can be largely categorized into two groups:
• Volatile fraction: constituting 90–95% of the oil and containing monoterpene and
sesquiterpene hydrocarbons with known derivatives (i.e., oxygenated, aliphatic alde-
hydes, alcohols, and esters fractions).
• The non-volatile residues: comprising only 1–10% of the oil, containing hydrocarbons,
fatty acids, sterols, carotenoids, wax, and flavonoids [37].
Several factors influence the quantity and quality of the extract, especially the compo-
sition of the soil, the plant organ, the phase of the vegetative cycle, and the climate, and
this is why it can be affirmed that composition is geographically related [38].
The presented results are consistent with those obtained by researchers [18], who
found a high inhibitory effect of anise essential oil on relevant bacteria (E. coli, E. faecalis, M.
luteus, S. typhi, and S. aureus) [18].
Al-Bayati [19] also confirms the strong inhibitory action of Pimpinella anisum essential
oil against bacteria and fungi, such as Staphylococcus aureus, Escherichia coli, and Candida
albicans. In the case of essential oils, antagonistic, additive, and synergistic effects were
ascertained. When these components’ effect equals the summation of the different effects,
the additive effect will emerge. In this study, carried out comparatively on the effectiveness
of Pimpinella anisum and Thymus vulgaris essential oils against P. aeruginosa, the authors
demonstrated that they were inactive at the highest concentration used (500.0 µg/mL) when
applied individually, while their association (1:1) impeded the growth of the pathogen P.
aeruginosa [19].
Synergism represents an overall activity greater than the total individual effects taken
separately. Conversely, the antagonistic effect is recorded if the activity of the components
in combination is lower, when compared to their effect applied separately [37].
There has been an increase in studies performed on the plant essential oils mechanisms.
However, compared to many studies on the characteristics of essential oils (EOs) and
their components and the number of studies investigating the specific target(s) of the
antimicrobial action of EOs and their components, a significant field of study remains.
The antimicrobial action of EOs is related to one of the most important characteristics of
them, respectively, their hydrophobicity leading to increased cellular permeability and,
consequently, leakage of cellular constituents. It is important to understand that a disrupted
cellular structure can affect other cellular structures in a cascade type of activity [37].
Plants 2023, 12, 2428 9 of 13

There are authors who claim that, in addition to the antibacterial activity, anise essential
oil also has noteworthy inhibitory activity against fungi, and the most active component
found was anethole [39].
Kosalec et al. [21] demonstrated that anise essential oil has strong antifungal activity
against dermatophytes and yeasts, the widest zone of inhibition appearing for Candida
parapsilosis, followed by Geotrichum spp, C. glabrata, and C. albicans [21].
The essential oils are complex mixtures with a great diversity of components and conse-
quently their promising antimicrobial activity is related to their composition, configuration,
amount, and possible interactions [40,41].
To date, various studies have been conducted on the essential oils and extracts of
Pimpinella anisum in order to ascertain the chemical compounds and pharmacological
properties of this plant. Various beneficial properties have been reported, including the
antimicrobial, antifungal, antiviral, antioxidant, and insecticidal effects of anise [42].
The strains that most commonly develop resistance were used; therefore, the obtained
results could be considered as significant. They open novel and noteworthy applications in
phytotherapy, in the frame of the One Health concept which strongly suggests the use of
essential oils as valuable phyto-resource alternatives in antibiotic resistance. The gathered
information can be applied in designing new antimicrobial associations or therapeutical
agents. To fully investigate the Pimpinella anisum characteristics, further in vivo models are
necessary and will remain to be studied soon.
In our case, analyzing the inhibition rate percentage (IRP), we perceived that in the case
of Gram-negative strains (Pseudomonas aeruginosa and Escherichia coli) and of the Candida
albicans levure, the IRP was greater in the case of the lower dilutions, in all these cases the
10 µL dilution generating inferior results.
In the case of the P. aeruginosa standard strain, the inhibition value revealed the anise
essential oil’s great potency, confirming that the antimicrobial potential of anise essential
oils varies between Gram-positive and Gram-negative bacteria, due probably to density
variations in the bacterial cell wall. As we know, essential oils are in preponderance
lipophilic, and this is why they are rapidly absorbed by Gram-positive bacteria due to their
dense layer (especially of lipophilic phospholipids in their cell walls).
This is why all studies tackling this issue could be considered of help. We consider that
this class of studies is performed to develop the associative features of phyto-therapeutic
means with other structures such as chemotherapeutics, sulfonamides, or antibiotics with
the aim of gathering new efficient associated phyto-structures especially where the efficacy
of anti-infectious classes is compromised by the resistance phenomena.

