Industrial Crops & Products 119 (2018) 255–259

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Industrial Crops & Products

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Short communication

Essential oil profiling of Ajowan (Trachyspermum ammi) industrial medicinal T

plant
⁎
Mehdi Soltani Howyzeha, Seyed Ahmad Sadat Nooria, , Vahid Shariati J.b
a
Department of Agronomy and Plant Breeding Science, College of Abouraihan, University of Tehran, P.O. Box: 3391653755, Tehran-Pakdasht, Iran
b
Plant Molecular Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), 14965/161, Tehran, Iran

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

Keywords: Ajowan (Trachyspermum ammi L.) is a highly valued medicinally important seed spice from Apiaceae family,
Inflorescence which contains essential oil, exhibiting a chemical composition that can be strongly altered with developmental
Metabolomics stages of the inflorescence. In order to investigate the changes of metabolite profile of Ajowan, the inflorescence
γ-Terpinene harvested in ten developmental stages. The oil of each inflorescence stage collected by hydrodistillation and
ρ-Cymene
analysed by GC/MS. The essential oil concentrations were in a range of 3.71% (stage 1)–5.08% (stage 8). In this
Thymol
study, fourteen components were identified, which thymol, γ-terpinene, and ρ-cymene comprised between 94%
Volatile oil
and 96% of the essential oil. The results showed that during the anthesis stages, the thymol percentage in the
essential oil of Ajowan was decreased, but the γ-terpinene percentage was increased. Hence, during the fruit
ripening stages, the thymol and ρ-cymene percentages were increased but the γ-terpinene percentage was de-
creased. There was a negative correlation of γ-terpinene, ρ-cymene, and thymol. The results suggested that
different harvest stages of Ajowan inflorescence have a significant effect on the essential oil concentration and
composition. Furthermore, the anthesis stage was an important turning point of the main component percentage
of the volatile oil composition of Ajowan inflorescence.

1. Introduction seed ripening in Ajowan.

Metabolite profiling (metabolomics) is essential in plant metabolite 2. Materials and methods

research and concentrate on the large group of metabolites analysis,
which is associated with a specific metabolic pathway or a group of
compositions, particularly in medicinal plants (Desai and Alexander,
2013; Wolfender et al., 2015). GC/MS technique is regularly and ef-
fectively used for the analysis of essential oils and has been extensively
applied to metabolite profiling in medicinal plants (Mirzahosseini et al.,
2017; Rubiolo et al., 2010; Rusanov et al., 2011).
Ajowan (T. ammi L. Sprague ex. Turill, syn. T. copticum Linn and
Carum copticum Benth and Hook), belonging to the family Apiaceae, is
an important annual herbaceous plant and a highly valued medicinally
seed spice (Malhotra and Vashishtha, 2005; Malhotra and Vijay, 2004).
Until now, research on Ajowan plant has been focused on its essential
oil concentration and composition (Baby et al., 2012; Mirzahosseini
et al., 2017; Zarshenas et al., 2014; Soltani Howyzeh et al., 2018). In
this study, we report the metabolite profiling of the chemical compo-
sition and oil content of different developmental stages using gas
chromatography/mass spectrometry (GC/MS), in order to recognize the
major changes in the essential oil composition during flowering and
2.1. Plant materials
Ajowan seeds were purchased from a medicinal plants bazaar in
Ahvaz, Iran. Ajowan seeds were identified by M. Soltani Howyzeh and a
voucher specimen (no: 20112) deposited in College of Aburaihan,
University of Tehran. Seeds were planted in an experimental field in
Ahvaz in January 2015. After the emergence of flower buds in May
2015 until fruit maturation stage in July 2015, in order to investigate
the changes of essential oil metabolite profile of T. ammi during flower
development, the process of inflorescence development, based on the
morphological traits, was divided into ten consecutive developmental
stages (∼5 days between each stage) (Fig. 1). The ten developmental
stages of inflorescence were descripted for the first time by the authors
as below.
Stage 1: Primary umbels are visible but umbellets are not developed
and are not visible in each umbel.
Stage 2: The umbellets of each umbel are visible but petals are not

⁎
Corresponding author.
E-mail address: noori@ut.ac.ir (S.A. Sadat Noori).

