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Composition and Antifungal Activity of Peppermint


(Mentha piperita) Essential Oil from Iran
a

Mohammad Moghaddam , Maryam Pourbaige , Heydar Kourosh Tabar , Nasrin Farhadi &
Seyed Mohammad Ahmadi Hosseini

Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad,


Mashhad, Iran
b

Department of Plant Diseases, Faculty of Agriculture, Tarbiat Modares University, Tehran,


Iran
c

Chemistry and Chemical Engineering Research Center of Iran, 657, Soheil Shiraz, Vanak,
Tehran, Iran
d

Department of Horticulture, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

Department of Horticulture, Faculty of Agriculture, Tarbiat Modares University, Tehran,


Iran
Published online: 25 Oct 2013.

To cite this article: Mohammad Moghaddam, Maryam Pourbaige, Heydar Kourosh Tabar, Nasrin Farhadi & Seyed Mohammad
Ahmadi Hosseini (2013) Composition and Antifungal Activity of Peppermint (Mentha piperita) Essential Oil from Iran, Journal
of Essential Oil Bearing Plants, 16:4, 506-512, DOI: 10.1080/0972060X.2013.813265
To link to this article: http://dx.doi.org/10.1080/0972060X.2013.813265

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TEOP 16 (4) 2013 pp 506 - 512

506
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Composition and Antifungal Activity of Peppermint


(Mentha piperita) Essential Oil from Iran
Mohammad Moghaddam 1*, Maryam Pourbaige 2, Heydar Kourosh Tabar 3,
Nasrin Farhadi 4 and Seyed Mohammad Ahmadi Hosseini 5

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Department of Horticulture, Faculty of Agriculture,


Ferdowsi University of Mashhad, Mashhad, Iran
2
Department of Plant Diseases, Faculty of Agriculture,
Tarbiat Modares University, Tehran, Iran
3
Chemistry and Chemical Engineering Research Center of Iran,
657, Soheil Shiraz, Vanak, Tehran, Iran
5
Department of Horticulture, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
5
Department of Horticulture, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
Received 25 Februray 2012; accepted in revised form 17 August 2012

Abstract: Essential oil of Mentha piperita was analyzed by GC and GC-MS and evaluated for in vitro
antifungal activity against Dreschlera spicifera, Fusarium oxysporum f.sp. ciceris and Macrophomina
phaseolina. Thirty-five components have been identified in the essential oil of M. piperita. The major compounds
identified in the oil were menthone (30.63 %), menthol (25.16 %), menthofuran (6.47 %), -phellandrene
(5.59 %), isomenthone (4.74 %), menthol acetate (4.61 %), pulegone (4.39 %), -caryophyllene (3.05 %),
neomenthol (2.83 %), 1,8-cineole (2.15 %). The antifungal assay was determined by agar dilution method with
five concentrations of peppermint oil (up to 1600 ppm). The oil was found to be effective against these fungal
pathogens under laboratory screening. The antifungal activities of the oil increased with an increase in the
concentration. Minimum effective concentrations of the oil against fungal pathogens were also different. The
fungistatic and fungicidal activity of the oil was determined. Results of fungistatic and fungicidal activities
showed that peppermint oil in these concentrations have no fungicide activity.
Key words: Mentha piperita; essential oil composition; antifungal activity; menthone; menthol.
Introduction
Pathogens fungi contaminate crops and foods
and cause significant yield reduction and
economic losses. Synthetic chemicals with
various degree of persistence are employed as
fungicide in crop protection. The use of such
fungicides, although increased crop production
but with some deterioration of environmental
quality and human health 1,2,3. Furthermore with
the target pathogen, pesticides may also kill
*Corresponding author (Mohammad Moghaddam)
E-mail: < m.moghadam@ferdowsi.um.ac.ir >

various beneficial organisms and their toxic forms


can persist in soil 4. The increasing incidence of
resistance among pathogens toward synthetic
chemicals is also a cause for serious concern 5.
Because of these problems in recent years, there
has been a clear tendency towards the utilization
of alternative methods for pest and disease control
in agriculture, that are less damaging to the
environment and human health. Research on plant
extracts and essential oils which may substitute
2013, Har Krishan Bhalla & Sons