4. Materials and Methods

The 100% pure anise Pimpinella anisum-Apiaceae (Umberlliferae) essential oil condi-
tioning (Aromatics International) used in the study (Batch no. ANS-103/09.20.2020) was
purchased from a local herbal store. According to the product’s information sheet, the
manufacturer extracted the essential oil by steam distillation from organically grown brown
pods of anise seed flowers, with a yield of 2–4%. Organoleptically, the oil has a grassy, sweet
aroma with a slightly spicy (licorice-like) smell; its color is pale yellow with a medium
viscosity.
The chromatographic and microbiological analysis of anise essential oil was carried
out within the Interdisciplinary Research Platform and the Toxicology—Pharmacology
Research Laboratory within the Faculty of Veterinary Medicine Timis, oara.

4.1. The GC/MS Compounds Analysis in P. anisum Essential Oil

The analysis of the anise essential oil sample taken in the study was carried out
using the Agilent Technology 7820A gas chromatograph, coupled with the Agilent MSD
5975 mass spectrometer (Agilent Scientific, Santa Clara, CA, USA) and with a DB WAX
capillary column (30 m × 250 pm × 0.25 µm). Helium was used with a mass flow rate of
Plants 2023, 12, 2428 10 of 13

1 mL × min. For the separation of compounds, the following oven program was used: at
40 ◦ C for 1 min, at 5 ◦ C for 1 min, and raised to 210 ◦ C for 5 min.
The temperatures for the injector and ion source were 250 and 150 ◦ C, respectively.
The injection volume was 1 µL of each pure, solvent-free oil or mixture at a ratio of 1:20.
The volatile compounds were identified using the National Institute of Standards
and Technology (NIST) spectrum library. This was conducted by comparing the mass
spectra with the ones stored in the NIST 02, (Wiley 275 libraries). The percentage value of
individual components was calculated based on GC peak areas without making use of any
correction factors.

4.2. Determination of Antimicrobial Activity of P. anisum Essential Oils

The antimicrobial activity was tested in triplicate. Bacterial strain suspensions in
BHI were used as a positive Control. Over each bacterial suspension well, the essential
oil of anise was used directly by adding in increasing amounts as 2, 4, 8, and 10 µL /
well. Plates were covered and left overnight at 37 ◦ C; then, the OD (optical density) was
measured at 590 nm using a Bio-Rad PR 1100 Elisa reader (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA).
The average values of determinations were expressed statistically by one-way ANOVA.
The differences were considered significant when p < 0.05 or lower.

