Essential oil composition and antioxidant activity of citron fruit ( Citrus medica var. macrocarpa Risso.) peel as relation to ripening stages

Abstract

Stages of maturity have decisive roles in determining the quality and quantity of essential oil (EO). In this regard, EO yield and composition and their antioxidant activity of citron fruit at four fruit maturity stages, i.e. the green mature (GM), intermediate (INT), yellow ripe (MAT) and overripe stage (OR) were studied. Obtained results showed significant effect of fruit maturity on most measured properties. The concentration of EO varied between 0.60 and 0.77% (v/w). The highest amount of limonene was 89.39% related to GM stage. The limonene decreased significantly during maturity. The highest antioxidant activity (76.08%) was measured at the OR stage, which is probably due to the presence of specific compounds in the EO and their synergistic effects. The phytochemical behaviors of this citron variety were different as relation to stages of fruit maturity. We can determine the ideal harvest period for maximum bioactive substances by recognizing these actions.

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Essential oil composition and antioxidant activity
of citron fruit (Citrus medica var. macrocarpa Risso.)
peel as relation to ripening stages
Askar Ghani, Nehleh Taghvaeefard, Mehdi Hosseinifarahi, Sarra Dakhlaoui &
Kamel Msaada
To cite this article: Askar Ghani, Nehleh Taghvaeefard, Mehdi Hosseinifarahi, Sarra Dakhlaoui
& Kamel Msaada (2022): Essential oil composition and antioxidant activity of citron fruit (Citrus
medica var. macrocarpa Risso.) peel as relation to ripening stages, International Journal of
Environmental Health Research, DOI: 10.1080/09603123.2022.2084514
To link to this article: https://doi.org/10.1080/09603123.2022.2084514
Published online: 18 Jun 2022.
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Essential oil composition and antioxidant activity of citron fruit
(Citrus medica var. macrocarpa Risso.) peel as relation to ripening
stages
Askar Ghani
a
, Nehleh Taghvaeefard
b
, Mehdi Hosseinifarahi
b,c
, Sarra Dakhlaoui
d
and Kamel Msaada
d
a
Department of Horticultural Science, Faculty of Agriculture, Jahrom University, Jahrom, Iran;
b
Department of
Horticultural Science, Yasuj Branch, Islamic Azad University, Yasuj, Iran;
c
Sustainable Agriculture and Food Security
Research Group, Yasuj Branch, Islamic Azad University, Yasuj, Iran;
d
Laboratory of Aromatic and Medicinal Plants,
Biotechnology Center in Borj Cedria Technopole, Hammam-Lif, Tunisia
ABSTRACT
Stages of maturity have decisive roles in determining the quality and
quantity of essential oil (EO). In this regard, EO yield and composition
and their antioxidant activity of citron fruit at four fruit maturity
stages, i.e. the green mature (GM), intermediate (INT), yellow ripe
(MAT) and overripe stage (OR) were studied. Obtained results showed
signicant eect of fruit maturity on most measured properties. The
concentration of EO varied between 0.60 and 0.77% (v/w). The highest
amount of limonene was 89.39% related to GM stage. The limonene
decreased signicantly during maturity. The highest antioxidant activ-
ity (76.08%) was measured at the OR stage, which is probably due to
the presence of specic compounds in the EO and their synergistic
eects. The phytochemical behaviors of this citron variety were dier-
ent as relation to stages of fruit maturity. We can determine the ideal
harvest period for maximum bioactive substances by recognizing
these actions.
ARTICLE HISTORY
Received 22 April 2022
Accepted 25 May 2022
KEYWORDS
Citrus macrocarpa L.;
ripening stages; essential oil;
linalool; limonene;
antioxidant activity
Introduction
One of the most important fruits of tropical and subtropical regions are Citrus species which belong
to the Rutaceae family (Vekiari et al. 2002; Abdelaali et al. 2018). Citron tree has been mentioned in
traditional books of Iranian folklore medicine, with the old name of “Otroj Kabir” or big citron and
scientific name is Citrus media var. macrocarpa Risso. This tree can be capable of bearing big fruits
which can often weigh as heavy as one kilogram. The peel essential oils (EO) of citrus fruits can be
considered as valuable by-products of process manufacturing, or they can be considered as main
products extracted from specific cultivars of citrus (Fagodia et al. 2017; Taghvaeefard et al. 2021).
