Food Bioscience 25 (2018) 28–31

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Chemical composition and antifungal activity of Anacyclus valentinus T

essential oil from Algeria
⁎
Abderrahmane Houichera, , Mahfoud Hamdib, Hind Hechachnab, Fatih Özogulc
a
Department of Agriculture, Faculty of Science, Laghouat University, BP 37 G, Ghardaïa Road, 03000 Laghouat, Algeria
b
Laboratory of Phytoprotection, Department of Agriculture, Faculty of Science, Laghouat University, BP 37 G, 03000 Laghouat, Algeria
c
Department of Fishing and Fish Processing Technology, Faculty of Fisheries, Cukurova University, 01330 Balcali, Adana, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: The chemical composition of Anacyclus valentinus essential oil from Algeria was determined and its antifungal
Anacyclus valentinus effect in vitro toward 7 toxigenic fungal strains was evaluated. Essential oil yield of 0.13% (v/w) was obtained
δ-3-carene from the aerial parts of A. valentinus using hydrodistillation. The GC/MS analysis identified 29 components of
Spathulenol which δ-3-carene (31%), spathulenol (14.2%), decanoic acid (5.5%), δ-cadinene (4.4%), anethole (3.4%) and
Fusarium graminearum
aromadendrene (3.3%) were the major compounds of this oil. The results of the antifungal activity showed that
Aspergillus parasiticus
the most sensitive fungal strains to A. valentinus oil were Fusarium graminearum, Aspergillus parasiticus, and
Penicillium expansum with lower minimal fungicidal concentration (MFC) of 1.25 μl/ml (v/v). The oil also had a
strong fungicidal effect against A. flavus and F. moniliforme at a MFC value of 2.5 μl/ml, while the oil con-
centration of 10 μl/ml was needed to show a fungicidal activity against A. ochraceus and P. citrinum. This study
suggested that A. valentinus oil is a potential candidate to be used as a safe biocontrol agent to prevent food crops
from fungal diseases and improve product quality and safety.

1. Introduction
The Anacyclus is a Mediterranean genus of mostly annual herbs,
belongs to the Asteraceae family, comprising about 12 species dis-
tributed mainly in the North West of Africa, but it is also found in
Southern Europe and the Middle East (Bremer & Humphries, 1993).
Among them, Anacyclus valentinus, which is an annual specie found in
different regions of Algeria, is commonly known as “ghertoufa” or va-
lence anacycle (Side Larbi, Meddah, Tir Touil Meddah, & Sonnet,
2016). In Algeria, A. valentinus has been traditionally used in folk
medicine (Selles et al., 2013) and as a food condiment in some parts of
the country (Side Larbi et al., 2016). It is a rich source of terpenoids and
flavonoids (Greger, 1978; Selles et al., 2013) and may have useful
biological activities.
Mycotoxin-producing fungi, especially within Aspergillus, Penicillium
and Fusarium species, are significant destroyers and contaminants of
agricultural products and seeds in the field, and during processing and
storage lead to significant economic losses and have serious health
hazards for humans and animals (Anjorin et al., 2013; Jimoh & Kolapo,
2008). Moreover, chemical control of several crop diseases mainly in-
volves the use of synthetic fungicides; however, the increasing concern
of authorities and consumers about the possible effects of toxic residues
on human health and the environment is leading to more strict control
on the use of these hazardous chemicals (Anjorin, Salako, & Makun,
2013; Marin, Ramos, & Sanchis, 2008). Thus, different strategies have
been proposed as alternatives to synthetic fungicides, among them the
biological control using essential oils from plants and other natural
products from bacteria and fungi which could be an alternative and
complement to existing methods of control (Fallik, 2008).
Essential oils or some of their components are largely used for their
antibacterial, antifungal and insecticidal activities, without showing the
same secondary effects as synthetic chemicals (Bakkali, Averbeck,
Averbeck, & Idaomar, 2008). Several essential oils of different Anacy-
clus species have been studied for their biological activities in vitro;
however, there are few reports on the antimicrobial activities of A.
valentinus essential oils, without characterizing their chemical compo-
sition. Therefore, the present study was carried out to determine the
chemical composition of A. valentinus essential oil from Algeria and
evaluate its antifungal effect in vitro toward 7 toxigenic fungal strains
belonging to Fusarium, Penicillium, and Aspergillus species.