Broth Micro Dilution Method

Among the most basic antimicrobial susceptibility testing methods is the broth mi-
crodilution method. This procedure tests 2-fold dilutions of the antimicrobial agent being
assessed in 96-well micro titer plates filled with a liquid growth medium. Each plate is
inoculated with microbes prepared in the same medium after dilution of the standardized
microbial suspension adapted to the McFarland scale of 0.5.
After inoculation, the 96-well microtiter plate is incubated under appropriate condi-
tions according to the microorganism tested.
The Minimum Inhibitory Concentration (MIC) is the lowest concentration of antimi-
crobial agent that completely inhibits the growth of the organism in the microdilution wells.
The broth microdilution method has been standardized by the Clinical and Laboratory
Standards Institute (CLSI) for testing aerobic bacteria, yeasts, and filamentous fungi. The
European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdi-
lution method is similar to that of CLSI, with modifications typically relating to some of
the test parameters, such as inoculum preparation, inoculum size, and MIC reading. The
reference strains (ATCC) for the in vitro testing used were Staphylococcus aureus (ATCC
25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Streptococcus
pyogenes (ATCC 19615), and Candida albicans (ATCC 10231), provided from the Microbiology
Lab facility, within the ULS “King Mihai I” from Timisoara.
All samples were run in triplicate. A fresh culture dilution of 10−3 with an inoculum
equivalent to a 0.5 McFarland standard was prepared and used. The ATCC strains used
were revitalized in Brain Heart Infusion (BHI) broth (Oxoid, CM1135) (Oxoid Ltd., Bas-
ingstoke, UK) overnight at 37 ◦ C and then plated on BHI agar for 24 h at 37 ◦ C. Dilution
was performed to an optical density (OD) of 0.5 McFarland standard (1.5 × 108 CFU/mL)
using BHI broth.
The resulting suspensions were assayed using a 96-well flat-bottom microdilution
plate with a usable volume of 200 µL. Using a Calibra 852 digital multi-channel pipette
(Socorex Isba S.A. Ecublens, Switzerland), 100 µL of suspension was placed in each well.

4.3. Statistical Analysis

The obtained results were presented as an arithmetic mean and SEM (Standard Error
of the Mean) and were subjected to statistical analysis using one-way ANOVA (analysis of
variance) with Bonferroni’s multiple comparison test. Statistically significant differences
Plants 2023, 12, 2428 11 of 13

were considered when p < 0.05 or lower. The GraphPad Prism 6.0 software (GraphPad
Software, San Diego, USA) was utilized for the analysis.

5. Conclusions
A chromatographic analysis of anise essential oil confirmed the massive presence
of polyphenolic compounds. The antimicrobial activity of Pimpinella anisum essential
oil was mainly related to its phenolic content, with propenyl-anisum, limonene, and
anisole being preponderant. To this aim, anise essential oil had certain antimicrobial
activity against both the Gram-positive and Gram-negative pathogens tested. The highest
percentages of inhibition were found in order on the Pseudomonas aeruginosa (109.84%) and
Staphylococcus aureus (41.21%) strains but also with excellent antifungal activity against
Candida albicans (72.04%).