The most common industrialized products obtained from citrus are seed oil, fruit peel essential oil
(EO), citrus flavonoids and pectin substances which make a considerable amount of income for the
producers (Negro et al. 2016; Ghani et al. 2021). The EO of citrus fruitsare commonly used as
flavoring agents in many food products such as non-alcoholic drinks, chocolates, gelatin, confec-
tionaries, desserts, cosmetics, perfumery, medicinal industries and remedial EOs (Zarrad et al. 2015;
Negro et al. 2016; Taghvaeefard et al. 2021). According to the available literature, citrus EOs can
CONTACT Kamel Msaada msaada.kamel@gmail.com Laboratory of Aromatic and Medicinal Plants, Biotechnology
Center in Borj Cedria Technopole, BP. 901, Hammam-Lif 2050, Tunisia
INTERNATIONAL JOURNAL OF ENVIRONMENTAL HEALTH RESEARCH
https://doi.org/10.1080/09603123.2022.2084514
© 2022 Informa UK Limited, trading as Taylor & Francis Group

contain limonene, citral, linalool, α-thujene, α-pinene, camphene, sabinene, ß-pinene, myrcene, α-
terpinene and p-cymene (Eldahshan and Halim 2016; Negro et al. 2016; Simas 2017; Fagodia et al.
2017).
Among these, the presence of limonene is more prominent than the other compounds and
constitutes nearly 90% of the EO in orange, grapefruit, citron and bitter-orange fruits (Vekiari et al.
2002; Negro et al. 2016; Fagodia et al. 2017). D-Limonene has anti-microbial properties and can be
responsible for the majority of antibacterial activity of citrus EOs against gram-positive bacteria,
besides enhancing the effectiveness of sodium benzoate (Colecio-Juarez et al. 2012; Eldashan and
Halim 2016; Mitropoulou et al. 2017). Furthermore, a high degree of anti-fungal activity of citral has
been reported (Fagodia et al. 2017; Simas et al. 2017, Mitropoulou et al. 2017). Identifying the best
harvesting time in medicinal plants can substantially assist in determining the quantity and quality
of active substance. With regard to many herbaceous plants, research has shown that specific times
of harvest that related with physiological behavior of medicinal plants can significantly determine
the amount and quality of active substances in plants (Msaada et al. 2007; Ghani et al. 2009). Also,
the growth stage can notably impress on secondary metabolites of medicinal trees and shrubs, and
the quality and quantity of their EOs are inevitably affected by fruit ripening or phenological stages.
Accumulation of EO in the finger citron through the process of ripening and their antioxidant
activity was reported (Wu et al. 2013). Since citrus fruits are produced in high amounts, and the
practice of extracting EOs from their peels has not been operational on an industrial scale, so far, it
is necessary to evaluate the conditions of extraction and the qualities of EOs so as to use them
optimally and economically (Espina et al. 2011). In another relevant research, it was revealed that
the highest amount of EO in the fruit peel of “Khasi” mandarin occurs at the intermediate stage of
maturity whereas the lowest amount of EO occurs at the overripe stage (Bhuyan et al. 2015).
Because citron fruit EOs have a wide range of applications in various industries, and fruit maturity
has an impact on EO quality and quantity, this study looked at the impact of different maturity
stages on fruit EO content, components, and antioxidant activity in Iranian citron fruit.
Material and methods
Plant materials and treatments
In this research, an experiment was conducted based on a randomized complete block design
(RCBD) with four treatments and three replications. The treatments consisted of different fruit
maturity stages (according to days after flowering), including the green mature stage (GM), peel
color change (INT), yellow-ripe stage (MAT) and overripe stage (OR) (Figure 1). The fruits of
Citrus medica var. macrocarpa Risso. variety were harvested at different ripening stages. The
orchard was located in Jahrom (south of the Fars province, Iran). The garden hosted the big citron
Figure 1. Superficial changes of flavedo from citron fruits at different harvest time (GM: green mature stage, INT: intermediate
stage, MAT: mature stage, OR: over ripe stage).
