Antifungal activity of phenolic monoterpenes and structure-related compounds against plant pathogenic fungi

Article abstract of DOI:10.1080/14786419.2017.1419232

The aim of this work is to explore the possibility of using the phenolic monoterpenes (PMs) as leading compounds with antifungal activity against plant disease. The in vitro antifungal activities of carvacrol and thymol against seven kinds of plant pathogenic fungi were evaluated on mycelium growth rate method, and the results showed that carvacrol and thymol exhibited broad spectrum antifungal activity. Structure requirement for the antifungal activity of PMs was also investigated. The preliminary conclusion was that phenolic hydroxyl and monoterpene were basic structures for the antifungal activity of PMs, and the position of phenolic hydroxyl showed less effect. Ester derivatives of carvacrol and thymol were more effective than carvacrol and thymol against plant pathogenic fungi. We suggested that carvacrol, thymol and their ester derivatives could potentially be used as new fungicide leading compounds.

Full text of DOI:10.1080/14786419.2017.1419232

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antifungal activity of phenolic monoterpenes
and structure-related compounds against plant
pathogenic fungi
Kaibo Wang, Shanshan Jiang, Te Pu, Liming Fan, Fawu Su & Min Ye
To cite this article: Kaibo Wang, Shanshan Jiang, Te Pu, Liming Fan, Fawu Su & Min Ye (2018):
Antifungal activity of phenolic monoterpenes and structure-related compounds against plant
pathogenic fungi, Natural Product Research, DOI: 10.1080/14786419.2017.1419232
To link to this article: https://doi.org/10.1080/14786419.2017.1419232
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2017.1419232
Antifungal activity of phenolic monoterpenes and structure-
related compounds against plant pathogenic fungi
Kaibo Wang1, Shanshan Jiang1, Te Pu, Liming Fan, Fawu Su and Min Ye
state Key laboratory for conservation and utilization of Bio-resources in Yunnan, Yunnan agricultural
university, Kunming, china
ABSTRACT
ARTICLE HISTORY
received 10 october 2017
accepted 12 december 2017
The aim of this work is to explore the possibility of using the phenolic
monoterpenes (PMs) as leading compounds with antifungal activity
against plant disease. The in vitro antifungal activities of carvacrol and
thymol against seven kinds of plant pathogenic fungi were evaluated
on mycelium growth rate method, and the results showed that
carvacrol and thymol exhibited broad spectrum antifungal activity.
Structure requirement for the antifungal activity of PMs was also
investigated. The preliminary conclusion was that phenolic hydroxyl
and monoterpene were basic structures for the antifungal activity
of PMs, and the position of phenolic hydroxyl showed less effect.
Ester derivatives of carvacrol and thymol were more effective than
carvacrol and thymol against plant pathogenic fungi. We suggested
that carvacrol, thymol and their ester derivatives could potentially be
used as new fungicide leading compounds.
KEYWORDS
carvacrol; thymol;
Phenolic monoterpenes;
antifungal activity; structure
requirement
1. Introduction
The two phenolic monoterpenes (PMs), Carvacrol (2-methyl-5-(1-methylethyl)-phenol) and
thymol (5-methyl-2-(1-methylethyl)-phenol), are widely found in aromatic plants such as
genus Origanum (Stefanakis et al. 2013), Thymus (Porte and Godoy 2008), Lippia
CONTACT Fawu su
su_faw@126.com; Min Ye
yeminpc@126.com
1these authors contributed equally to this work.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1419232.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
K. WANG ET AL.
(Cervantesmartínez 2014) and Satureja (Yousefzadi et al. 2012; Wesolowska et al. 2015).
PMs and the PMs-rich essential oils are generally considered to have good biological activ-
ities, such as antibacterial, antioxidant, antifungal, insecticidal and acaricidal activity
(Friedman 2014; Suntres et al. 2015). The antimicrobial activity can potentially be applied to
keep food fresh (Rattanachaikunsopon and Phumkhachorn 2010; ), store fruits (Pérez-Alfonso
et al. 2012) and inhibit fungal infection (Taha and Azeiz 2010). The PMs are generally pro-
duced as a chemical defence mechanism against phytopathogenic microorganisms and
used as potential antifungal active ingredients against plant pathogen fungi (Vázquez et al.
2001; Soković et al. 2002; Numpaque et al. 2011).
It is reported that, the action mechanism of the antifungal effect of PMs may cause by
impairing biosynthesis of ergosterin and disrupting membrane integrity of fungi (Ahmad
et al. 2011), which are essential process to the vital activity of most pathogenic fungi.
Therefore, it attracted our interests to study the inhibitory effect of PMs against plant
pathogenic fungi in agriculture, and the antifungal activities of carvacrol and thymol were
evaluated by mycelium growth rate method against seven species of fungi which cause
widely damage to crops. In previous studies, the phenolic hydroxyl group of PMs is con-
sidered to be essential for their antibacterial activity (Ultee et al. 2002; Ben et al. 2006;
Veldhuizen et al. 2006). Because of the different physiological regulatory mechanisms, the
agent may show different effect and action mechanism against different microorganisms.
Aim to confirm the structure requirement for the antifungal activity of PMs, we investigated
the influence of removing or changing the substituting group of PMs on their antifungal
activity.
In the present work, the hyphae growth inhibition ratios of PMs and their structure-related
compounds (Figure 1) had been evaluated by mycelium growth rate method. Essential struc
-
ture for the antifungal activity of PMs was preliminarily investigated by comparing the
hyphae growth inhibition ratios of target compounds against each target pathogen,
respectively.
Figure 1. chemical structures of PMs and structure-related compounds used in this study.