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/plants12132428/s1, Figure S1: GC-MS analysis - The peaks
of the compounds from Pimpinella anisum essential oil.
Author Contributions: Conceptualization, E.D. and R.T.C.; methodology, E.D. and D.M.O.; software,
F.M. and C.S.D.; validation, E.D., E.T. and R.T.C.; formal analysis, C.S.D. and M.F.; investigation,
E.D.; resources, D.M.O.; data curation, E.D. and E.T.; writing—original draft preparation, C.S.D. and
R.T.C.; writing—review and editing, R.T.C. and M.F.; visualization, F.M. and C.S.D.; supervision,
E.D.; project administration, E.D.; funding acquisition, R.T.C. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by USV “Regele Mihai I” from Timis, oara, Romania, from Project
financing excellence in CDI, code: 6PFE.
Data Availability Statement: All data are included in the present manuscript.
Acknowledgments: This paper is in the frame of COST Action CA18217—European Network for
Optimization of Veterinary Antimicrobial Treatment (ENOVAT).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Saibi, S.; Belhadj, M.; Benyoussef, E.H. Essential oil composition of Pimpinella anisum from Algeria. Ann. Chem. Lett. 2013,
2, 401–404. [CrossRef]
2. Muselin, F. Noţiuni de Biologia Plantelor Pentru uz Veterinar; Mirton Publisher: Timisoara, Romania, 2016; ISBN 978-973-52-1611-5.
(In Romanian)
3. Eugenia, D.; Cristina, R.T. Elemente de Terapie Alternativă şi Complementară în Medicina Veterinară; Solness Publisher: Timisoara,
Romania, 2015; ISBN (13)978-973-729-451-7. (In Romanian)
4. Sayed-Ahmad, B.; Talou, T.; Saad, Z.; Hijazi, A.; Merah, O. The Apiaceae: Ethnomedicinal family as source for industrial uses.
Ind. Crops Prod. 2017, 109, 661–671. [CrossRef]
5. Rebey, I.B.; Wannes, W.A.; BenKaab, S.; Bourgou, S.; Saidani Tounsi, M.; Ksouri, R.; Fauconnier, M.L. Bioactive compounds and
antioxidant activity of Pimpinella anisum L. accessions at different ripening stages. Sci. Hort. 2019, 246, 453–461. [CrossRef]
6. Omidbaigi, R.; Hadjiakhoondi, A.; Saharkhiz, M. Changes in content and chemical composition of Pimpinella anisum L. oil at
various harvest time. J. Essent. Oil Bear. 2003, 6, 46–50. [CrossRef]
7. Orav, A.; Raal, A.; Arak, E. Essential oil composition of Pimpinella anisum L. fruits from various European countries. Nat. Prod.
Res. 2008, 22, 227–232. [CrossRef]
8. Arslan, N.; Gürbüz, B.; Bayrak, A.; Gümüscü, A. Variation in essential oil content and composition in Turkish anise (Pimpinella
anisum L.) populations. Turk. J. Agric. Forest. 2004, 28, 173–177. Available online: https://journals.tubitak.gov.tr/agriculture/vol2
8/iss3/4 (accessed on 27 April 2023).
9. Ozcan, M.M.; Chalchat, J.C. Chemical composition and antifungal effect of anise (Pimpinella anisum L.) fruit oil at ripening stage.
Ann. Microbiol. 2006, 56, 353–358. [CrossRef]
10. Rodrigues, V.M.; Rosa, P.T.V.; Marques, M.O.M.; Petenate, A.J.; Meireles, M.A.A. Supercritical extraction of essential oil from
aniseed (Pimpinella anisum L.) using CO2 : Solubility, kinetics, and composition data. J. Agric. Food Chem. 2003, 51, 1518–1523.
[CrossRef]
11. Tabanca, N.; Demirci, B.; Özek, T.; Kirimer, N.; Baser, K.H.C.; Bedir, E.; Khan, I.K.; Wedge, D.E. Gas chromatographic-mass
spectrometric analysis of essential oils from Pimpinella species gathered from Central and Northern Turkey. J. Chromat A 2006,
1117, 194–205. [CrossRef]
Plants 2023, 12, 2428 12 of 13