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A. GHANI ET AL.

(Citrus medica L. var. macrocarpa Risso.) in Qotb-Abad which was geographic characteristics of 28°
40ʹ 29ʺ.43 northern latitudes and 53° 36ʹ 27ʺ.17 east longitude (with an altitude of 1046.4 m above
sea level). The trees were approximately six years old. The fruits were harvested on various dates, i.e.
23 September (GM), 25 November (INT), 28 December (MAT), and 22 February (OR) coincides
with 166, 226, 261 and 317 days after flowering, respectively (Figure 1). The physiochemical features
of gardens soil are also provided (Table 1).
Fruit harvest and drying process
In each growth stage, the fruits were harvested from four different sides of each tree (i.e. North,
South, East and West). Furthermore, in each replication 20 fruits were harvested from three at
different heights (i.e. 0–70 cm, 70–140 cm and 140–210 cm). The fruit samples were mixed and
shuffled thoroughly for randomness. The fruits were taken to the laboratory and were thoroughly
washed with water. The thin, outer layer of the fruit (i.e. flavedo) was peeled manually. After the
separation, the fruit peels were cut and divided into small pieces (2–3 cm). To dry up completely,
they were left in the shade at room temperature (30 ± 2°C) for 7 days. After drying, the peel was
separately packaged and refrigerated to prevent the occurrence of unwanted changes to the peels by
the environment.
Isolation of essential oils
For citron fruits, each replication, 100 g of dried fruit peel (flavedo) was processed by the Clevenger-
type apparatus in order to extract the EO for two hours after the samples had reached boiling point
and EO yield was expressed as volume/weight (v/w). The EO yield was calculated by multiplying the
EO percentage in flavedo biomass (dry matter) in fruit unite, in each growth stage.
Essential oil analysis by Gas Chromatography/Mass Spectrometer (GC/MS)
Identifying the components of EOs involved the use of the gas chromatography (GC) and gas
chromatography/mass spectrometry (GC/MS) analysis. To identify the constituents of the essential
oils, Agilent Technologies-7890A gas chromatograph was used. The type, length, diameter, and
thickness of the column were HP-5-MS, 30 m, 0.22 mm, and 0.25 μm, respectively. The temperature
program of the column was 60–210°C at a rate of 4°C/min. Nitrogen carrier gas was used at a flow rate
of 0.5 mL/min. Gas chromatograph connected to a mass spectrometer (GC/MS) was an Agilent
Technologies-5975C model. The column type was HP-5 MS, 30 m in length, 0.25 mm in diameter,
and 0.25 μm in thickness. The temperature program was 280°C and helium carrier gas was used at
a flow rate of 1 mL/min. The relative percentage of each component of EO was determined based on
chromatogram peak area and compared with the total area by using the normalization method of the
GC/FID peak areas. Retention indices were determined using retention times of n-alkanes (C8-C25)
that were injected after the volatile oil under the same chromatographic conditions. The retention
indices for all components were estimated according to the method using n-alkanes as standard. The
compounds were identified by comparison of retention indices (RI, HP-5 MS) with those reported in
the literature and by comparison of their mass spectra with the Wiley GC-MS Library and Adams
library (Adams 2001).
Table 1. Soil properties of cultivated region from citron fruit.