NATURAL PRODUCT RESEARCH
3
2. Results and discussion
2.1. Chemistry
The reaction route for synthesising ether and ester derivatives of PMs (compound 1a, 1b,
2a and 2b) is outlined in Figure 2. The preparations of 1a-1b and 2a-2b were performed
according to the reported methods, respectively (Narkhede et al. 2008; Mathela et al. 2010).
All the synthesised compounds were identified on the basis of 1H-NMR,13C-NMR and
HR-ESI-MS analyses.
2.2. Antifungal activity
The hyphae growth inhibition ratios of carvacrol and thymol against seven kinds of fungi
are listed in Table 1. The two PMs, carvacrol and thymol, played a similar role in antifungal
activity against target pathogens. The inhibition ratios of the two PMs against target path-
ogens at high concentration were significantly higher than that at low concentration. And
just as predicted, the two PMs showed broad antifungal spectrum, they had various degrees
of inhibition effect against both Oomycetes (Phytophthora capsici and Phytophthora nicoti-
anae) and Hyphomycetes (Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Pyricularia
grisea, and Rhizoctonia solani). And, the two PMs showed rather good antifungal activity
against B. cinerea and R. solani, the inhibition ratio of carvacrol was higher than 90% against
B. cinerea at 50 μg mL−1, which was equal to the commercial fungicide chlorothaloni.
The inhibition ratios of PMs structure-related compounds (p-cymene, o-cresol, m-isopro-
pylphenol, 2,5-xylenol) and four synthesised compounds against five kinds of Hyphomycetes
are listed in Table 2. The compounds without methyl or isopropyl groups (o-cresol, m-iso-
propylphenol and 2,5-xylenol) showed lower antifungal activity than carvacrol and thymol.
P-cymene, a monoterpene without hydroxyl group, was also lower effective than carvacrol
and thymol. The antifungal activity of PMs had been changed by introducing substituent
groups to their phenolic hydroxyl groups. As generally observed, ester derivatives (com-
pound 1a and 1b) showed higher inhibition effect against target pathogens, and ether
derivatives (compound 2a and 2b) showed negative effects against target pathogens. The
hyphae growth inhibition ratios of compound 1a and 1b against B. cinerea and R. solani were
higher than 90% at 50 μg mL−1, which were equal to or better than the commercial fungicide
chlorothaloni.
Figure 2. synthesis of carvacrol and thymol ether and ester derivatives.

