Industrial Crops & Products 138 (2019) 111584

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Industrial Crops & Products

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Effects of cadmium and lead on seed germination, morphological traits, and T

essential oil composition of sweet basil (Ocimum basilicum L.)
Bahman Fattahia, Kazem Arzania, , Mohammad Kazem Souria, Mohsen Barzegarb
⁎

a
Department of Horticultural Science, Tarbiat Modares University (TMU), P.O. Box 14115-336, Tehran, Iran
b
Department of Food Science and Technology, Tarbiat Modares University (TMU), P.O. Box 14115-336, Tehran, Iran

ARTICLE INFO ABSTRACT

Keywords: The negative impact of contaminated soil with heavy metals on plant and human health is an important global
Environmental pollution concern. The aim of this study was to explore the influence of cadmium (Cd) and lead (Pb) on seed germination,
Heavy metals morphological traits, and essential oil (EO) composition of sweet basil (Ocimum basilicum L.). Two months prior
Medicinal plant to the experiment, soils pre-treated using Cd (0, 5, 10, 20) and Pb (0, 100, 200, 400) in mg kg soil−1. Seeds were
Stress
sown in the pots containing the contaminated soil under greenhouse conditions at 26 ± 6 °C and 60–70 %RH.
Vegetable crop
The amount and composition of EO were determined using Gas chromatography–mass spectrometry (GC-MS).
The contaminated soil had a negative impact on seed germination, leaf area, and flowering, stem growth and
plant dry mass. The extracted EO yield was varied (P > 0.05) among the applied treatments from 0.28 to 0.39%
(v/w). The GC-MS analysis of the EO identified the presence of 38 compounds. The major identified components
of the EO were included estragole (18.80–50.32%), 2, 6- octadienal (3.2–11.95%), caryophyllene oxide
(0.98–10.69%), caryophyllene (0.42–5.70%), phthalic acid (1.43–47.89%), and geranial (2.60–9.43%). The
canonical correspondence analysis (CCA) showed a positive correlation between the plant height and phthalic
acid contents. In conclusion, sweet basil cultivation in the Cd and Pb contaminated soils could cause undesirable
effects on the seed germination and morphological traits, but might be have a positive influence on the EO yield,
composition and phytoremediation of the soil.

1. Introduction Sweet basil is considered as an essential fresh vegetable and a

Contamination of arable soils with heavy metals is a global chal-
lenge in many parts of the world. In addition, environmental and public
health issues are the main concerns of human health regarding heavy
metal pollution. This pollution can have negative effects on the vege-
table crops and also consumers (Wuana and Okieimen, 2011). Chemical
fertilizers and municipal waste can result in the heavy metal build-up in
arable soil in big cities (Fattahi et al., 2017). Cadmium (Cd) and lead
(Pb) are major toxic heavy metals with an unknown biological role in
human life (Fattahi et al., 2017). Besides, it has been reported that Cd
and Pb are toxic to any biological system even at the low concentrations
(Shahid et al., 2017). The general toxic effects of Cd and Pb on plants
include inhibition of shoot and root growth, and reduction in photo-
synthesis, and further crop yield (Lone et al., 2008; Majer et al., 2002).
Moreover, accumulation of these heavy metals in different tissues of the
affected plants, particularly in the fresh vegetables can simply transfer
them into the human food chain, causing several health problems such
as brain and kidney diseases (Lone et al., 2008; Majer et al., 2002).
medicinal plant with various uses. According to some research results,
basil species have antibacterial, antioxidant, antifungal, anti-in-
flammatory, and many other beneficial effects on consumer health
(Tanrıkulu et al., 2018; Juliani et al., 2002). In addition, phenolic
compounds such as rosemarinic and cynamic acids, flavonoids and
anthocyanins, essential oils, and saponines are among the major bio-
chemical’s of sweet basil (Filip et al., 2017; Jayasinghe et al., 2003). As
reported by Chalchat and Özcan (2008), basil leaves and flowers have 1
and 0.5% oil, respectively. Estragole, 1,8-cineole, methyl eugenol, and
linalole are recognized as the main oil components of sweet basil (Azzaz
et al., 2018; Al Abbasy et al., 2015). Also, it has been reported that
estragole is one of the predominant compounds in basil oil that has anti-
inflammatory and anti-edematogenic activities (Rodrigues et al., 2016).
Different levels of Cd (0, 2.5, & 5 mg L−1) and Zn (0, 10, &
20 mg L−1) on sweet basil plants showed that large amounts of these
metals were accumulated in the shoots and roots after increasing their
concentrations in the soil. However, the content was considerably
higher in the roots than shoots for both Cd and Zn (Chaiyarat et al.,