Location Soil texture Sand (%) Silt (%) Clay (%) EC (ds/m)
“Big citron” garden Sandy-loam 75.4 17.8 6.8 6.17
K (ppm) P (ppm) N (%) OC (%) pH
“Big citron” garden 460 34.6 0.05 0.77 7.72
INTERNATIONAL JOURNAL OF ENVIRONMENTAL HEALTH RESEARCH
3

DPPH radical-scavenging
The radical scavenging capacity of EO was determined by a spectrophotometric method based on
the reduction of a methanol solution of DPPH (1,1-diphenyl-2-picrylhydrazyl) using the method of
Oke et al. (2009) with slight modifications. At first, 100 μL of each EO was added to 5 mL of
a 0.004% methanol solution of DPPH. The mixture was shaken vigorously and left to stand at room
temperature for 30 minutes in the dark conditions. The absorbance of the samples was read against
a blank at 517 nm. Inhibition of free radical, DPPH, in percent (I%) was calculated according to the
following formula:
I%¼Ablank Asample
Ablank 100
Statistical analysis
The experiment was carried out in triplicate. JMP software (version 8) was used for statistical
analysis and the averages were compared by using Tukey test (HSD) at 5% probability. Also,
relationships between factors were specified by Pearson’s correlation test. A principal component
analysis (PCA) and cluster analysis were performed in order to discriminate between maturity
stages on the basis of their essential oil composition (Table 2).
Results and discussion
Essential oil yield
According to the obtained results, there was a significant effect on the yield of essential oil during the
ripening period. However, the big citron shows significant changes in its EO content during its
ripening. The fruit maturity stage has significant effect on the percentage of EO, and the EO yield was
affected by the ripening stage. The highest yield of EO (0.12 mL per fruit) was measured in yellow-ripe
(MAT) stage (Figure 2(a)). Since secretory glands are located in the flavedo section, the EO percentage
and yield were lower in the Citrus medica L. var. macrocarpa Risso. The density of the secretory glands
in the peel, i.e. in the flavedo, can be influential in defining the amount of EO. However, there have
been no comprehensive researches on diversity of different citrus species with regard to their EO yield.
The difference between the amounts of EO in various species of citrus, during their various stages of
maturity and ripening, have been reported by several researchers (Mejri et al. 2022). The available
reports indicate that such differences in the EO amount are mostly because of genetic variations and
differences in environmental conditions especially the climatic ones. Achieving the best harvest time
from each species or variety needs to study accurately. In China, the qualitative and quantitative
differences among the EO were analyzed in C. medica var. “Sarcodaytylis”. In the mentioned research,
changes in the EO were evaluated at three maturity stages (GM, INT and MAT). The results showed
that the amount of EO increases during ripening (Wu et al. 2013). The highest EO yield and content
(0.126 mL in fruit, 0.43% v/w) extracted from grapefruit are reported at yellowish green stage (YGS)
and yellow stage (YS) (Ghani et al. 2021). In Tunisia, a study involved evaluating changes in the EO of
four citrus species at three stages of maturity, i.e. the green mature stage, the half-ripe stage and the
ripe stage (Bourgou et al. 2012). It was reported that the fruits EO of bitter-orange decreased initially
during the maturity stage, but then increased notably during the ripening stage. So that, the highest
amount of EO was recorded in ripening stage and the lowest content was recorded in the half-ripe
stage. In comparison, however, lemons have reportedly shown the highest amount of EO during their
green mature stage and the amount of EO decreases. This is while two species of citrus, i.e. the
Maltaise orange and mandarin, usually show their highest amount of EO at the half-ripe stage
(Bourgou et al. 2012). These significant changes in the specifications of EO can be attributed to the
genetic diversity of different citrus species. Other researchers have also reported such changes in EO
4
A. GHANI ET AL.

during maturity (Wu et al. 2013; Bhuyan et al. 2015). However, it should be noted that the climatic
conditions of each region can significantly influence the EO content. The harvest time is also
important in terms of the climatic specifications. Different results were reported in two cases of
research on citrus cultivars in Tunisia where the locations varied in terms of humidity in the country
(Hosni et al. 2010; Bourgou et al. 2012; Mejri et al. 2022).