https://doi.org/10.1016/j.indcrop.2018.04.022
Received 19 December 2017; Received in revised form 8 April 2018; Accepted 9 April 2018
Available online 24 April 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
M. Soltani Howyzeh et al.
Fig. 1. Stages of T. ammi inflorescence development used in the present study. A) Side view. B) Top view. Numbers show the inflorescence stages in tandem.
visible.
Stage 3: The umbellets are fully developed, and the petals are
visible.
Stage 4: The petals are fully opened and in the middle of them red
colour is visible. The red colour comes from the anther of the stamen.
Anthers in this stage are not opened.
Stage 5: Anthesis happens and the red colour in the middle of petals
is not observable. The filaments of the stamens are visible and anthers
start to pollinations.
Stage 6: The anthesis of all umbellets is completed and the white
petals start to fall.
Stage 7: The petals are fully fallen, and green fertilized ovaries
256
Industrial Crops & Products 119 (2018) 255–259
appear. Above each ovary, the remains of style and stigma are visible.
Stage 8: Only dark-green fruits are visible. Each fruit is swollen and
juicy.
Stage 9: The fruits ripened and the colour the fruits become light
green to some yellow.
Stage 10: The fruits fully ripened and the inflorescence dried. The
colour of the fruits becomes brownish.
2.2. Essential oil isolation
The harvested samples from each inflorescence stage (3 replica-
tions) were dried at room temperature and ground to a coarse powder
M. Soltani Howyzeh et al. Industrial Crops & Products 119 (2018) 255–259

Fig. 2. Profile plot and means comparison of essential oil concentrations correspond to ten developmental stages of Ajowan inflorescence. The stages with the same
letter, are not significantly different at P ≤ 0.05 probability level.

Table 1
Chemical composition of essential oil of Ajowan inflorescence stages.
No Essential oil constituents (%) Inflorescence stages RIa Identification

1 2 3 4 5 6 7 8 9 10

1 α-Thujene 0.46 0.36 0.34 0.37 0.33 0.38 0.34 0.38 0.35 0.34 930 MS, RI
2 α-Pinene 0.12 0.09 0.13 0.11 0.14 0.11 0.11 0.12 0.13 0.11 939 MS, RI
3 Sabinene 0.31 0.16 0.12 0.21 0.19 0.24 0.22 0.20 0.21 0.20 975 MS, RI
4 β-Pinene – – – 0.24 0.22 0.32 0.48 0.61 0.64 0.71 979 MS, RI
5 Myrcene 0.88 0.62 0.57 0.68 0.51 0.55 0.56 0.54 0.51 0.53 991 MS, RI
6 δ-3-carene 0.05 – – – – – – – – – 1012 MS, RI
7 α-Terpinene 0.50 0.38 0.28 0.29 0.27 0.20 0.43 0.33 0.25 0.23 1017 MS, RI
8 ρ-Cymene 13.87 9.54 10.38 11.72 12.45 11.65 10.31 14.59 16.83 19.51 1025 MS, RI
9 β-Phellandrene 1.20 0.62 0.60 0.67 0.58 0.60 0.58 0.50 0.50 0.52 1030 MS, RI
10 γ-Terpinen 70.39 63.44 60.33 56.56 63.35 74.75 63.18 52.81 55.23 45.87 1060 MS, RI
11 Terpinolene 0.07 – – 0.05 – – 0.07 – – – 1089 MS, RI
12 Terpinene-4-ol 0.13 0.13 0.13 0.11 0.10 – 0.07 0.07 – 0.10 1177 MS, RI
13 Thymol 11.35 23.96 25.58 27.65 18.20 10.13 17.48 29.15 24.41 30.77 1290 MS, RI
14 Carvacrol 0.30 0.25 0.21 0.32 0.29 0.24 0.22 0.25 0.31 0.33 1299 MS, RI
15 unknown 0.16 0.15 0.15 0.10 0.10 0.23 0.13 0.33 0.16 0.17 – –
16 unknown 0.09 0.29 0.43 0.64 0.56 0.60 0.94 0.08 0.38 0.38 – –
17 unknown 0.14 – 0.56 0.19 0.18 – 0.14 – 0.09 0.11 – –
Total (%) 99.9 100 100 95.3 100 97.5 99.9 99.8 100 100

a
The retention index of compounds on HP-5MS column was also determined.