⁎
Corresponding author.
E-mail address: arzani_k@modares.ac.ir (K. Arzani).

https://doi.org/10.1016/j.indcrop.2019.111584
Received 1 February 2019; Received in revised form 16 June 2019; Accepted 17 July 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

2011). Furthermore, Zheljazkov et al. (2006) found that increasing Besides, for uniform distribution of heavy metals, the soil was passed
copper (Cu) concentration (from 20 to 60 & 150 mg L−1) significantly several times through the 2 mm sieve. The moisture content of the soil
reduced plant dry mass, plant height (PH), and oil yield in dill plants as was adjusted at 60–65% field capacity (FC). Then, the soil was in-
compared to the control. They indicated that the concentration of cubated for two months at 24 ± 4 °C. Thereafter, the soil of each
150 mg L−1 was phytotoxic and inhibited dill plant growth. Moreover, treatment was placed into the black plastic pots containing 8–10 kg dry
plant leaf area and dry weight of the aerial parts in peppermint were soil and the seeds were sown in 1 cm depth. A total amount of 15 plants
reduced under Cd and Pb treatments as compared to the control plant per pot were allowed to grow while the remaining plants were re-
(Amirmoradi et al., 2012). As stated by Kranner and Colville (2011), moved. The pots were then kept under the greenhouse conditions at
heavy metals can induce toxicities at different growth stages of the 26 ± 6 °C temperature and 60–70% RH. The pots were irrigated in a
plants starting from germination. According to them, heavy metals such day intervals with purified tap water based on 80% FC. Therefore, the
as Cd, Cu, and arsenic (As) can inhibit seed germination even at the low plants were left to grow under the aforementioned conditions for three
concentrations. Likewise, Peralta et al. (2001) indicated that Lucerne months.
seed germination was very susceptible to the presence of heavy metals
as all the germination indices reduced under heavy metal treatments.
The effect of contaminated soil with different Cd and Pb levels on 2.2. Germination experiment
the leaf colorophyl pigments and photochemical efficiency of photo
system ІІ (Fv/Fm ratio) of sweet basil was evaluated and reported by The germination of sweet basil seeds was prepared for evaluation
Fattahi et al. (2017). This study was performed to evaluate the impact using Cd and Pb treatments in the different experiment, under the la-
of different Cd and Pb levels on seed germination and some morpho- boratory conditions. The basil seeds were first disinfected with sodium
physiological traits as well as EO composition of the sweet basil. In hypochlorate for 20 min and then were placed on a tissue paper in the
addition, the canonical correlation of the morphological traits and the Petri dishes each containing 20 seeds. The tissue papers were wetted
oil compositions under Cd and Pb treatments were examined. with different Cd (0, 1, 2, 4, 8, & 16 mg L−1) and Pb (0, 5, 10, 20, 40, &
80 mg L−1) solution concentrations (Table 1). Petri dishes were sealed
with cellophane covers to prevent water loss and seed infestations, then
2. Materials and methods placed in a germinator at 24 °C and 64% RH. The germinated seeds
were counted every day, so the minimum radicle length emergance
2.1. Experimental setup (RE) at 2 mm was considered as the germination count. The seeds
germination counts were continued for 20 days. The quantity and