⁎
Corresponding author.
E-mail address: a.houicher@yahoo.fr (A. Houicher).

https://doi.org/10.1016/j.fbio.2018.07.005
Received 27 November 2017; Received in revised form 11 July 2018; Accepted 11 July 2018
2212-4292/ © 2018 Elsevier Ltd. All rights reserved.
A. Houicher et al. Food Bioscience 25 (2018) 28–31

2. Materials and methods Table 1

Composition of Anacyclus valentinus essential oil characterized using GC/MS.
2.1. Plant material No. Components Retention % Composition Identificationb
time (min)a Anacyclus
Anacyclus valentinus samples (aerial parts) were collected from the valentinus
region of Hassi R’mel, Laghouat, Algeria during the flowering stage
1 δ-3-Carene 11.81 31.0 MS
from April to June, 2015. After transportation to the laboratory, the 2 Limonene 14.86 1.7 MS
samples were air dried at room temperature (< 30 °C) in the dark and 3 trans-Ocimene 16.72 1.9 MS
stored in paper bags until use, a maximum of two months. The plant 4 allo-ocimene 19.98 1.3 MS
was then confirmed by the botanists of the Department of Agriculture, 5 Naphthalene 21.64 0.8 MS
6 Estragole 22.25 1.4 MS
Laghouat University, Laghouat, Algeria.
7 1-Methyldodecylamine 22.59 0.6 MS
8 Adrenalone 23.32 0.2 MS
2.2. Analysis of essential oil using GC/MS 9 Cuminaldehyde 23.61 2.5 MS
10 Carvone 23.70 2.7 MS
11 Anethole 24.93 3.4 MS
To obtain essential oil, the dried samples were subjected to hydro-
12 Thymol 25.47 2.1 MS
distillation for approximately 4 h using a Clavenger-type apparatus 13 Decanoic acid 28.15 5.5 MS
(Schott Duran, Mainz, Germany), using the British Pharmacopoeia 14 Geranyl acetate 28.30 1.9 MS
method (British Pharmacopoeia, 1990). A Perkin Elmer Clarus instru- 15 Aromadendrene 28.56 3.3 MS
ment model 500 GC/MS (Yokohama, Japan), equipped with a SGE 16 α-Curcumene 30.16 2.3 MS
17 Acenaphthylene 30.60 2.9 MS
capillary column (60 m × 0.25 mm ID, BPX5, film thickness 0.25 µm,
18 β-Patchoulene 31.12 0.7 MS
Austin, TX, USA), was used to determine the chemical composition of 19 δ-Cadinene 32.62 4.4 MS
the oil obtained. The GC/MS analysis was carried out with the following 20 Spathulenol 34.11 14.2 MS
conditions: flow rate of carrier gas (helium, purity 99.95%) 1.5 ml/min; 21 Agarospirol 34.41 1.2 MS
splitless injection mode with ionization voltage 70 ev; mass spectra 22 Cis-α-Copaene-8-Ol 34.57 1.7 MS
23 α-Bisabolol 35.09 1.5 MS
range of 35–425 m/z; column oven temperature programmed at 24 Zizanyl Acetate 35.40 1.0 MS
60–250 °C (4 °C/min), maintained at 250 °C (10 min), and injector 25 β-Ionone 35.84 1.0 MS
temperature 240 °C. The injection volume of diluted sample (1% n- 26 Trimethyl-decalin 39.99 0.9 MS
hexane, v/v) was 1 μl. The oil components were identified by com- 27 Nerolidol 42.07 0.4 MS
28 Eicosane 47.68 0.5 MS
parison of their mass spectra with those from NIST and Wiley libraries
29 Tricosane 54.06 0.3 MS
(http://chemdata.nist.gov and https://www.wiley.com) or reported in Total identified 93.3
the literature (Adams, 2007). The relative percentage of the oil con- Monoterpenes 35.9
stituents were calculated from the peak area of the chromatogram using hydrocarbons
the software provided by the manufacturer and assuming peak areas for Oxygenated 14.0
monoterpenes
different compounds were all proportional to their molecular con- Sesquiterpenes 10.7
centration. hydrocarbons
Oxygenated 19.0
2.3. Antifungal activity sesquiterpenes
Others 13.7
Oil yield (%) 0.13