Essential oil composition
The analysis of variance showed how the ripening stage affects the components of EO in the
C. medica var. macrocarpa Risso variety and the ripening stages has a significant effect on
the major constituents of EO (Table 2). The comparison of means values showed how the
ripening stage affects the components of EO in this variety (Table 2). Monoterpene hydro-
carbons (the maximum and minimum content equal to 92.10 and 78.96% in green mature
(GM) and peel color change (INT) stages were measured, respectively) and oxygenated
monoterpenes (the highest and lowest amount equal to 18.98 and 6.59% in INT and GM
stages were detected, respectively) were the dominant groups in the essential oils of macro-
carpa variety. Limonene is considered as the most important and major component of EO in
this variety that strongly influenced by ripening stage in such a way that the highest amount
of limonene (89.39%) was recorded in the GM stage of fruit ripening (Figure 3). In the INT
stage, when the fruit was changing color, the amount of limonene decreased (reaching
76.49%). However, as the maturity progressed further into the yellow-ripe stage, the amount
of limonene increased slightly (and reached 83.43%) but decreased again at the overripe
stage (78.96%) (Figure 3). There was a significant difference in terms of the amount of
limonene in the different stages of maturity (Table 2). Two other important compounds in
the citron EO were neral and geranial. These components were also influenced by maturity.
Accordingly, both components were at their lowest amount (2.20 and 2.88% respectively) at
the GM stage, whereas the highest amounts (5.87 and 7.69% respectively) were observed in
the INT stage. As the maturity progressed (MAT stage) the amount of these compounds
decreased and thereafter remained constant (Table 2). Other components which showed
changes in their concentrations were myrcene, linalool and nerol (Table 2). The trend of
changes was observed to be similar in linalool and nerol, such that both components showed
their lowest amounts at GM stage and an increase was observed during the GM stage. As the
ripening stage began, the amounts of linalool and nerol decreased slightly but reached their
maximum amounts at the OR stage. Other changes in the components of citron EO are
listed in Table 2. In relevant research on finger citron EO, the highest amount of limonene
b
b
a
b
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
GM INT MAT OR
Essential oil yield (%, v/w)
Maturity stages
b
cc
a
0
10
20
30
40
50
60
70
80
90
GM INT MAT OR
Antioxidant activity (%)
Maturity stages
ab
Figure 2. Change in essential oil yield (a) and antioxidant activity (b) at different fruit maturity (GM: green mature, INT:
intermediate, MAT: yellow-ripe, OR: over ripe stage).
INTERNATIONAL JOURNAL OF ENVIRONMENTAL HEALTH RESEARCH
5

Table 2. Change in essential oil constituents of Citrus medica L. var. macrocarpa Risso. at different fruit maturity.
NO Components RI
Stage of maturity
F PGM INT MAT OR