This experiment was conducted under greenhouse conditions at the quality traits for seed germination were evaluated as follows (Fattahi
Department of Horticultural Science, Tarbiat Modares University et al., 2011):
(TMU), Iran. Sweet basil seeds of a local population were purchased Germination start (GS) = T2 – T1 (1), where T1 is the day of seed
from Pakan Seed Co., Isfahan, Iran. The suitable fertile soil with the sowing, and T2 is the day of beginning of first seed germination;
following physical and chemical characteristics was used in the ex- Germination percentage = (n/N)×100 (2), where n denotes to the total
periment. The soil texture was silty loam with 23, 29 and 48% clay, number of germinated seeds and N shows the number of total seeds
sand and silt, respectively. The pH of the soil was 7.6 with 3.28 dS m−1 sowed; Also, the Germination rate (GR) was calculated using the fol-
EC and 0.07%, 0.68%, and 199 mg.kg-1 and 7.5 mg.kg-1 for nitrogen lowing equation as described by Burgert and Burnside (1972):Germi-
(N), carbon (C), potassium (K) and phosphorus (P), respectively. The nation rate (GR) = ∑(number of germinated seeds till n-1)/n (3), where n
concentration of cadmium and lead in the soil was 0.49 and 0.97 mg.kg- is incubation days.
1
respectively.
Note that, two months prior to seed sowing, different concentrations
of Cd (0, 5, 10 and 20 mg kg−1 dry soil) and Pb (0, 100, 200 and 2.3. Determination of morphological traits
400 mg kg−1 dry soil) were uniformly applied into the soil. The related
codes for the applied treatments presented in Table 1. In order to pre- Plants were grown for three months and thereafter various morpho-
pare the contaminated soil for the experiment, nitrate salts of Cd and Pb physiological traits were measured. Morphological traits were recorded
were dissolved in distilled water (DW) and sprayed into the soil. at the flowering stage, including plant height (PH, in cm), the sec-
ondary stems length (SSL, in cm), leaf length (LL, in cm), leaf width
Table 1 (LW, in cm), root and shoot fresh and dry weights (RFW, SFW, RDW, &
The related codes for the applied treatments on sweet SDW, in gr), internodes length (IL, in cm), flowering stem length (FSL,
basil (Ocimum basilicum L.). in cm), collar diameter (CD, in mm), stem diameter (SD, in mm), and
leaf area index (LAI in cm2). The leaf area for each treatment was
Treatments Related Codes
calculated using a digital leaf area meter (Nguy-Robertson et al., 2012).
Cadmium 0 Control
Cadmium 5 Cd5
Cadmium 10 Cd10 2.4. Soil and plant analysis for cadmium and lead
Cadmium 20 Cd20
Lead 0 Control
The pre-contaminated soil for the experiment was analyzed for Cd
Lead 100 Pb100
Lead 200 Pb200 and Pb before treatments applications. Also, at the end of the experi-
Lead 400 Pb400 ment and after destructive plants harvest, the pot soil was re-analyzed
Cadmium 1 Cd1 for the amount of Cd and Pb based on the DTPA (diethylene triamine
Cadmium 2 Cd2
penta-acetic acid) method that described previously by Beckett (1989).
Cadmium 4 Cd4
Cadmium 8 Cd8 Note that, for Cd and Pb measurements in the leaves and roots, plants
Cadmium 16 Cd16 materials were first oven dried at 60 °C for 72 h and then ground
Lead 5 Pb5 powdered. Tissue powder (0.5 g) was digested in 25 ml nitric acid
Lead 10 Pb10 (65%), and Cd and Pb concentrations were determined according to the
Lead 20 Pb20
method described by Sekabira et al. (2011) using atomic absorption
Lead 40 Pb40
Lead 80 Pb80 spectrometry (Flow Injection Analysis System 400AA 100; Perkin-
Elmer, Waltham, MA, USA).

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B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

2.5. Essential oil extraction (EO)

The EO extraction was carried out in 50 g dried shoot samples after

hydro-distillation using the Clevenger-type apparatus for 4 h. The EO
extract was kept in the sealed vial under the dark at 4 °C until time of
analysis. The amount of EO content (% v/w) was recorded based on the
EO oil volume per air-dried sample (Fattahi et al., 2016).

2.6. Essential oil analysis

Essential oil samples were analyzed using gas chromatograph (GC),

according to the method described in detail for the type, column etc. by
Fattahi et al. (2016). A 1 μL of the sample was dissolved in diethyl ether
as a solvent then injected in the GC. Retention indices (RI) were cal-
culated at the same conditions using the retention times of the injected
n-alkenes (C6-C24). In order to identify the compounds, their RI was
compared with those reported in the literature by Davies (1990) and
Adams (2007). The mass spectra of the compounds were obtained using
Agilent MSD Chemstation Libraries according to the installation of the
national institute of standards and technology (NIST). The compound
percentages were calculated using the area normalization method, ex-
cluding the response factors (Fattahi et al., 2016).

2.7. Statistical analyses

The data were subjected to analysis of variance (ANOVA) using

statistical analysis system software (SAS) version 9.02. The significant
differences were calculated using the least significance difference (LSD)
and the differences were considered statistically significant at P ≤ 0.05
(Varasteh et al., 2012). In addition, canonical correspondence analysis
Fig. 1. Effects of cadmium (Cd) and lead (Pb) on A; germination percentage B;
(CCA) was used to study phytochemical components as influenced by germination rate and C; germination start of sweet basil (Ocimum basilicum L.).
morphological traits (Fattahi et al., 2016). The CCA and related scatter Mean values of four replications ± standard error of the mean.
plots were monitored by the Paleontological Statistics software (PAST).

3.2. Morphological traits

3. Results and discussion
The results of soil analysis showed that the soil used in this ex-
periment had a relatively neutral pH with a low level of heavy metals
3.1. Effect of Cd and Pb on germination indices
such as Cd and Pb. In addition, there was a significant difference
(P < 0.05) among the applied treatments in terms of morphological
The different Cd and Pb concentrations were applied in-vivo in order
traits. The effects of different Cd and Pb concentrations on sweet basil
to monitor the effect of these elements on the percentage, rate, and basil
growth are presented in Table 2. The variation coefficient was the
seeds germination (Fig. 1). Germination of basil seed in control treat-
lowest for PH (CV = 6.21%) while it was the highest for IL
ment began after 4.66 days from the beginning of the experiment and
(CV = 32.46%). The IL ranged from 3.82 to 7.90 cm, with minimum
an increase in Cd and Pb concentrations led to an increase in germi-
and maximum values recorded for Cd5, Pb100, respectively. The LAI
nation time to 7.66 and 10 days (Fig. 1A).
per plant varied from 190.25 to 211.25 cm2, with the maximum value
Germination percentage of the control treatment was 81.33%.
found in the control.
However, it reduced to 4% by increasing the Cd concentration up to
Note that, if basil is cultivated for fresh consumption, the high
16 mg L-1. In addition, using Pb concentration up to 80 mg L−1 sig-
secondary stem length (SSL) is considered as an undesirable trait.
nificantly decreased seed germination percentage to 9.33% (Fig. 1B). In
However, cultivation of basil under the uncontaminated soil can be
other words, there was a general decrease in germination rate by in-
important in producing high quality vegetables. The SSL with minimum
creasing the concentrations of both elements since the highest germi-
and maximum values were recorded in control (2.14 cm) and Pb400
nation rate was observed in control (1.69 seed per day). Besides, the
(5.79 cm), respectively. Flowering stem in the control appeared later
lowest germination rate was detected in 16 mg L−1 Cd. Moreover, using
than the other treatments, which was due to the impact of con-
Pb at 40 and 80 mg L−1 reduced the germination rate (0.19 seed per
taminated soil for earlier flowering. Heavy metals stress is probably
day) as compared to the control treatment (Fig. 1C). It has been re-
believed to produce the ethylene biosynthesis enzyme, ACC-Synthase,
ported a similar results in the reduction of seed germination rate along
so ethylene stimulates flower buds in the plants (Apelbaum and Yang,
with the increase in the seed abscisic acid (ABA), following the increase
1981). In addition, early flowering from plants subjected to drought
in the Cd, Pb, and Hg application concentrations (Munzuroğlu et al.,
stress condition reported by Siddique et al. (2003). Note that, the crisp
2008). In addition, several researchers reported that using heavy metals
and freshness of the vegetables considered as qualitative trait. In the
can inhibit seed germination (Munzuroglu and Geckil, 2002; Li et al.,
present research, stem diameter (SD) in Cd and Pb treatments was more
2005; Deng et al., 2016). Furthermore, when plants grow in low con-
than control treatment (Table 2). Although, the stem dry weight (SDW)
centrations of heavy metals such as Cd, Cu, and As, their seeds dor-
was the highest and lowest in the control (15.2 g) and Cd10 (7.9 g)
mancy were prolonged under the room temperature (Kranner and
treatments, respectively (Table 2). It has been reported that the effect of
Colville, 2011).

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B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

Table 2
Means comparisons of morphological traits (Mean values of four replications ± standard error of the mean) of sweet basil (Ocimum basilicum L.) under cadmium (Cd)
and lead (Pb) concentrations (mg. kg−1 soil).
R Characteristics* Unit Control Cd5 Cd10 Cd20 Pb100 Pb200 Pb400 CV (%)

1 PH cm 27.07 ± 0.49 ab 25.98 ± 0.14b 27.20 ± 0.35ab 28.41 ± 1.38ab 26.18 ± 0.99ab 28.75 ± 1.05a 26.27 ± 1.12ab 6.21
2 SSL cm 2.14 ± 0.52c 3.84 ± 0.79bc 5.00 ± 0.55ab 4.18 ± 0.22ab 4.48 ± 0.78ab 4.27 ± 0.65ab 5.79 ± 1.83a 30.82
3 IL cm 5.93 ± 0.44ab 3.82 ± 0.18b 4.21 ± 0.41ab 5.59 ± 0.47ab 7.90 ± 3.38a 5.24 ± 0.63ab 6.24 ± 0.48 ab 32.46
4 FSL cm 4.98 ± 0.18b 9.60 ± 0.33a 8.73 ± 0.22a 8.93 ± 1.28a 8.65 + 0.13 a 8.33 ± 0.81a 7.92 ± 1.54a 22.00
5 LL cm 2.35 ± 0.02ab 1.73 ± 0.01c 1.84 ± 0.06c 2.50 ± 0.15ab 2.01 ± 0.13bc 2.43 ± 0.22ab 2.55 ± 0.31a 15.66
6 LW cm 1.30 ± 0.02a 0.88 ± 0.01b 0.95 ± 0.04b 1.29 ± 0.09a 1.00 ± 0.07a 1.26 ± 0.12a 1.30 ± 0.12a 14.59
7 CD mm 1.21 ± 0.05c 1.37 ± 0.04bc 1.58 ± 0.19abc 1.97 ± 0.07a 1.77 ± 0.19ab 1.79 ± 0.15a 1.89 ± 0.16a 16.18
8 SD mm 0.91 ± 0.02c 1.09 ± 0.01c 1.38 ± 0.22ab 1.54 ± 0.07a 1.47 ± 0.22ab 1.42 ± 0.12ab 1.61 ± 0.14a 19.11
9 SDW gr 15.2 ± 0.95a 9.50 ± 1.31bc 7.90 ± 0.29c 10.77 ± 0.52b 8.92 ± 0.39bc 10.35 ± 0.90bc 9.07 ± 1.31bc 18.34
10 LAI cm2 211.25 ± 1.31a 198.00 ± 3.62b 191.25 ± 1.93b 190.25 ± 3.52b 203.75 ± 4.15b 202.50 ± 3.52b 201 ± 2.19b 10.37

*
PH = plant height; SSL = secondary stems length; IL = internode length; FSL = flowering stem length; LL = leaf length; LW = leaf width; CD = collar diameter;
SD = stem diameter; SDW = shoot dry weight; LAI = leaf area index.

Cd and Pb in EO chemical compounds in peppermint reduced leaf area
and dry weight of the aerial parts in Cd and Pb treatments in com-
parison with control (Amirmoradi et al., 2012).
3.3. Soil and plant analysis for cadmium and lead
Results showed that by increasing Cd and Pb concentrations in the
soil, the higher content of these metals was observed in the leaf and root
samples (Fig. 2a, 2b). In addition, Cd and Pb content in the leaf were
higher than in the root samples. Also, lead concentration in plant tissues
was higher than Cd concentration (Fig. 2). It has been reported that the
world allowable standard levels of Cd and Pb in basil leaves are 0.1 and
0.3 mg.kg−1 plant dry weight, respectively (Sharma and Prasad, 2010).
In the present research, Cd and Pb concentrations in the leaf and root
tissues of basil plants were higher than the standard. So, it is not sug-
gesting for fresh consumption, those basil plants that grown under
heavy metals polluted soils. The higher concentrations of Cd and Pb in
the leaf sample tissues that recorded from this experiment will suggest
possible using basil plant for the purpose of phytoremediation of con-
taminated soils. It has been shown in the other experiment that some of
medicinal plants can be used for phytoremediation of contaminated
soils (Zheljazkov, et al. 2006). The high uptake rates of arsenic by
different basil species (O. basilicum, O. tenuiflorum and O. gratissimum)
showed high arsenic concentration in the leaves and roots, suggested
that basil plantation might be a suitable plant for arsenic remediation
(Siddiqui et al., 2013).
3.4. The percentage of EO
The EOs isolated among the samples in this experiment was yellow
in color and showed over the range of 0.28-0.39% (v/w). In addition,
the amount of EO was varied among the different treatments
(P > 0.01). The highest and lowest amount of EO was belonged to the
Cd20 (0.39%) and control samples (0.28%), respectively. An increase in
Cd and Pb concentration enhanced the EO yield. The average EO per-
centage for different treatments shown in Fig. 3. Padalia et al. (2017)
reported 0.53% EO yield that obtained from the basil at flowering stage.
The report from the other experiment indicated that, the effect of 80%
FC water stress in comparison with the 100% FC resulted to the increase
in the basil EO content, while the 60% FC treatment reduced EO yield
extract (Sirousmehr et al., 2014).
3.5. EO composition
The identified chemical components of sweet basil are provided in
Table 3. In the studied samples about 38 of these components re-
presented about 90.14–97.74 % of the oil (Table 3). A sample GC-MS
chromatogram of sweet basil and mass spectra of some components are
shown in Fig. 4. Oxygenated monoterpene and sesquiterpene hydro-
carbons formed the major part of the studied EO yield. Ladwani and
Salman (2018) reported the similar results regarding oxygenated
monoterpene (60.76%) and sesquiterpene hydrocarbons. In another
study, monoterpene containing compounds were found as the pre
dominant components of basil EO (more than 70%). In addition, Telci
et al. (2006) evaluated the variation of basil oil chemical composition

Fig. 2. Cadmium (Cd) and lead (Pb) concentrations in soil, leaf and root tissues of basil plants in Cd treatments (A) and Pb treatments (B) after destructive plants
harvest. Mean values of four replications ± standard error of the mean.

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B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

and observed that CCA oxygenated monoterpene (63.7–85.6%) was the

Fig. 3. The quantity of essential oil (EO) of sweet basil (Ocimum basilicum L.)
grown under cadmium (Cd) and lead (Pb) treatments. Mean values of four re-
plications ± standard error of the mean.
major compound of the EO. In the present research, the elevation of Pb
contamination from 0 to 400 mg kg−1 of the soil increased the number
of oxygenated monoterpene compounds from 40.6 to 61.8%. Moreover,
as shown in Table 3, after Cd treatment, Cd5 (42.74) and Cd10 (55.24)
showed higher monoterpenic compounds than Cd20 (26.87%). It has
been reported that the application of Pb (0, 50, 100, 500, and
1000 ppm) enhanced oxygenated monoterpenic compounds in Cymbo-
pogon citrates (Lermen et al., 2015). In addition, in the present research
an increase in Cd and Pb concentration led to the reduction of the
amounts of sesquiterpene hydrocarbons. Also, results obtained in the
control plants indicated that estragole, phthalic acid, 2,6- octadienal,
caryophyllene oxide, and caryophyllene are the main components of
the sweet basil EO yield. In addition, in the present research the in-
crease in soil Pb from 0 to 400 mg kg−1 has resulted in 110% increase in
estragole production. Despite, the proven role of estragole as medicinal
treatment, its higher concentration on rodent animals showed the car-
cinogen effects. However, the carcinogenic effect of estragole on human
has not been yet reported (Clarke, 2008). Estragole in the presence of
other EO components has less toxicity, and can be used without pro-
blem for its inflammatory effects as cream or ointment application

Table 3
Essential oil components percentage of sweet basil (Ocimum basilicum L.) grown under cadmium (Cd) and lead (Pb) concentrations (mg. kg−1 soil).
R Compounds Empirical Formula RI Control Cd5 Cd10 Cd20 Pb100 Pb200 Pb400

1 α- Thujene C10H16 845 0.84 0.61 0.52 0.23 0.93 0.71 0.52
2 α-Pinene C10H12O 928 2.46 0.51 0.41 0.25 0.61 0.53 0.39
3 1- Octan-3-ol C8H16O 963 0.84 0.27 0.14 0.12 0.14 0.22 0.32
4 6-Methyl-5- hepten C8H14O 995 0.49 0.35 0.21 t 0.22 0.12 t
5 β-myrcene C10H16 981 t 0.58 t t 1.21 t t
6 β-pinene C10H16 984 0.54 0.2 t 0.10 1.32 0.25 0.20
7 L-Limonene C10H16 1028 t 0.2 0.12 t 1.14 0.13 t
8 1,8- Cineole C10H18O 1032 0.22 0.1 t 0.12 1.13 t 0.15
9 Octanal C8H16O 1005 0.19 0.12 0.10 0.10 1.70 0.11 t
10 Octanol C8H18O 1010 0.10 0.21 0.25 0.33 0.40 0.58 0.36
11 Linalool C10H18O 1099 0.18 0.42 0.25 0.38 0.20 0.32 0.38
12 Fenchone C10H16O 1093 t – t t 2.12 0.38 t
13 Heptadienal C7H10O 1124 t – 0.1 t 2.18 0.21 0.15
14 Estragole C10H12O 1213 23.69 24.87 41.99 18.80 27.76 28.78 50.32
15 2,6- Octadienal C8H12O 1232 6.93 11.95 11.20 4.06 4.26 14.76 3.2
16 Nerol C10H18O 1234 2.48 2.27 2.19 1.68 2.59 2.42 2.20
17 Fenchole C10H18O 1242 t 0.10 0.15 0.18 0.15 – –
18 Cyclohexanedim ethanol C8H16O2 1254 0.22 0.25 – – t t –
19 Geraniol C10H18O 1252 2.32 4.52 3.21 2.53 2.13 0.91 1.93
20 Geranial C10H16O 1274 9.43 8.95 6.92 2.60 6.24 8.54 6.31
21 Neryl acetate C12H20O2 1366 0.22 1.89 0.62 0.85 0.45 0.25 0.44
22 Geranyl acetat C12H20O2 1368 0.39 0.97 0.46 0.61 0.61 0.55 0.48
23 α-Copaene C15H24 1369 0.54 0.91 t t t 0.62 t
24 Methyl eugenol C11H14O2 1442 2.47 2.37 3.29 0.45 0.94 1.50 1.79
25 Caryophyllene C15H24 1451 5.70 3.98 4.67 1.21 1.79 3.20 0.42
26 β- Farnesene C12H20O2 1459 – 0.68 0.67 0.21 0.24 0.92 1.32
27 β- Selinene C15H24 1491 2.28 0.94 1.16 0.23 1.42 0.83 0.33
28 Germacrene D C15H24 1495 0.66 t 1.06 t 1.45 0.42 t
29 β- Bisabolene C15H24 1498 0.96 0.48 2.40 t – 0.88 0.49
30 γ-Cadinene C15H24 1514 t 0.16 0.21 0.25 1.31 0.33 1.44
31 Cis-α-Bisabolene C15H24 1544 2.58 2.15 1.85 1.33 0.32 1.81 0.45
32 Caryophyllene oxide C15H24O 1584 7.39 10.69 7.41 0.99 1.21 0.98 9.28
33 Farnesole C15H26O 1592 t 0.21 0.41 1.67 1.21 0.42 0.65
34 Neophytadiene C20H38 1605 0.52 0.65 1.2 3.13 1.75 1.85 1.92
35 Humulene epoxide II C15H24O 1610 2.66 1.26 t 0.32 t 0.21 2.27
36 Oxabicyclo dodeca C15H24O 1625 0.21 2.41 1.30 1.12 0.56 1.77 2.15
37 2-Pentadecanone C15H30O 1642 0.17 0.22 0.14 0.12 0.12 t t
38 Phthalic acid C8H6O4 1680 20.06 8.32 1.43 47.89 20.33 14.57 0.42
Monoterpene hydrocarbons – – 1.38 1.59 0.52 0.45 4.6 0.71 0.52
Oxygenate monoterpene – – 40.60 42.74 55.24 26.87 44.72 42.26 61.8
Sesquiterpene hydrocarbons – – 12.72 12.51 12.65 1.81 4.50 8.09 2.71
Oxygenate Sesquiterpene – – 3.43 10.90 8.63 4.22 3.10 3.38 14.35
Diterpede hydrocarbons 0.52 0.65 1.20 3.13 1.75 1.85 1.92
Others – – 39.09 26.38 17.80 55.38 31.47 32.79 8.98
Total (%) – – 97.74 94.77 96.04 91.86 90.14 90.08 90.28
Eo quantity % (v/w) – – 0.28 0.34 0.34 0.39 0.30 0.32 0.38

RI*: retention index; t: trace (0.1 >).

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B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

Fig. 4. Gas chromatography–mass spectrometry (GC-MS) peaks and mass spectra spectrum of some essential oil (EO) major components of sweet basil (Ocimum
basilicum L.).

6
B. Fattahi, et al. Industrial Crops & Products 138 (2019) 111584

(Yadav et al., 2013). The safe range of 40–87% chavicol (estragole) in Table 4
basil oil has been reported by Clarke (2008). Basil plants containing Canonical coefficients, Eigen-values and variance for three canonical corre-
22–88% estragole can be effectively used for aromatherapy (Clarke, spondence analysis (CCA) sets between phytochemicals with morphological
2008). In addition, estragole reported that has anti insect activity, and traits of sweet basil (Ocimum basilicum L.).
can be used as a natural insecticide (Ling Chang et al., 2009). Traits CCA sets of Morphological factors
In present research, all other EO compounds are listed in Table 3. In
addition, the obtained data showed that the raise in the Cd con- 1 2 3

centration in the soil was led to the increase of the compounds such as Essential oil compounds
linalool, octanol, nerol, neryl acetate, γ-cadinene, caryophyllene oxide, Estragole 1.61 −0.56 −1.09
farnesol, and neophytadiene. Besides, it decreased compounds likeα- Nerol 0.84 1.57 −1.48
thujene, α-pinene, 1, 8- cineole, octanal, geranial, methyl eugenol, β- 2,6- Octadienal 0.21 3.80 −2.36
Geraniol 0.38 1.73 4.24
Selinene, cis-α-bisabolene, humulene epoxide II, and phthalic acid
Geranial −0.34 0.86 2.50
(Table 3). Furthermore, increasing Pb concentration enhanced the Methyl eugenol 0.41 2.91 1.15
compounds such as octanol, linalool, nerol, neryl acetate, car- Caryophyllene −1.13 4.92 1.13
yophyllene oxide, neophytadiene, and oxabicyclododeca while de- Cis-α-Bisabolene −1.40 4.25 0.57
Caryophyllene oxide 3.66 2.54 1.14
creasing other compounds like α-pinene, 1- Octan-3-ol, 6-methyl-5-
Phthalic acid −3.00 0.13 −1.75
hepten, β-pinene, 1,8- cineole, octanal, geranial, geranyl acetat, methyl Control 0.20 0.01 0.04
eugenol, caryophyllene, β- selinene, germacrene D, β- bisabolene, cis-α- Cd5 0.12 0.17 0.08
bisabolene, and phthalic acid compared the control treatment (Table 3). Cd10 0.26 0.10 0.01
Prasad et al. (2011) examined the influence of heavy elements on basil Cd20 −0.46 −0.12 −0.17
Pb100 −0.04 −0.14 0.01
oil found that treatment of Cr (chromium), Cd, Pb, and Ni (nickel) in-
Pb200 −0.01 0.07 −0.03
creased methyl chavicol whereas decreasing the amount of linalole in Pb400 0.32 −0.11 0.03
sweet basil. The Cr treatment in Ocimum tenuiflorum L. increased the Morphological factor
biosynthesis of the eugenol (Amirmoradi et al., 2012(. However, Plant Height −0.53 −0.01 −0.75
Sub Stems Length 0.59 −0.27 −0.10
Siddiqui et al. (2013) found no specific process on arsenic treatment in
Internodes’ Length −0.23 −0.84 −0.11
Ocimum tenuiflorum L., Ocimum basilicm L., and Ocimum gratissimum L. Flowering Stem Length 0.68 0.04 −1.46
Leaf Length −0.06 −0.58 −1.12
Leaf Width −0.19 −0.47 −0.97
3.6. Principal component analysis (PCA)
Collar Diameter 0.51 −0.84 −2.12
Stem Diameter 0.71 −0.96 −2.23
The PC1 and PC2 scatter plots were applied to monitor the phyto- Dry Weight Shoot −1.01 0.03 1.43
chemical distance (Fig. 5). The scatter plots showed the phytochemical Leaf Area Index −0.07 −0.31 0.25
distances among the samples within the plot, influenced by their re- Eigen-value 0.031 0.009 4.750
Variance (%) 76.3 23.7 0.001
lationships, the studied populations were divided into three groups. The
EO of the control, Pb100, and Cd20 formed an individual treatment
separated from other samples and characterized by higher phthalic acid
presented a positive amount of nerol (0.84) while showing a negative
(38). In treatment, Cd5 and Pb200 formed other isolated groups with
amount of phthalic acid (-3.00) (Table 4). In other words, the basil
higher 2, 6- octadienal (15). The rest of the EO compounds formed in
plant with higher SSL and FSL had high nerol versus low phthalic acid
another group (Fig. 5).
contents. In the second canonical sets, treatments with low IL, SD, and
CD had higher caryophyllene and cis-α-bisabolene contents. The third
3.7. The CCA among morphological and EO traits canonical is demonstrated in Table 4. The scatter plots of CC1 and CC2
were employed to determine more associations between the morpho-
The CCA was employed to explore the influence of morphological logical and phytochemical traits. As it is illustrated in Fig. 6, a positive
characters on the phytochemical composition. The first studied cano- correlation was found between PH of the morphological trait and
nical sets of EO to morphological traits (more than 76%) indicated that phthalic acid contents. Estragole positively correlated with plants
the treatments with positive and notable values of SSL and FSL

Fig. 5. Classification of seven treatments applied on sweet basil (Ocimum basilicum L.) based on the essential oil (EO) chemical composition with biplot of first two
components.

7
B. Fattahi, et al.
Fig. 6. Canonical correspondence analysis biplot in the percentage of the major essential oil (EO) components with the morphological traits of sweet basil (Ocimum
basilicum L.).
containing higher SSL. In addition, methyl eugenol, 2, 6- octadienal,
geraniol, and nerol had a negative correlation with the IL (Fig. 6).
4. Conclusion
The growth of sweet basil was significantly affected by the con-
taminated soil with Cd and Pb. In addition, sweet basil cultivation in
the contaminated soils and in the fields that are irrigated with waste
water can cause undesirable effects on the seed germination and also
plant morpho- physiological traits. In such situation, plants producing
with higher SSL and early flowering which are not suitable for fresh
consumption. However, Basil cultivation in contaminated soils by
heavy metals could have a positive influence on phyto- remediation.
The amount of EO yield produced under the stresses of heavy metals,
and the EO components that have a high correlation with the amount of
Cd and Pb contaminations.
Declaration of Competing Interest
There are no conflicts of interest.
Acknowledgments
We would like to thank Tarbiat Modares University (TMU) for fi-
nancial support. This work was supported under PhD Student Grant
Program by TMU. In addition, greenhouse and laboratory facilities
provided by Pomology Lab., Department of Horticultural Science at
TMU are acknowledged.
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