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Mohammad Moghaddam et al., / TEOP 16 (4) 2013 506 - 512


the use of agrochemical or which may contributes
to the development of new compounds is
extremely important. Numerous studies have
examined the antifungal action of essential oils
against phytopathogenic fungi 6,7,8,9,10,11,12.
Mentha piperita is a perennial herbaceous plant
belonging to the family Laminacea. It is native
of the Mediterranean region. Peppermint oil is
the most popular and widely used essential oil
employed in food, pharmaceutical and cosmetic
industries 13. The oil also possesses biological
activity against numerous organisms, including
fungi 14,15,16, bacterial 15,17, nematodes18 and insect
pest 19. The chemical composition of peppermint
has been the subject of numerous studies 13,14,20,21.
To our knowledge, effect of essential oil of M.
piperita has not reported on Dreschlera spicifera,
Fusarium oxysporum f.sp. ciceris and
Macrophomina phaseolina. The aim of the
present work is determine chemical composition
essential oil of M. piperita cultivated in Iran and
evaluation effect of this oil on three plant
pathogens above.
Materials and methods
Plant material
The leaves of Mentha piperita were harvested
from plant grown in experimental field of
Zardband Medicinal Plants Production Company
located in north of Tehran, at flowering stage.
Isolation of the essential oil
Air-dried leaves of the plant were subjected to
hydro-distillation for 4h using a Clevenger-type
apparatus, according to the methods recommended by the European Pharmacopoeia22. The oil
was dried over anhydrous Na2So4 and stored in
dark at 4C.
Gas chromatography (GC)
A Varian GC 3800 system was used for GC
analysis, fitted with a fused methylsilicone CP
Sil 5 CB column (60 m 0.25 mm ID., 0.25 m
film thickness). Oven temperature was
programmed from 36 to 250C at 4.5C/min.
Injection was performed at 280C in the split ratio
1:100; 0.1 l of sample was injected. A flow of 1
ml/min carrier gas helium was used. Flame

507

ionization detection (FID) was performed at


280C.
Gas chromatography-mass spectrometry (GCMS)
Analyses were carried out in a Fison 8000 gas
chromatograph fitted with a fused methylsilicone
CP Sil 5 CB column (60 m 0.25 mm ID., 0.25
m film thickness), coupled to a Trio 1000 mass
detector. Column temperature was programmed
from 36 to 250C at 4.5C/min. Injection was
performed at 280C. Helium was used as carrier
gas (1 ml/min). Mass spectra were recorded in
the scan mode at 70 eV (25-500C); 0.5 ml of the
sample was injected by the splitless technique
(1/100).
The components of oil were identified by
comparison of their mass spectra with those
assembled via a Wiley 5 mass spectra computer
library or with authentic compounds. Data
obtained were confirmed by comparison of
retention indices, either with those of authentic
compounds or with the data published in the
literature 23,24. Component relative concentrations
were obtained directly from GC peak areas.
Fungal species
The fungi obtained from the culture collection
at Plant Pathology Agricultural Research Institute, Tehran, Iran and maintained on a suitable
medium until used. Fungi plant pathogens in these
tests were Dreschlera spicifera, Fusarium
oxysporum f.sp. ciceris and Macrophomina
phaseolina.
Antifungal assay
Activities of oil were tested against three fungi
pathogens above using a range of different
concentration (100, 200, 400, 800, and 1600 ppm)
on Potato Dextrose Agar (PDA). The fungitoxicity was calculated according to the method
of Zambonelli et al.25. The oil were dissolved in
ethyl alcohol and 5% Tween 20, and added to
the culture medium at a temperature of 40-45C,
then poured into Petri dishes (90 mm). The fungi
were inoculated as soon as the medium had
solidified. Disks (5 mm) of mycelial material,
taken from the edge of seven-day-old fungal