12. Tort, N.; Honermeier, B. Investigation on the ratio of methylchavicol and transanethole components in essential oil of anis
(Pimpinella anisum L.) from different regions of Turkey. Asian J. Chem. 2005, 17, 2365–2370. Available online: https://asianpubs.
org/index.php/ajchem/article/view/13980/13954 (accessed on 27 April 2023).
13. Hekmat AL-Hmadi, H.; El Mokni, R.; Joshi, R.K.; Ashour, M.L.; Hammami, S. The Impact of Geographical Location on the
Chemical Compositions of Pimpinella lutea Desf. Growing in Tunisia. Appl. Sci. 2021, 11, 7739. [CrossRef]
14. Koeduka, T.; Baiga, T.J.; Noel, J.P.; Pichersky, E. Biosynthesis of t-anethole in anise: Characterization of t-anol/isoeugenol synthase
and an O-methyltransferase specific for a C7-C8 propenyl side chain. Plant. Physiol. 2009, 149, 384–394. [CrossRef]
15. Lavaee, F.; Moqadas, A.; Modarresi, F.; Nowrouzi, M. The Effect of Pimpinella anisum and Origanum vulgare Extracts Against
Streptococcus sanguinis, Streptococcus mutans, and Streptococcus salivarius. J. Dent. Shiraz 2022, 23, 113–120. [CrossRef]
16. Al-Balawi, A.N.; Elmetwalli, A.; Baraka, D.M.; Alnagar, H.A.; Alamri, E.S.; Hassan, M.G. Chemical Constituents, Antioxidant
Potential, and Antimicrobial Efficacy of Pimpinella anisum Extracts against Multidrug-Resistant Bacteria. Microorganisms 2023,
11, 1024. [CrossRef]
17. Gulcin, I.; Oktay, M.; Kirecci, E.; Kufrevioglu, O.I. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum
L.) seed extracts. Food Chem. 2003, 83, 371–382. [CrossRef]
18. Abdel-Reheem, M.A.T.; Oraby, M.M. Antimicrobial, cytotoxicity, and necrotic ripostes of Pimpinella anisum essential oil. Ann.
Agric. Sci. 2015, 60, 335–340. [CrossRef]
19. Al-Bayati, F.A. Synergistic antibacterial activity between Thymus vulgaris and Pimpinella anisum essential oils and methanol
extracts. J. Ethnopharmacol. 2008, 116, 403–406. [CrossRef]
20. Sukhdev, S.H.; Suman, P.S.K.; Gennaro, L.; Dev, D.R. Extraction Technologies for Medicinal and Aromatic Plants, International
Centre for Science and High Technology. 2004, Italy. Available online: https://www.unido.org/sites/default/files/2009-10/
Extraction_technologies_for_medicinal_and_aromatic_plants_0.pdf (accessed on 27 April 2023).
21. Kosalec, I.; Pepeljnjak, S.; Kuatrak, D. Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L.,
Apiaceae). Acta Pharmaceutica 2005, 55, 377–385.
22. Puvaca, N.; Milenkovic, J.; Galonja Coghill, T.; Bursic, V.; Petrovic, A.; Tanaskovic, S.; Pelic, M.; Ljubojevic-Pelic, D.; Miljkovic, T.
Antimicrobial Activity of Selected Essential Oils against Selected Pathogenic Bacteria: In Vitro Study. Antibiotics 2021, 10, 546.
[CrossRef]
23. Dumitrescu, E.; Muselin, F.; Dumitrescu, C.S.; Orasan-Alic, S.A.; Moruzi, R.F.; Doma, A.O.; Mohamed, A.E.; Cristina, R.T. Juniper
communis L. Essential Oils from Western Romanian Carpathians: Bio-Structure and Effective Antibacterial Activity. Appl. Sci.
2022, 12, 2949. [CrossRef]
24. Pourgholami, M.H.; Majzoob, S.; Javadi, M.; Kamalinejad, M.; Fanaee, G.H.; Sayyah, M. The fruit essential oil of Pimpinella anisum
exerts anticonvulsant effects in mice. J. Ethnopharmacol. 1999, 66, 211–215. [CrossRef] [PubMed]
25. Al-Mofleh, I.A.; Alhalder, A.A.; Mossa, J.S.; Al-Soohalbani, M.O.; Rafatullah, S. Aqueous suspension of anise Pimpinella anisum
protects rats against chemically induced gastric ulcers. World J. Gastroenterol. 2007, 13, 1112–1118. [CrossRef] [PubMed]
26. Iannarelli, R.; Marinelli, O.; Morelli, M.B.; Santoni, G.; Amantini, C.; Nabissi, M.; Maggi, F. Aniseed (Pimpinella anisum L.) essential
oil reduces pro-inflammatory cytokines and stimulates mucus secretion in primary airway bronchial and tracheal epithelial cell
lines. Ind. Crops Prod. 2018, 114, 81–86. [CrossRef]
27. Tas, A.; Özbe, H.; Atasoy, N.; Altug, M.E.; Ceylan, E. Evaluation of analgesic and antiinflammatory activity of Pimpinella anisum
fixed oil extract. Indian. Vet. J. 2006, 83, 840–843. Available online: https://www.researchgate.net/publication/236232426_
Evaluation_of_analgesic_and_antiinflammatory_activity_of_Pimpinella_anisum_fixed_oil_extract (accessed on 27 April 2023).
28. Tas, A. Analgesic effect of Pimpinella anisum L. essential oil extract in mice. Indian. Vet. J. 2009, 86, 145–147. Available
online: https://www.researchgate.net/publication/287854483_Analgesic_effect_of_Pimpinella_anisum_L_essential_oil_extract_
in_mice (accessed on 27 April 2023).
29. Lee, H.S. p-anisaldehyde: Acaricidal component of Pimpinella anisum seed oil against the house dust mites Dermatophagoides
farinae and Dermatophagoides pteronyssinus. Planta Med. 2004, 70, 279–281. [CrossRef]
30. Samaneh, R.H.; Malek, H.A. Anti-proliferative effect of the extracts and essential oil of Pimpinella anisum on gastric cancer cells.
J. Herbmed Pharmacol. 2016, 5, 157–161. Available online: http://herbmedpharmacol.com/Article/JHP_1049_20160707042447
(accessed on 27 April 2023).
31. Kahloula, K.; Slimani, M.; Houari Adli, D.E.; Rachdi, S.; Boumediene, D. Neuro beneficial effects of Pimpinella anisum against lead
exposure. Int. J. Green. Pharm. 2013, 7, 18–24. [CrossRef]
32. Acimovic, G.; Korac, J.; Jacimovic, G.; Oljaca, S.; Djukanovic, L.; Vuga-Janjatov, V. Influence of ecological conditions on seeds
traits and essential oil contents in anise (Pimpinella anisum L.). Not. Bot. Horti Agrobot. Cluj. Napoca 2014, 42, 232–238. [CrossRef]
33. Gende, L.B.; Maggi, M.D.; Fritz, R.; Eguaras, M.J.; Bailac, P.N.; Ponzi, M.I. Antimicrobial activity of Pimpinella anisum and
Foeniculum vulgare essential oils against Paenibacillus larvae. J. Essential Oil Res. 2009, 21, 91–93. [CrossRef]
34. Ullah, H.; Mahmood, A.; Ijaz, M.; Tadesse, B.; Honermeier, B. Evaluation of anise (Pimpinella anisum L.) accessions with regard to
morphological characteristics, fruit yield, oil contents and composition. J. Med. Plants Res. 2013, 7, 2177–2186. [CrossRef]
35. Hasimi, A.; Tolan, V.; Kizil, S.; Kilinc, E. Determination of essential oil composition, antimicrobial and antioxidant properties of
anise (Pimpinella anisum L.) and cumin (Cuminum cyminum L.) seeds. J. Agric. Sci. 2014, 20, 19–26. [CrossRef]
36. Mohammed, S.; Albulushi, A.; Al Saidi, H.; Amaresh, N.; Mullaicharam, A.R. Study of physicochemical properties, antibacterial
and GC-MS analysis of essential oil of the aniseed (Pimpinella anisum Linn.) in Oman. J. Pharmacogn. Phytochem. 2014, 2, 24–33.
Plants 2023, 12, 2428 13 of 13