1 (E)-2-Hexenal 848 0.03 ± 0.00c 0.15 ± 0.00a 0.04 ± 0.01c 0.07 ± 0.01b 135.80 0.000***
2 α-Pinene 931 0.39 ± 0.01a 0.30 ± 0.01b 0.36 ± 0.02a 0.40 ± 0.01a 35.20 0.000***
3 Sabinene 971 0.11 ± 0.00b 0.10 ± 0.00b 0.11 ± 0.01b 0.15 ± 0.01a 22.53 0.000***
4 ß-Pinene 976 0.04 ± 0.02b 0.06 ± 0.02b 0.07 ± 0.01b 0.41 ± 0.14a 19.76 0.000***
5 Myrcene 988 1.87 ± 0.01a 1.52 ± 0.01c 1.63 ± 0.04b 1.54 ± 0.03c 109.02 0.000***
6 Limonene 1027 89.39 ±0.39a 76.49 ±0.45d 83.43 ±1.46b 78.86 ±1.59c77.02 0.000***
7 (E)-ß-Ocimene 1045 0.18 ± 0.01a 0.10 ± 0.07c 0.14 ± 0.00b 0.17 ± 0.02a 2.73 0.113ns
8 γ-Terpinene 1057 0.09 ± 0.07b 0.12 ± 0.05b 0.11 ± 0.02b 0.76 ± 0.35a 9.91 0.004**
9trans-Linalool oxide 1073 0.03 ± 0.00b 0.10 ± 0.04a 0.04 ± 0.00b 0.04 ± 0.00b 9.48 0.005**
10 Linalool 1099 0.38 ± 0.01b 1.54 ± 0.05a 1.00 ± 0.01ab 1.79 ± 0.61a 12.63 0.002**
11 trans-p-Mentha-2,8-dien-1-ol 1121 0.03 ± 0.00c 0.17 ± 0.00a 0.10 ± 0.01b 0.11 ± 0.01b 187.79 0.000***
12 cis-Limonene oxide 1135 0.01 ± 0.01c 0.10 ± 0.00a 0.06 ± 0.01b 0.08 ± 0.03ab 15.85 0.000***
13 Citronellal 1151 0.11 ± 0.00c 0.30 ± 0.02b 0.27 ± 0.02b 0.41 ± 0.01a 365.65 0.000***
14 Terpinen-4-ol 1177 0.03 ± 0.00d 0.10 ± 0.00a 0.05 ± 0.00c 0.08 ± 0.00b 520.58 0.000***
15 α-Terpineol 1191 0.18 ± 0.00d 0.52 ± 0.01a 0.27 ± 0.02c 0.36 ± 0.04b 109.04 0.000***
16 trans-Dihydro carvone 1202 0.01 ± 0.01b 0.13 ± 0.05 a 0.05 ± 0.00b 0.08 ± 0.01ab 11.67 0.002**
17 trans-Carveol 1219 0.08 ± 0.00c 0.43 ± 0.00a 0.25 ± 0.02b 0.29 ± 0.01b 333.50 0.000***
18 Nerol 1228 0.36 ± 0.06d 1.26 ± 0.08b 0.75 ± 0.12c 1.56 ± 0.05a 118.69 0.000***
19 Neral 1238 2.20 ±0.20c 5.87 ±0.13a 3.75 ±0.48b 3.69 ±0.04b95.20 0.000***
20 Geraniol 1254 0.22 ± 0.03c 0.90 ± 0.08b 0.67 ± 0.06bc 1.76 ± 0.39a 30.06 0.000***
21 Geranial 1269 2.88 ±0.23c 7.69 ±0.17a 4.87 ±0.69b 4.92 ±0.01b84.67 0.000***
22 Neryl acetate 1363 0.05 ± 0.01c 0.06 ± 0.01c 0.14 ± 0.00b 0.30 ± 0.03a 167.65 0.000***
23 Geranyl acetate 1382 0.07 ± 0.00b 0.07 ± 0.01b 0.14 ± 0.00b 0.39 ± 0.11a 20.10 0.000***
24 n-methyl-Methyl anthranilate 1408 0.05 ± 0.01b 0.10 ± 0.02a 0.07 ± 0.01b 0.07 ± 0.01b 11.53 0.002**
25 cis-a-Bergamotene 1413 0.01 ± 0.01b tr tr 0.17 ± 0.00a 811.86 0.000***
26 trans-α-Bergamotene 1434 0.20 ± 0.00a 0.09 ± 0.00c 0.17 ± 0.01b tr 748.99 0.000***
27 ß-Bisabolene 1506 0.28 ± 0.01a 0.14 ± 0.01b 0.25 ± 0.02a 0.28 ± 0.02a 68.20 0.000***
28 α-Bisabolol 1685 0.03 ± 0.01a 0.04 ± 0.00a 0.04 ± 0.00a 0.18 ± 0.11a 5.18 0.027*
Grouped compounds
Monoterpene hydrocarbons 92.10 ± 2.01a 78.96 ± 1.58 d 86.01 ± 3.62b 82.47 ± 1.25c 51.25 0.000***
Oxygenated monoterpenes 6.59 ± 0.58d 18.98 ± 0.95a 12.26 ± 1.20c 15.65 ± 1.02b 925.12 0.000***
Sesquiterpene hydrocarbons 0.48 ± 0.08a 0.23 ± 0.05c 0.42 ± 0.04b 0.45 ± 0.02a 251.32 0.000***
Oxygenated sesquiterpenes 0.03 ± 0.00b 0.04 ± 0.00b 0.04 ± 0.01b 0.18 ± 0.02a 94.82 0.000***
Others 0.08 ± 0.01d 0.24 ± 0.02a 0.10 ± 0.01c 0.14 ± 0.01b 840.02 0.000***
Total identified (%) 99.30 ± 0.10a 98.45 ± 0.05b 98.83 ± 0.13b 98.89 ± 0.03b 47.01 0.000***
a
RI: The retention Kovats index were determined on HP-5 capillary column.