4
K. WANG ET AL.

NATURAL PRODUCT RESEARCH
5
Table 2. antifungal activity of PMs structure-related compounds against five kinds of hyphomycetes.
Hyphae growth inhibition ratios (%) (n = 3)
Concentration
(μg∙mL−1
)
A. solani
B. cinerea
F. oxysporum
P. grisea
R. solani
Compounds
p-cymene
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
9.68 0.94
10.22 3.85
0.25 1.76
0.51 2.24
−0.38 0.38
28.70 1.51
0.63 2.72
1.15 0.38
15.20 4.81
8.12 7.69
11.29 9.62
10.99 1.23
17.26 3.08
18.12 3.32
88.48 0.75
39.51 8.15
39.86 4.53
42.63 10.94
12.08 1.73
19.93 9.97
12.89 0.67
9.24 5.79
12.74 3.40
0.53 2.75
0.46 3.99
4.78 5.48
o-cresol
15.36 5.47
73.99 1.03
19.16 8.24
76.24 1.33
52.41 4.90
73.13 2.25
44.10 12.23
27.17 13.04 12.18 0.08
m-isopropyl-
phenol
2,5-xylenol
0.71 4.47
36.28 2.42
2.86 0.01
6.44 2.14
10.37 1.01
41.60 4.25
1.51 1.94
35.54 6.25
0.64 2.27
3.53 0.73
7.77 4.57
12.43 5.42
45.13 2.49
50.63 4.26
3.25 3.62
5.01 0.45
5.54 1.99
26.81 4.37
24.94 0.99
58.10 3.57 96.30 3.57
5.45 3.59 37.77 12.00
48.83 3.26 96.65 3.07
0.37 1.87
2.87 2.49
0.95 1.71
1.83 0.61
1a
67.88 8.13 100.00 0.00
1b
12.14 4.47
62.22 9.87
3.14 4.07
3.78 2.72
4.38 2.84
3.79 1.23
57.89 0.87
72.44 2.97
46.15 1.33
99.63 0.64
3.00 0.35
22.17 6.06
2.17 1.25
2.35 1.54
60.77 1.77
76.65 1.67
2a
3.98 2.08
20.30 2.60
2.38 4.76
15.33 4.06
2b
chlorothalonil*
50.53 1.16 95.14 0.80
78.90 0.86 97.22 1.35
Note: the compound which showed higher inhibition ratio were highlighted in bold.
*chlorothalonil were used as the positive controls.
The in vitro antifungal activities of PMs and their ester derivatives were equal to or better
than the commercial fungicide chlorothalonil against B. cinerea and R. solani. We suggested
that carvacrol, thymol and their ester derivatives could potentially be used as new fungicide
leading compounds. The earliest idea of choosing both Oomycetes and Hyphomycetes as
target fungi was to make a more reliable conclusion for the antifungal spectrum of PMs.
Instead, the two PMs showed antifungal effect against two kinds of Oomycetes. As is well
known, ergosterol biosynthesis is absent in physiological progress of Oomycetes. The results
are conflicted with the conclusion in the study of PMs against several resistant Candida
strains (Ahmad et al. 2011). Past studies have also explained the mechanisms of PMs inhibit
fungi growth by studying their effect onTOR (Target of Rapamycin) pathway (Rao et al. 2010).
Additional antifungal assay and physiological experiment will be considered to investigate
the specific mechanism of PMs on fungi growth in our further study.
In this study, we determined phenolic hydroxyl and alkyl group of PMs were essential for
their antifungal activity and the position of phenolic hydroxyl on benzene ring showed less
effect. Previous studies suggested that hydroxyl and alkyl group of PMs provide appropriate
hydrophobicity, and the hydroxyl group is essential for their hydrogen bonding and pro-
ton-release ability which are important for their ability to cause the cell death (Ultee et al.
2002; Ben et al. 2006), and it is reported that thymol ester derivatives show higher activity
in comparison to thymol, whereas the carvacrol ester derivatives are much less active than
carvacrol (Mathela et al. 2010). However, the antifungal assay results were different in our
study, ester derivatives of PMs without a free hydroxyl group were even more effective than
their parent molecule. While we could come to just a preliminary conclusion according to
limited compounds, more derivatives of PMs should be synthesised to make a completely
structure–activity relationships study.