37. Faleiro, M.L. The mode of antibacterial action of essential oils. In Science against Microbial Pathogens: Communicating Current Research
and Technological Advances; Méndez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2011; pp. 1143–1156. Available online: https://
dsagrow.com/wp-content/uploads/2020/05/The-mode-of-antibacterial-action-of-essential-oils.pdf (accessed on 27 April 2023).
38. Burt, S. Essential oils: Their antibacterial properties and potential applications in foods—A review. J. Food Microbiol. 2004,
94, 223–253. [CrossRef]
39. Shukla, H.S.; Tripathi, S.C. Antifungal substance in the essential oil of anise (Pimpinella anisum L.). Agric. Biol. Chem. 1987,
51, 1991–1993. Available online: https://www.jstage.jst.go.jp/article/bbb1961/51/7/51_7_1991/_pdf (accessed on 27 April
2023). [CrossRef]
40. Lis-Balchin, M.; Deans, S.G.; Eaglesham, E. Relationship between bioactivity and chemical composition of commercial essential
oils. Flav Frag. J. 1998, 13, 98–104. [CrossRef]
41. Dorman, H.J.D.; Deans, S.G. Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. J. Appl. Microbiol. 2000,
88, 308–316. [CrossRef]
42. Shojaii, A.; Abdollahi Fard, M. Review of Pharmacological Properties and Chemical Constituents of Pimpinella anisum. Int. Sch.
Res. Not. 2012, 2012, 510795. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.