b
In each row, means with similar letter are not significant at %5 level of Tukey test.
c
GM: green mature stage, INT: intermediate stage, MAT: mature stage, OR: over ripe stage.
d
Components less than 0.05% was not reported.
NS: not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
6
A. GHANI ET AL.

was 36.37% in the green mature stage, but decreased (32.07%) in the semi-ripe stage and
through maturity, its value increased slightly again (33.84%) (Wu et al. 2013), which is
consistent with the results of the present study. Increasing of limonene (90.92% and 93.98%)
measured of grapefruit during fruit maturity are reported from Iran (Ghani et al. 2009). In
various cases of research on citrus peel EOs, limonene has been identified as the main
compound. In a study on four citrus species, limonene appeared as the dominant compo-
nent of the EO in bitter orange fruits and varied from 67.90% to 90.95% during maturity.
The highest amount was recorded at the overripe stage. On the other hand, the amount of
limonene in lemon (C. limon) was highest at the first and third stages of maturity (GM and
MAT) as compared to the second stage (INT). In orange fruit, changes in limonene levels
during maturity did not follow a significant trend. In mandarin, the highest level of
limonene was measured at the ripe stage and the lowest level was observed at the second
stage (half-ripe) (Bourgou et al. 2012). In another study, limonene was reported as the main
component in mandarin EO in France (52.20–96.20%) (Lota et al. 2000). The high levels of
limonene in the EOs of the species and citrus varieties indicate that the composition is
dominant in citrus. In some species, even more than 90% of citrus EOs is comprised of
limonene, indicating that citrus peel can be a major source of limonene for industrial
purposes and health promoting (Fagodia et al. 2017; Zarrad et al. 2015; Simas et al. 2017).
In the present study, the big citron contained neral, geranial, myrcene, linalool and nerol as
the major compounds in its EO. However, the prominent compounds of small citron were
limonene, linalool, linalyl acetate, γ-terpinene and α-terpineol. The results reported by Wu
et al. (2013), showed that the highest amount of γ-terpinene (25.23%) was measured in the
essential oil of finger citron when harvested at the second stage (color-changing stage) and
the lowest amount was recorded in the GM stage (Wu et al. (2013). Research on Iranian
citron showed that the amount of γ-terpinene was much lower (approximately 4–6%), and
that its highest amount was measured in the GM stage, but which decreased during
maturity. The amounts of ß-pinene and 3-carene in Iranian varieties were much lower
than those in Chinese varieties. This is due to the differences in cultivars and climatic
conditions. On the other hand, the amounts of neral and geranial compounds in finger
citron cultivars were less than the amounts observed in Iranian cultivars (i.e. ranging from
1.04 to 1.60% and 1.42 to 2.26%, respectively). In the finger citron, the amount of linalool
was negligible (0.17%) and was not affected by maturity (Wu et al. (2013). According to
Figure 3. Limonene content fluctuation in citron EOs as relation to fruit maturity (GM: green mature, INT: intermediate, MAT:
yellow-ripe, OR: over ripe stage).
INTERNATIONAL JOURNAL OF ENVIRONMENTAL HEALTH RESEARCH
7

a relevant study in Tunisia, the amount of linalool was measured in four citrus species
during maturity. The amount of linalool was very slight in the different species except in
lemon which had a substantially higher amount of linalool (1.59%) (Bourgou et al. 2012).