The antifungal susceptibility testing of A. valentinus essential oil was
done using a broth macrodilution method M38-A (CLSI Clinical and a
Retention time (min) in elution order from the SGE column.
b
Laboratory Standards Institute, 2002) with 7 toxigenic fungal strains. MS: NIST and Wiley libraries, and the literature.
One type strain was from the Central Bureau of Fungal Cultures (CBS)
culture collections of micro-organisms (Utrecht, The Netherlands) (As- Madrid, Spain) plates, and the minimal fungicidal concentrations (MFC)
pergillus parasiticus CBS 100926), and two type strains were from the were evaluated after 72 h incubation at 35 °C.
Agricultural Research Service (ARS) culture collection (United States
Department of Agriculture, Washington, DC, USA) (Aspergillus flavus
NRRL 3251, and Aspergillus ochraceus NRRL 3174). Four type strains 3. Results and discussion
were from the Belgian Coordinated Collections of Micro-organisms
(Catholic University of Leuven, Belgium) (Fusarium moniliforme MUCL An essential oil yield of 0.13% (v/w) was obtained from the aerial
53645, Fusarium graminearum MUCL 53452, Penicillium expansum parts of Anacyclus valentinus using hydrodistillation (Table 1). Several
MUCL 29192, and Penicillium citrinum MUCL 31475). In all experi- studies have extensively analyzed the essential oils of different species
ments, RPMI-1640 medium (Roswell Park Memorial Institute medium, of Anacyclus. However, little information is available on the chemical
Sigma R6504, St. Louis, MO, USA) adjusted to pH 7.0 with MOPS characterization of the essential oils obtained from different A. va-
(morpholinopropane-1-sulfonic acid, Sigma M3183) buffer was used. lentinus. In one study (Side Larbi et al., 2016), a yield of 0.63% was
Briefly, serial doubling dilutions from 0.04 to 20 μl/ml were prepared in obtained from the aerial parts of A. valentinus grown in Algeria, which is
DMSO (dimethyl sulfoxide, Sigma 34943), and the final concentration noticeably higher than that obtained from the current samples. Table 1
of the DMSO was ≤ 1%. For each fungal strain, inoculum was prepared also shows the qualitative and quantitative compositions of A. va-
from 7 to 14 day cultures, adjusted to the density of a 0.5 McFarland lentinus oil. The GC/MS analysis identified 29 components, corre-
standard at 530 nm wavelength using a spectrophotometer (Jenway, sponding to more than 93.3% of the total sample. δ-3-Carene, spathu-
6405 UV/VIS, Essex, UK) and diluted 1:100 to obtain a working sus- lenol, decanoic acid, δ-cadinene, anethole, and aromadendrene were
pension within the range of 0.4–5 × 104 CFU (colony-forming units)/ the major compounds of A. valentinus oil. Other constituents were also
ml. Then, the test tubes were inoculated in triplicate, including two identified in this oil with minor concentrations. Side Larbi (2016) re-
control tubes (sterility and growth) per strain. After incubation at 35 °C ported that the main components of A. valentinus grown in Algeria were
for 48 h/72 h, the minimal inhibitory concentrations (MIC) were de- germacrene (15.5%), trans-chrysanthenyl acetate (12.4%), trans-thu-
termined visually. From each negative tube, 20 μl aliquots were then jone (7.3%), eudesma-4(15),7-diene-1-β-ol (6.2%), and β-biotol (5.1%).
transferred onto Sabouraud dextrose agar (Eur-Pharm, 1024.00, The composition of essential oils depends on several factors including