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Mohammad Moghaddam et al., / TEOP 16 (4) 2013 506 - 512


cultures, were placed at the center of each Petri
dish. The controls set were prepared similarly by
inoculating fresh medium with ethyl alcohol + 5
% Tween 20 and aqueous solutions were used as
second controls. The Petri dishes with the
inoculum were placed in incubator under controlled temperature condition of 252C. The
efficacy of treatment was evaluated after 2, 3 and
7 days. The percentage of inhibition of mycelial
growth was calculated from the mean values of
colony diameter of treated and control (ethyl
alcohol + 5 % Tween 20). The fungicidal activity
of the oils was determined using the technique
of Thompson 26. In disks where no growth was
observed (total inhibition = 100) the mycelial
disks were transferred into fresh plates of PDA
in order to determine, after 72h, whether the
inhibition was of a fungistatic or fungicidal
nature.

Statistical analyses
The experiment was repeated three times for
each fungus and for each dose. The significance
of the findings was determined by variance
analysis (ANOVA) by using SPPS 10.0 software
package.
Results and Discussion
The results of the oil analyses are given in Table
1. The essential oil content of peppermint leaves
was 1.38 % (w/w), based on the dry weight of
the plant. Thirty-five compounds were identified
in the essential oils of M. piperita. The major
compounds were menthone (30.63 %), menthol
(25.16 %), menthofuran (6.47 %), -phellandrene
(5.59 %), isomenthone (4.74 %), menthol acetate
(4.61 %), pulegone (4.39 %), -caryophyllene
(3.05 %), neomenthol (2.83 %), 1,8-cineole (2.15
%), germacrene D (1.87 %), trans-sabinene

Table 1. Percentage composition of Mentha piperita oil.


No.

Component

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

-Thujene
-Pinene
Sabinene
-Pinene
3 Octanol
Myrcene
-Terpinene
p-Cymene
1,8-Cineole
-Phellandrene
(Z)--ocimene
-Terpinene
trans-Sabinene hydrate
Linalool
enthone
Isomenthone
Menthofuran
Neomenthol
Menthol
Isomenthol
-Terpineol
Pulegone
Piperitone
Neomenthol acetate

508

RIa

Percentage

922
930
965
970
978
981
1007
1010
1019
1020
1024
1048
1053
1082
1142
1149
1153
1155
1168
1174
1177
1219
1229
1261

0.05
0.85
0.62
1.26
0.05
0.17
0.09
0.14
2.15
5.59
0.02
0.17
1.53
0.14
30.63
4.74
6.47
2.83
25.16
0.24
0.36
4.39
0.48
0.28

Mohammad Moghaddam et al., / TEOP 16 (4) 2013 506 - 512

509

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table 1. (continued).

No.

Component

25
26
27
28
29
30
31
32
33
34
35

Menthol acetate
Isomenthol acetate
-Bourbonene
-Elemene
-Caryophyllene
(Z)--farnesene
-Humulene
Germacrene D
Germacrene A
Caryophyllen oxide
Viridiflorol
Total

RIa

Percentage

1280
1295
1388
1390
1425
1446
1457
1483
1496
1578
1590

4.61
0.14
0.23
0.15
3.05
0.39
0.13
1.87
0.4
0.19
0.4
99.97

RI= Retention Index

hydrate (1.53 %) and -pinene (1.26 %). The oil


composition was partially different from other
studies. The findings on the major components
of M. piperita oil were in agreement with the
previous reports 14,20 except for -phellandrene,
menthol acetate and neomenthol. According to
Giamperi et al.14 and Pino et al 20 limonene was
occurred in appreciable amounts in oil from Italy
(4.2 %) and Mexico (2 %), whereas it wasnt
detected in our study. Also menthyl acetate found
the major compound in essential oil of Mexico
and Morocco 27 whereas it wasnt detected in oil
from Italy and our study. The difference in the
oil composition may be due to the climatic
condition and geographic factors or the
occurrence of a chemotype 21.
The results obtained in assays of the antifungal
activity of the oil of M. piperita are shown in
Table 2. Variance analysis revealed a significant
effect in relation to observation times, does, fungi
and corresponding interactions. The oil of M.
piperita significantly inhibited the growth of the
fungus compared to the control. The results
showed that the antifungal activity of the oil
increased with an increase in concentration. The
minimum concentration of the oil required to
inhibit the mycelial growth of test fungi was
difference. It is evident that the inhibitory effect
of the oil on mycelial growth of fungi varied

