Essential oils of Tunisian Pinus radiata D. Don, chemical composition and study of their herbicidal activity

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 ... n 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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Corresponding author: AMRI Ismail 
Laboratory of Biotechnology and Nuclear Technology 
National Center of Nuclear Science and Technology (CNSTN) 
Sidi Thabet Technopark, Ariana, Tunisia 
E-mail: amri_amri@live.fr 
Tel.: 0021652832495.