29
A. Houicher et al.
Table 2
Antifungal activity of Anacyclus valentinus essential oil determined using a broth
macrodilution method.
Strains Anacyclus valentinus
MIC (μl/ml) MFC (μl/ml)
Fusarium graminearum MUCL 53452 1.25 1.25
Fusarium moniliforme MUCL 53645 2.5 2.5
Aspergillus parasiticus CBS 100926 1.25 1.25
Aspergillus ochraceus NRRL 3174 2.5 10
Aspergillus flavus NRRL 3251 2.5 2.5
Penicillium expansum MUCL 29192 1.25 1.25
Penicillium citrinum MUCL 31475 5 10
climate, plant part used, place of growth and harvesting time, natural
variation, storage conditions, and analysis parameters in compound
identification (Pauli & Schilcher, 2009).
Table 2 shows the results of the antifungal activity of A. valentinus
essential oil. The most sensitive fungal strains to A. valentinus essential
oil were F. graminearum, A. parasiticus, and P. expansum with MIC and
MFC values of 1.25 μl/ml (v/v). A potent inhibitory effect was observed
with this oil against F. moniliforme, A. ochraceus, and A. flavus, whereas
P. citrinum was effectively inhibited at MIC value of 5 μl/ml (v/v). The
oil also had a strong fungicidal effect against A. flavus and F. mon-
iliforme, while the oil concentration of 10 μl/ml (v/v) was sufficient to
show a fungicidal activity against A. ochraceus and P. citrinum. In the
literature, there is only one study (Si Moussa, Belabid, Tadjeddine,
Bellahcene, & Bayaa, 2010) that investigated the antifungal effect of A.
valentinus essential oil against phytopathogenic fungal species. The
authors reported that A. valentinus essential oil at the concentration of
1000 ppm (µg/ml) totally inhibited microconidia germination and the
mycelial growth of Fusarium oxysporum f. sp. albedinis. Essential oils and
extracts of other Anacyclus sp. have been reported to also show variable
degrees of antimicrobial activities against different Candida species and
bacteria (Hammami et al., 2013; Selles et al., 2013; Zardi-Bergaoui
et al., 2008), but no report was found about their effects toward plant
pathogenic fungi. In fact, the fungicidal effects of essential oils depend
on several factors, mainly the major constituents of the oil and the
target microorganisms. The most abundant groups of compounds in
essential oils are terpenes which are known to be active against a wide
range of microorganisms, including fungi and yeast. In this study, A.
valentinus oil is mainly composed of δ-3-carene (31%) and spathulenol
(14.2%) and its antifungal activity could be related to the pre-
dominance of these compounds. δ-3-Carene was found to be the most
active constituent against Aspergillus, Candida and dermatophyte fungi
(Cavaleiro, Pinto, Gonçalves, & Salgueiro, 2006). Cosentino et al.
(2003) reported that δ-3-carene was the potent compound that had a
strong activity against a wide range of foodborne pathogens and spoi-
lage microorganisms. Several authors (Fontenelle et al., 2008;
Ngassapa, Runyoro, Vagionas, Graikou, & Chinou, 2016; Wenqiang,
Shufen, Ruixiang, & Yanfeng, 2006) have also shown the antimicrobial
efficacy of essential oils of different plants in which spathulenol is the
main compound of these oils. In addition, the other major compounds
of the oil such as decanoic acid, δ-cadinene, anethole, and aromaden-
drene are known to have antifungal activities (Cheng, Lin, & Chang,
2005; Karapinar, 1990; Mulyaningsiha, Sporera, Zimmermann,
Reichlinga, & Wink, 2010; Sjogren, Magnusson, Broberg, Schnurer, &
Kenne, 2003). However, minor constituents may also contribute to this
activity taking into account the possible synergistic or potentiating ef-
fects between oil compounds. In general, essential oil constituents have
different modes of antifungal activity from one fungal strain to another
(Bakkali et al., 2008). The essential oils have the ability to penetrate
and disrupt the fungal cell wall and cytoplasmic membranes through a
permeabilization process, which results in the malformation of im-
portant structures and leads to cell death (Swamy, Akhtar, & Sinniah,
30
Food Bioscience 25 (2018) 28–31
2016). All these observations may explain the effectiveness of A. va-
lentinus oil against the fungi tested.
4. Conclusions
While the essential oil of A. valentinus was obtained at low yield, it
showed strong fungicidal activity against all fungi tested which could
be related to the predominance of δ-3-carene and spathulenol in this oil.
Considering that essential oils are generally recognized as safe for
human health and the environment, the present findings suggested that
A. valentinus oil is a potential candidate to be used as a safe biocontrol
agent to prevent food crops from fungal diseases and improve product
quality and safety. However, the antifungal efficiency of essential oils in
combination needs to be determined with further studies, together with
toxicological and regulatory investigations of these natural products
prior to their application as biocontrol agents.
Acknowledgements
The authors wish to thank Dr. Mohamed Kouidri and Dr. Tayeb
Berramdane (Laghouat University) for the botanical identification of
the plant material used in this study.
Conflict of interest
The authors declare that there are no conflicts of interest in this
research study.
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