Antioxidant activity of EO
The ripening phases had a significant effect on the antioxidant activity of the EOs. The
highest levels of antioxidant activity in the citron fruits was 76.08%, which were recorded at
the OR stage. There was no significant difference between the first and third stages of
maturity (GM and MAT), and the lowest level of antioxidant activity was recorded at the
INT stage (64.12 ± 0.43). From the second stage to the third maturity stage (INT to MAT),
the antioxidant activity decreased in this variety, but increased again at the OR stage
(Figure 2(b)). The antioxidant activity of finger citron was measured at three stages of
maturity (GM, INT and MAT) by using two methods (DPPH and H
2
O
2
). In both methods,
the highest antioxidant activity was observed in the GM stage, but declined through
maturity (Wu et al. 2013). The mentioned research is in contradiction to the current results
of this research. Variations in the biological activity, as attributed to the EOs obtained from
medicinal plants, may be due to their different chemical compositions, resources, phenolo-
gical stage (Azizi et al. 2010; Mohtashami et al. 2013; Afshari and Rahimmalek 2018)
conditions of cultivation, drying method (Ennajar et al. 2010) maturity or phenological
stage (Ghani et al. 2009; Saharkhiz et al. 2009, Dong et al. 2019) harvest time (Ozkan et al.
2010) and evaluation method. The antioxidant activity of any essential oil largely depends
on the dominant components of the essential oil. In certain circumstances, the compounds
may exhibit synergistic or antagonistic relations, with regard to the presence of minor
compounds in the EO (Wu et al. 2013; Dakhlaoui et al. 2022). Principal component analysis
(PCA) and cluster analysis (CA) results (Figure 4(a,b)) showed the existence of three groups.
In the macrocarpa variety, the INT and OR stages are in the same group and the other
stages are in two distinct groups, which shows that in this variety, after the fruit maturity to
very ripe stage, minor changes occur in terms of quantity and quality of essential oil
components. The present study revealed that fruits of lemon showed their highest
MG
MAT
OR
INT
MG
MAT
OR
INT
Euclidean distances
4,0 4,5 5,0 5,5 6,0 6,5 7,0
Linkage Distance
MAT
OR
INT
MG
a b
Figure 4. Relative position of maturity stages based on their EOs composition (Table 2) in the space defined by the three principal
components analysis (PCA, Figure 4 a) and cluster analysis (CA, Figure 4 b) (GM: green mature stage, INT: intermediate stage,
MAT: mature stage, OR: over ripe stage).
8
A. GHANI ET AL.

antioxidant activity at the OR stage, which is analogous to the decrease in limonene content.
Other compounds such as nerol, linalool, and linalyl acetate may have a synergistic relation-
ship with limonene, thereby causing the increase in antioxidant activity of EO at the OR
stage.
Conclusions
With regard to some traits, the quantitative and qualitative changes in the essential oil
indicated significant variations with ripening stages. EO analysis indicated that this variety
was limonene chemotype. On the other hand, the highest EO yield in macrocarpa variety can
be obtained in MAT stage (0.12 ± 0.01%). Since essential oils of citron fruits can potentially be
used in the food industry and for pharmaceutical purposes, it is very important to consider
the exact stage at which the extraction of EO would be most optimum (with considering the
quality of the essential oil). This can help reduce the costs of EO extraction. The qualitative
features of the essential oil are of prime importance. Limonene was the major component of
the essential oil in the citron varieties. Limonene can be extracted at the highest rate in GM
stage (89.39 ± 0.6%). The highest antioxidant activity of the EO was measured at the OR stage
(76.08 ± 0.51%), which is probably due to the presence of specific compounds in the EO and
their synergistic effects.
Acknowledgment
The authors are very grateful from Mrs. Bahmanzadegan for their cooperation (GC/MS guidance) in this project.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The author(s) reported there is no funding associated with the work featured in this article.
ORCID
Askar Ghani
http://orcid.org/0000-0002-0691-4351
Nehleh Taghvaeefard http://orcid.org/0000-0002-9576-6273
Mehdi Hosseinifarahi http://orcid.org/0000-0002-7398-6902
Sarra Dakhlaoui http://orcid.org/0000-0002-1538-0582
Kamel Msaada http://orcid.org/0000-0003-4826-8041
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