6
K. WANG ET AL.
3. Experimental section
3.1. Antifungal assays
The in vitro antifungal activities of target compounds were determined by the mycelium
growth rate method according to the methods described in paper (Kim et al. 2003). The
panel of microorganisms included five kinds of Hyphomycetes (A. solani, B. cinerea, F.
oxysporum, P. grisea, and R. solani) and two kinds of Oomycetes (P. capsici and P. nicotianae).
These strains were separately maintained on Potato Dextrose Agar (PDA) or Carrot Decoction
Agar (CA) slants at −10 °C. All of the strains used for antifungal activity assays were incubated
on Petri dishes containing the PDA medium in advance. Each test compounds were dissolved
in acetone, and the result solution was added to PDA flat-medium (1/100, Vacetone/VPDA) at
40~60 °C, and then cooled to room temperature to prepare the poisoned medium. At the
same time, medium supplemented with equivalent quantities of acetone were set up as
control, and chlorothalonil was used as a positive control group. Afterwards, a mycelium
agar disc (5 mm diameter) of the tested strains were inoculated to poisoned and non-poi-
soned medium, and all Petri dishes were incubated at constant temperature of 25~26 °C in
dark, each compound was repeated three times.
Percentage mycelial inhibitionn = [(C − T)∕C] × 100
Where C is the mean colony diameter for the control sets and T is the mean colony diameter
for the treatment sets. The data from each part were analysed using percentage, mean,
standard deviation (n = 3) statistical methods based on the IBM SPSS statistics 22.0
software.
3.2. Chemistry
The reagents (carvacrol, thymol, p-cymene, o-cresol, m-isopropylphenol, 2,5-xylenol) and
solvents for reaction were purchased from Aladdin (China) or Sinopharm Chemical Reagent
Co., Ltd (China). Solvents for extraction and chromatography were of technical grade and
distilled prior to use. Reactions were monitored by thin layer chromatography (TLC) using
silica gel coated glass slides (silica gel 60 GF 254, Qingdao Haiyang Chemical, China).
Detections were conducted under UV (254 nm). 1H- and 13C-NMR spectra were recorded on
a Bruker Avance III 500 NMR spectrometer. The chemical shifts (δ) were reported in ppm
with reference to internal TMS, and coupling constants (J) were given in Hz. ESI-MS spectra
were recorded on a Thermo Fisher Scientific TSQ Endura MS.
3.2.1. Synthesis of 5-isopropyl-2-methylphenyl acetate (1a)
Acetyl chloride (86.35 mg, 1.1 mmol, 1.1 equiv) in 1.1 mL THF was added to a solution of
carvacrol (150 mg, 1 mmol, 1.0 equiv) and triethylamine (111 mg, 1.1 mmol, 1.1 equiv) in
anhydrous THF (1 mL) over 1 min at 0 °C. The reaction mixture was stirred at 0 °C for 15 min
and then at room temperature for 3 h. After the reaction was completed, 10 mL of water was
added to the reaction mixture, and extracted with EtOAC (2 × 25 mL). The organic layer was
washed with water, and dried over anhydrous sodium sulphate. The solvent was evaporated
in vacuo, and the crude product was purified by silica gel colunm chromatography (Petroleum
ether : EtOAC = 10:1) to give compound 1a in 86% yield.

NATURAL PRODUCT RESEARCH
7
3.2.2. Synthesis of 2-ethoxy-4-isopropyl-1-methylbenzene (2a)
To a solution of carvacrol (150 mg, 1 mmol, 1.0 equiv) in DMF (2 mL) potassium carbonate
(207 mg, 1.5 mmol, 1.5 equiv) and bromoethane (130 mg, 1.2 mmol, 1.2 equiv) were added.
The reaction mixture was stirred at room temperature for 10 h. The progress of reaction was
monitored by TLC. After completion, 10 mL of water was added to the reaction mixture, and
extracted with EtOAC (3 × 25 mL). The organic layer was washed with water, brine and dried
over anhydrous sodium sulphate. The solvent was evaporated in vacuo, and the crude prod-
uct was purified by silica gel colunm chromatography (Petroleum ether : EtOAC = 10:1) to
give compound 2a in 90% yield.
Ester and ether derivatives of thymol (Compound 1b and 2b) were prepared as described
for compound 1a and 2a, with yields of 93% and 88%, respectively.
4. Conclusions
The antifungal activities of PMs and their structure-related compounds against several plant
pathogenic fungi were tested by mycelium growth rate method. We suggested that carvacrol,
thymol and their ester derivatives could potentially be used as new fungicide leading com-
pounds. We determined hydroxyl and alkyl group of PMs were essential for their antifungal
activity. It was important for synthetic research work of PMs derivatives to get higher anti-
fungal activity compounds.
Supplementary material
Spectral data relating to this article can be found in the online versions as Appendix.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by Project of the National Natural Science Foundation of China (grant
number 31460487) and Project of the Yunnan Provincial Natural Science Foundation of China (grant
number 2016FB065).
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