by an electrical grinding mill and then soaked (50 g) in fresh distilled
water (500 ml) for essential oil extraction. Hydrodistillation was done
by using a Clevenger-type apparatus for about 4 h (Niazian et al., 2017;
Noori et al., 2017). The essential oil extraction was done in agreement
with a method recommended by the British Pharmacopoeias (British
Pharmacopoeia, 1988). The accumulated oil was dried over anhydrous
sodium sulphate and kept at 4 °C.
2.3. Gas chromatography–mass spectrometry analysis (GC/MS)
scan time of 0.4 s and mass range of 40–460 amu was used. 1 μl of di-
luted samples (1/100; v/v, in methanol) was manually injected in the
splitless mode. The interface temperature was 290 °C. Helium gas was
selected as the carrier with the same flow rate (1 ml/min) for GC/FID.
The programme of oven temperature was initiated at 40 °C, held for
1 min, and then raised up to 250 °C at a rate of 3 °C/min. The oil
compounds were identified by comparing their retention indices (RI),
mass spectra fragmentation with NIST (National Institute of Standards
and Technology) Adams library spectra, Wiley 7 n.1 mass computer
library, and with those reported in literatures (Adams, 2007).

The Ajowan oil sample analysis was done by using GC/MS. The GC/
MS analysis was performed on a GC/MS apparatus using the HP
(Agilent Technology): 7890 Network GC System gas chromatograph
connected to a mass detector (5973 Network Mass Selective Detector).
The gas chromatograph was equipped with an HP-5MS capillary
column (fused silica column, 30 m × 0.25 mm i.d., Agilent
Technologies) and an EI mode with ionization energy of 70 eV with a
2.4. Data analysis
The analysis of one-way variance and the calculations of correla-
tions were done using the statistical analysis system (SAS) computer
software (SAS Institute Cary, NC, USA, 1988). For mean comparison,
the Duncan’s multiple range tests and standard error (SE) were used at

257
M. Soltani Howyzeh et al. Industrial Crops & Products 119 (2018) 255–259

Fig. 3. Changes of main essential oil ‘s components in the ten stages of Ajowan inflorescence.

Table 2
The correlations between constituents of essential oil and its content in the ten stages of Ajowan inflorescence.
Traits α-Thujene α-Pinene Sabinene β-Pinene Myrcene α-Terpinene ρ-Cymene β-Phellandrene γ Terpinen Thymol Carvacrol E.O.C (uL/g)

α-Thujene 1
α-Pinene −0.17 1
Sabinene 0.77** −0.22 1
β-Pinene −0.34 0.03 0.08 1
Myrcene 0.86** 0.05 0.61* −0.58 1
α-Terpinene 0.57 0.29 0.42 −0.39 0.71* 1
ρ-Cymene 0.03 0.22 0.26 0.71** −0.16 −0.31 1
β-Phellandrene 0.86** 0.04 0.68* −0.58 0.96** 0.69* −0.11 1
γ Terpinen 0.45 −0.54 0.39 −0.62 0.39 0.29 −0.63* 0.52 1
Thymol −0.47 0.47 −0.61 0.39 −0.37 −0.31 0.36 −0.55 −0.93** 1
Carvacrol 0.16 0.26 0.38 0.30 0.20 −0.16 0.70* 0.18 −0.42 0.24 1
E.O.C (uL/g) −0.13 −0.008 −0.44 −0.19 −0.18 0.14 −0.56 −0.32 0.04 0.23 −0.50 1

** and * significant at P ≤ 0.01 and P ≤ 0.05 probability levels, respectively. E.O.C: Essential oil content.

p ≤ 0.05 to establish significant differences.
3. Results and discussion
3.1. Inflorescence stages and essential oil concentrations
The concentrations of essential oil in ten stages of the Ajowan in-
florescence were significantly different. Stage 8 had the maximum es-
sential oil concentration (5.08%), and stage 1 had the lowest essential
oil concentration (3.71%) (Fig. 2). During the developmental stages of
Ajowan inflorescence, the essential oil concentration increased from
stage 1 to stage 2 and then approximately fixed until stage 8, but after
that started to decrease (Fig. 2). This showed that during fruit ripening
the essential oil concentration decreased. The concentration of essential
oil reported in previous studies was in the range of 2%–6.0%
(Mirzahosseini et al., 2017; Niazian et al., 2017; Rahimmalek et al.,
2017; Zarshenas et al., 2014). Different harvest stages significantly
changed the essential oil concentration and its composition in medic-
inal plants (Omidbaigi et al., 2003; Zehtab-Salmasi et al., 2001).
3.2. Essential oil composition
The components of essential oil of the inflorescence stages of
Ajowan were determined (Table 1). In this study, fourteen components
of the essential oil of ten stages of Ajowan inflorescence were identified
by GC/MS analysis. The thymol, γ-terpinene, and ρ-cymene comprised
between 94% and 96% of the essential oil components (Table 1). On the
other hand, the rest components only comprised less than 4–6% of the
essential oil of Ajowan in ten developmental stages of the inflorescence.
The changes of the main essential oil‘s components during devel-
opmental stages of inflorescence were shown in Fig. 3. The thymol
percentage in the essential oil were gradually raised from stage 1 to
stage 4, then it reduced to the minimum amount (10.13%) at stage 6,
after that it increased until stage 8, at stage 9 there is a slight decrease
and then it raised to the maximum amount (30.7%) at stage 10 (Fig. 3).
It obviously seen that during anthesis stages (stage 5 and stage 6) the
thymol percentage in the essential oil of Ajowan was decreased (Fig. 3).
It has been reported earlier that the different growth stage affected the
concentration of the essential oil in different parts of plants (Singh
et al., 1989). The analysis of essential oil from the aerial parts of Ar-
temisia campestris var. glutinosa in different phenological status showed
some quantitative differences before and after anthesis (Juteau et al.,
2003).
The γ-terpinene percentage in the essential oil of Ajowan vice versa
the thymol percentage, was gradually decreased from stage 1 to stage 4,
then it raised to about the maximum amount (74.75%) at stage 6, after
that it reduced until stage 8, and at stage 9, there is a slight increase,
and then it diminished to the minimum amount (45.87%) at stage 10
(Fig. 3). The γ-terpinene percentage increased during the anthesis
stages (stage 5 and stage 6), unlike the thymol percentage (Fig. 3). The
Ρ-cymene percentage during developmental stages of Ajowan in-
florescence changed from 9.54% at stage 2–19.51% at stage 10 (Fig. 3).

258
M. Soltani Howyzeh et al.
The increase of γ-terpinene percentage during anthesis stages (stage 5
and stage 6) may be due to the attraction of pollinators. The effect of
flower volatiles on pollinators has been studied with consideration on
the volatile characteristics, floral scent profiles and ecological interac-
tion of the plant with the environment (Pichersky et al., 1994;
Pragadheesh et al., 2017; Steenhuisen et al., 2012).
3.3. Correlation of essential oil ‘s components
The correlations between the amounts of the components of the
essential oil and the oil content were calculated in Table 2. The ρ-
cymene as one of the main constituents of the essential oil had positive
and significant correlations with β-pinene (0.71) and carvacrol (0.70),
also it negatively correlated with γ-terpinene (−0.63). The results of
correlations analysis also showed a high negative correlation between
thymol and γ-terpinene (−0.93). The negative correlation of γ-terpi-
nene with ρ-cymene and thymol probably could be due to the fact that
γ-terpinene is the precursor of ρ-cymene and thymol formation. The
thymol and ρ-cymene formation were done by the bioconversion of γ-
terpinene (Carvalho Filho et al., 2006; Galata et al., 2014).
4. Conclusion
Metabolite profiling of the essential oil of the medicinal plant T.
ammi (L.) during the developmental stages of the inflorescence showed
that essential oil concentrations were significantly different. In this
study, fourteen components of the essential oil of ten stages of Ajowan
inflorescence were identified by GC/MS analysis. Thymol, γ-terpinene,
and ρ-cymene comprised between 94% and 96% of the essential oils
components. The changes of the main essential oil‘s components during
developmental stages of inflorescence showed that during anthesis
stages (stage 5 and stage 6) the thymol percentage in the essential oil of
Ajowan decreased. The γ-terpinene percentages in ten stages of Ajowan
inflorescence had a reverse trend with thymol percentage. The results of
this study prove the performance of metabolite profiling in order to
determine the major variation in the composition and content of the
essential oil during the developmental stages of Ajowan inflorescence
and illuminate the relationships between them.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors are grateful to Dr. M.H Asare, the secretary of science
and technological development staff of medicinal plants and traditional
medicine of Islamic Republic of Iran for his kindness supports of
Ajowan project. Also the authors would like to thank Mr. H. Naseri, Mr.
S. Ebadi, Mr. A. Alinejati and Ms. H. Naderi for kindly help in culti-
vation, growing and gathering the plant materials and also for oil ex-
traction of inflorescence samples.
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