among the fungal species. These results are in


agreement with the other studies 7,8,11 which found
differences between fungi in resistance to essential oils. The sensitivity of the various pathogens
may depend on the morphological and physiological characteristics of the fungus hyphae. In
general, percentages of inhibition depend on the
day of observation, dose and fungus. After 7 days
(Table 2), at a concentration of 100 ppm significant difference wasnt found on Macrophomina
phaseolina with control whereas Fusarium
oxysporum f.sp. ciceris and Dreschlera spicifera
showed significant difference. It was found that
800 ppm and 1600 ppm on Dreschlera spicifera
and 1600 ppm on Fusarium oxysporum f.sp.
ciceris were completely inhibitory. Results
fungistatic and fungicidal activities showed that
peppermint oil in these concentrations have no
fungicide activity.
It has been established that the composition of
essential oils will depends on the plant species,
the chemotypes and the climatic conditions;
therefore their antimicrobial activity could vary
28,29
. Inhibition of the growth of these fungal
pathogens may be due to major component such
as menthone, menthol and menthofuran.
Furthermore, it is possible that the other minor
components may act together synergistically in
each oil as has already been suggested5.

C= Dreshlera spiciferaLSD (P=0.01)


B= Macrophomina phaseolina
A= Fusarium oxysporum f.sp. ciceris

2.3 0.15
00.00 4.70.76
15.31.24 1.20.69 13.22.53
45.81.28 57.90.39
572.13
73.10.56 80.51.05 1000.00
1000.00 93.40.18 1000.00
00.00
00.00
00.00
8.40.87 1.40.39 7.62.41
290.56 2.81.04 20.36.21
66.32.4 91.50.35 65.54.89
87.60.86 94.91.03 1000.00
1000.00 1000.00 1000.00
00.00
00.00
00.00
13.90.78 3.40.73 10.81.72
32.50.98 12.41.17 22.11.26
72.46.27 95.30.83 67.85.12
1000.00 97.10.80 1000.00
1000.00 1000.00 1000.00
00.00
00.00
00.00
100
200
400
800
1600
Control

Day2
B

Percent inhibition of mycelial growth


Day3
A
B
C
Concentration
(ppm)

Table 2. Antifungal activity of Mentha piperita oil

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Day7
B

Mohammad Moghaddam et al., / TEOP 16 (4) 2013 506 - 512

510

Baruah et al.9 compared antifungal properties


of essential oils from Chymbopogon martini
var.motia. Eucalyptus citriodora, Cinnomomum
tamala and M. piperita against Fusarium moniliforme and found that minimum inhibition of
fungal growth was created by M. piperita.
Giamperi et al.14 showed differences in antifungal
activity of essential oils and obtained that peppermint oil had 100 % inhibition on Phytophtora
cinnamomi, Pyrenochaota lycopersici and
Verticillium dahiae with 800 ppm and 91.8 %
inhibition at 1600 ppm for Trichophyton mentagrophytes. Comparing results of Macrophomina
phaseolina with those of Dwivedi et al.12 showed
that peppermint oil was more effective than some
higher plant but was less than Tranchyspermum
ammi.
In our study, by comparing the results of
percentage inhibition of mycelial growth for
every fungus, a difference between the tolerances
against the oil can be observed. Macrophomina
phaseolina showed a greater tolerance within the
selected concentration range (Table 2). This may
be duo to produces more enzymes by Macrophomina phaseolina whish catalyzes the
oxidation and thus inactivation of the added oil.
Inhibition of the growth of these fungal pathogens
may be due to emulsion damaged the cell wall
and cell membrane to various degrees duo to
different capacity to penetrate oil into the chitinbased cell walls of fungal hyphae.
Acknowledgment
The authors would like to thank to H. Zamyad,
Department Plant Breeding, Tarbiat Modares
University and Dr. N. Yazdani Department
Horticulture, University of Tehran, for their
assistances.

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511

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