Antioxidant Potential of Pine Needles: A Systematic Study on the Essential Oils and Extracts of 46 Species of the Genus Pinus

Abstract

The antioxidant activity of the essential oils, as well as of the organic and hydroethanolic extracts, of the fresh needles of 54 pine taxa was evaluated using the peroxy-oxalate and luminol chemiluminescence assays. Among all evaluated essential oils, P. canariensis and P. attenuata displayed the highest levels of activity. P. contorta var. murrayana, followed by P. nigra var. caramanica, exhibited the highest antioxidant capacity among the organic extracts, while the most active hydroethanolic extract was that of P. nigra subsp. nigra. Based on the overall levels of activity, the latter taxon was selected for phytochemical analysis targeting the isolation of the bioactive constituents. As such, the organic extract of P. nigra subsp. nigra was subjected to chromatographic separations to yield 11 secondary metabolites (1−11) that were evaluated for their antioxidant activity. Nonetheless, the isolated compounds were found to be less active than the crude extract, thus suggesting the potential role of synergism.

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Article
Antioxidant Potential of Pine Needles: A Systematic Study on
the Essential Oils and Extracts of 46 Species of the Genus Pinus †
Aikaterini Koutsaviti 1, Samer Toutoungy 2, Rouba Saliba 2, Sofia Loupassaki 2, