Little is known about the phytotoxic effects of essential oils from Pine species, GC and GC/MS analysis of Pinus
radiata D. Don essential oil obtained from needles, resulted in the identification of 49 components comprising 97.6 %
of the oil totality. The composition was l dominated by monoterpene hydrocarbons (86.4 %) with β-pinene (40.2 %),
limonene (25.5 %) and α-pinene (15.2 %) were the major compounds. On the herbicidal activity, the oil strongly
inhibited seed germination and seedling growth of all tested weeds in a dose dependent manner with the effect being
significantly more effective on dicots (Sinapis arvensis L. and Trifolium campestre Schreb) than monocots (Phalaris
canariensis L.).
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Cite this paper: Vietnam J. Chem., 2021, 59(2), 247-252 Article
DOI: 10.1002/vjch.202000103
247 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH
Essential oils of Tunisian Pinus radiata D. Don, chemical composition
and study of their herbicidal activity
Amri Ismail¹
*
, Kouki Habiba¹, Mabrouk Yassine¹, Hanana Mohsen
2
, Jamoussi Bassem
3
,
Hamrouni Lamia
4
1
Laboratory of Biotechnology and Nuclear Technology, National Center of Nuclear Science and Technology
(CNSTN), Sidi Thabet Technopark, Ariana, Tunisia
2
Plant Molecular Physiology Laboratory, Center of Biotechnology of Borj-Cedria, BP 901, 2050
Hammam-Lif, Tunisia
3
Chemistry Laboratory, Higher Institute of Education and Continuous Training, 43 Rue de la Libertelo
2019 Le Bardo, Tunisia
4
Laboratory for Forest Ecology, National Institute for Research in Rural Engineering, Water and
Forests, BP 10, 2080 Ariana, Tunisia
Submitted June 24, 2020; Accepted February 21, 2021
Abstract
Little is known about the phytotoxic effects of essential oils from Pine species, GC and GC/MS analysis of Pinus
radiata D. Don essential oil obtained from needles, resulted in the identification of 49 components comprising 97.6 %
of the oil totality. The composition was l dominated by monoterpene hydrocarbons (86.4 %) with β-pinene (40.2 %),
limonene (25.5 %) and α-pinene (15.2 %) were the major compounds. On the herbicidal activity, the oil strongly
inhibited seed germination and seedling growth of all tested weeds in a dose dependent manner with the effect being
significantly more effective on dicots (Sinapis arvensis L. and Trifolium campestre Schreb) than monocots (Phalaris
canariensis L.).
Keywords. Essential oils, Pinaceae, herbicidal activity, weeds.
1. INTRODUCTION
In the last decades, scientists have been carried out
to control plant diseases, particularly by the
development of chemical pesticides. Although
efficient, however their excessive applications in the
crop lands and environment to avoid harmful pests
resulted in an increased risk of enhanced pest
resurgence and development of resistance,
toxicological implications to non-target organisms
and increased environmental pollution.
[1]
In fact,
combating pollution and their harmful effects is a
necessity for the intervention of scientists. For this,
we must replace these synthetic pesticides with
biological molecules, which are safer and do not
induce any toxicological effects on the environment.
The biological control of plant pest and diseases
must be by using plant secondary metabolites, which
play an important role in plant resistance to pests
and several diseases. Therefore, screening plant
essential oils and plant extracts for their biological
activities could lead to discovery of new molecules
for pest control.
[2]
Pinus radiata D. Don belongs to
the Pinaceae family that comprises about 250
species which are divided into three subgenera,
based on needles, seeds and cones characters:
Ducampopinus, Strobus and Pinus. Pinus is the
largest genus of conifers occurring naturally.
[3]
Essential oil composition of P. radiata has been
previously studied by recent reports in Greece and
Italy.
[4,5]
According to these reports, the major
components of this oil were determined as β -pinene
(16.8-35.21 %) and α-pinene (11.06-21.9 %). The
antioxidant and radical-scavenging activities of P.
radiata oil have been evaluated by means of 1,1-
diphenyl-2-picrylhydrazyl assay, β-carotene
bleaching test and luminol-photochemiluminescence
assay. The antimicrobial properties of the oil were
tested on five food-spoilage yeasts.
[5]
Moreover,
knowing that production of essential oils depend on
several factors that geographical origin and genetic
background and to the best of our knowledge, the
Vietnam Journal of Chemistry Amri Ismail et al.
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 248
chemical composition of Tunisian P. radiata
essential oil has not been published and there is no
report on the herbicidal effects of P. radiata oil,
therefore the aims of this study are, in a first step, to
study the chemical composition of the essential oil
of P. radiata growing in Tunisia, and in a second
step, we evaluated their herbicidal activity against
the germination and seedling growth of weeds.
2. MATERIALS AND METHODS
2.1. Plant Material
The needles of Pinus radiata D. Don were collected
during month of October from the Souinet arboreta
of the National Institute of Researches on Rural
Engineering, Water and Forests. Five batches of
needles were taken from different trees were
harvested, and mixed to have a homogeneous
sample. The experimental site is characterized by a
humid climate at an altitude of 492 m. this site is
located in Ain Draham, governorate of Jendouba, in
the north of Tunisia. The plant was identified by Dr
Lamia HAMROUNI, National Institute of
Researches on Rural Engineering, Water and
Forests, TUNISIA and a sample (PR-1109) was
submitted to the herbarium division of the Institute.
2.2. Isolation of the essential oils
Three replications of 100 g of air-dried and finely
grounded needles were submitted to
hydrodistillation for 3 hours with 500 ml distilled
water using a Clevenger type apparatus. The volatile
oils were collected and dried over anhydrous sodium
sulfate and stored in sealed glass brown vials in a
refrigerator at 4 °C. Yield based on dried weight of
the sample was calculated (w/w %).
2.3. Gas chromatography and mass spectrometry
analysis
2.3.1. Gas chromatography analysis with FID
detection
The essential oils were analyzed using a Hewlett
Packard 5890 II GC equipped with Flame Ionization
Detector (FID) and HP-5 MS capillary column (5 %
phenyl/95 % dimethyl polysiloxane: 30 m×0.25 mm
id, film thickness 0.25 μm). The carrier gas was
nitrogen with a flow rate of 1.2 mL/min. Injector
temperature was set at 250 °C and at 280 for
detector. The oven temperature was kept at 50 °C for
1 min then programmed from 50 °C to 250 °C at 5
°C/min then, held isothermal for 4 min. Samples
were diluted in hexane (1/100V/V). Volumes of 1μL
were injected in the splitless mode. The relative
percentage of each component was calculated
electronically from FID area percent data.
2.3.2. Gas chromatography analysis with MS
detection
Analysis of P. radiata essential oil was carried out
using a Hewlett Packard 5890 II GC, equipped with
a capillary column HP-5 MS (30 m×0.25 mm, film
thickness 0.25μm) and mass selective detector HP
5972. The oven temperature was kept at 50 °C for 1
min then programmed from 50 °C to 250 °C at 5
°C/min and subsequently, held isothermal for 4 min.
The carrier gas was Helium at a flow rate of 1.2
mL/min. In GC/MS detection, an electron ionization
system with a scan time of 1.5 s and mass range 40-
300 amu with ionization energy of 70 eV was used.
Injector was set at 250 and transfer line temperatures
at 280 °C. Samples diluted in hexane (1/10 V/V) of
1μl were injected in the splitless mode. The
identification of oil components was based on mass
spectra (compared with Wiley 275.L, 6th edition
mass spectral library) or with standard compounds
and experienced by comparing their retention index
with those of authentic compounds or based on the
results published in the literature.
[6,7]
Other
confirmation was done from data generated from a
series of n-alkanes retention indices (C9-C28) on
HP-5 MS capillary column.
2.4. Seed germination and seedling growth
experiments
Mature seeds of weeds of Sinapis arvensis L.,
Phalaris canariensis L. and Trifolium campestre
Schreb were collected from parent plants growing in
fields in July. Seeds were sterilized with 15%
sodium hypochlorite for 20 min. Then, they were
rinsed with distilled water. Next, oils were dissolved
in tween–water solution 1 % (V/V). The final
concentrations of treatments were (0, 1, 2, 3, 4 and
6μL/mL). Solutions of 8 ml were transferred on the
layers of filter paper placed in the Petri dish.
Afterward, 20 seeds from each weed were placed on
the filter paper. Petri dishes were closed with an
adhesive tape and were incubated at 25 °C on a
growth chamber equipped with 12 h of light.
[8]
The
number of germinated seeds and seedling lengths
were measured after 10 days and all tests were
arranged in a completely randomized design with
three replications by treatment.
2.5. Statistical analysis
Vietnam Journal of Chemistry Essential oils of Tunisian Pinus radiate
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 249
Data of seed germination and seedling growth were
subjected to one-way analysis of variance (ANOVA)
using the SPSS 13.0 software package. Differences
between means were evaluated by Student -
Newman-Keuls test and means with values of P ≤
0.05 were considered significantly different.
3. RESULTS AND DISCUSSION
The hydro-distillation of dried Pinus radiata needles
gave yellowish essential oil (yield 0.23 %, w/w).
The chromatographic analysis showed a mixture of
components belonging four subclass of compounds
with a significant fraction of hydrocarbonated
sesquiterpenes and monoterpenes. The list of the
compounds, in order of their elution on the apolar
HP-5 MS column, and the quantitative data, are
reported in table 1.
49 oil compounds were identified accounting for
97.67 % of the total oil. Monoterpene hydrocarbons
displayed the highest contribution (86.44 %)
amongst which β-pinene (40.23 %), limonene (25.5
%) and α-pinene (15.22 %) were the most abundant.
Whereas oxygenated monoterpenes were
represented only by 3.88 %. In comparison with
monoterpenes, sesquiterpenes were relatively weak
(7.35 %); with 5.55 % of sesquiterpene
hydrocarbons and oxygenated sesquiterpenes were
the poorest fraction (1.8 %). The essential oil of P.
radiate was previously investigated in Greece and
Italy. The obtained results were in agreement with
our data; it was shown that β-pinene (16.8-35.21 %)
and α-pinene (11.06-35.21 %) were the major
components. However, limonene is most abundant
in oils from Tunisian P. radiate (25.5 %) than the
sample of Greece (4.42 %) and Italy (12.6 %).
[4,5]
These differences could be related to the
environmental factors (climate and soils), the genetic
diversity and the extraction conditions.
Table 1: Chemical composition of P. radiata essential oil
Peaks Compounds R.I.a R.I.b Area (%) Identification
1 tricyclene 926 1014 0.1 MS, RI
2 α-thujene 931 1030 0.1 MS, RI
3 α-pinene 939 1033 15.22 MS, RI, Co-inj
4 α-fenchene 950 1059 0.1 MS, RI
5 camphene 950 1068 0.4 MS, RI, Co-inj
6 β-pinene 976 980 40.23 MS, RI
7 β-myrcene 991 1152 1.39 MS, RI, Co-inj
8 α-phellandrene 1007 1160 0.25 MS, RI
9 δ-3-carene 1011 1148 1.46 MS, RI
10 α-terpinene 1016 1183 0.1 MS, RI
11 p-cymene 1026 1258 0.1 MS, RI
12 limonene 1033 1032 25.5 MS, RI
13 (Z)-β-ocimene 1040 1230 0.77 MS, RI
14 δ-terpinene 1062 1236 0.1 MS, RI, Co-inj
15 α-terpinolene 1088 1280 0.62 MS, RI
16 linalool 1098 1547 0.34 MS, RI
17 α-fenchol 1098 1571 0.1 MS, RI
18 (Z)-pinocarveol 1141 - 0.34 MS, RI
19 camphor 1143 1473 0.11 MS, RI
20 δ-terpineol 1163 1662 0.88 MS, RI
21 carvone 1198 1636 0.1 MS, RI
22 pinocarvone 1164 - 0.11 MS, RI
23 myrtenol 1176 1586 0.43 MS, RI
24 terpen-4-ol 1179 1571 0.16 MS, RI
25 α-terpineol 1196 1673 0.88 MS, RI
26 verbenone 1204 1733 0.13 MS, RI
27 (Z)-carveol 1219 - 0.14 MS, RI
Vietnam Journal of Chemistry Amri Ismail et al.
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 250
Peaks Compounds R.I.a R.I.b Area (%) Identification
28 iso-bornyl acetate 1278 1562 0.16 MS, RI
29 α-cubebene 1354 1458 0.17 MS, RI
30 α-ylangene 1372 1485 0.15 MS, RI
31 β-cububene 1409 1564 0.21 MS, RI
32 α- caryophyllene 1419 1588 0.2 MS, RI
33 β- caryophyllene 1420 1567 0.52 MS, RI
34 β-gurjenene 1423 1537 0.21 MS, RI
35 α-cedrene 1432 1428 0.14 MS, RI
36 β-farnasene 1464 - 0.59 MS, RI
37 germacrene-D 1480 1721 1.34 MS, RI, Co-inj
38 valencene 1490 - 0.12 MS, RI
39 (E)-α-bisabolene 1498 1715 0.15 MS, RI
40 α-murrolene 1499 1738 0.24 MS, RI
41 epi-zonarene 1501 1688 0.19 MS, RI
42 Δ-cadinene 1502 1772 0.78 MS, RI
43 α-amorphene 1527 1752 0.21 MS, RI
44 (Z)-nerolidol 1544 2032 1.07 MS, RI
45 germacrene-B 1552 1845 0.33 MS, RI
46 spathunelol 1576 2144 0.16 MS, RI
47 α-cadinol 1653 2225 0.14 MS, RI
48 farnesol 1724 2351 0.16 MS, RI
49 manoyl oxide 1993 2350 0.27 MS, RI
Yield (w/w) % 0.23
Total identified 97.67
Monoterpenes hydrocarbons
Oxygenated monoterpenes
86.44
3.88
Sesquiterpenes hydrocarbons 5.55
Oxygenated sesquiterpenes 1.8
RI: Retention Index, MS: mass spectrometry, Co-inj: co-injection,
a
Apolar HP-5 MS column,
b
Polar HP Innowax
column.
Herbicidal activity
Table 2 shows that the essential oil strongly
inhibited the germination and seedling growth of
tested weeds in a dose dependent manner with the
effect being significantly more effective on dicots
(S. arvensis and T. campestre) than monocots (P.
canariensis). In fact, at lower concentrations (from 1
to 3 μL/mL for dicots and from 1 to 4 μL/mL P.
canariensis), we noted a partial inhibition in
germination and seedling growth of weeds.
However, at high concentrations (4 μL/mL for dicots
and 6 μL/mL for P. canariensis), we noted a total
inhibition in germination and seedling growth of all
tested weeds. These results are in agreement with
literature.
[9]
Indeed, in recent reports, we have shown the
phytotoxic potential of some species essential oils
belonging different families that Pinaceae,
Cupressaceae and Anacardiaceae family.
[9-12]
According to these studies, Pine species are known
to possess a potent herbicidal activity. Recently, we
have demonstrated that P. Pinea and P. patula
displayed inhibitory effects against germination and
seedling growth of Sinapis arvensis, Lolium rigidum
and Raphanus raphanistrum.
[11-13]
Indeed, in our
present study, P. radiata oil was rich in
monoterpenes, especially α, β-pinene and limonene
which are known for their phytotoxic
effects.
[14]
Abrahim et al. (2001) have demonstrated
the phytotoxic of monoterpenes and it have shown
that exposure of seedlings to α-pinene reduce
seedling growth by increasing the lipid peroxydation
and causing oxidative damage in root, leading to
disruption of membrane integrity, uncoupling of
oxidative phosphorylation by acting as a
Vietnam Journal of Chemistry Essential oils of Tunisian Pinus radiate
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 251
protonophoric agent, and by inhibition of electron transfer chain.
[14]
Table 2: Herbicidal activity of P. radiata essential oil against germination and seedling growth of weeds
Weeds
Dose
(µL/ml)
Germination (%)
Seedling Growth (mm)
Aerial parts Roots
S. 0 98.33±2.88 a 12.23±1.12 a 12.63±0.7 a
arvensis 1 71.66±10.4 b 10±1 b 10.16±1.04 b
2 28.33±7.63 c 7.33±1.04 c 6.56±0.81 c
3 8.33±2.88 d 3.86±0.32 d 3.56±0.28 d
4 0±0 d 0±0 e 0±0 e
6 0±0 d 0±0 e 0±0 e
T. 0 91.66±7.63 a 9.73±0.75 a 11.93±0.81 a
campestre 1 65±5 b 8.5±0.5 b 8.83±0.76 b
2 33.33±5.77 c 5.96±0.45 c 5.8±0.72 c
3 13.33±2.88 d 2.6±0.36 d 2.6±0.36 d
4 0±0 e 0±0 e 0±0 e
6 0±0 e 0±0 e 0±0 e
P. 0 93.33±5.77 a 13.16±1.75 a 14.83±0.76 a
canariensis 1 78.33±2.88 b 7.16±1.04 b 12.16±1.25 b
2 53.33±7.63 c 4.66±0.76 c 9.5±0.86 c
3 33.33±7.63 d 3.56±0.51 c 5.33±0.76 d
4 15±5 e 2.86±0.8 c 2.36±0.4 e
6 0±0 f 0±0 d 0±0 f
4. CONCLUSION
Essential oils of Pinus radiata obtained by
hydrodistillation were composed of monoterpene
hydrocarbons. Our results showed that P. radiata
displayed a phytotoxic effect against germination
and seedling growth of weeds. According to our
knowledge, this is the first report regarding the
herbicidal activity against these three weeds.
However, the development of natural herbicides
would help to decrease the negative impact of
synthetic pesticides such as resistance and
environmental pollution. Based on our preliminary
results, the essential oils of forest trees particularly
pine species could be suggested as alternative
herbicides. But, other studies are required to study
the applicability, safety and allelopathic effects of
oil against crops.
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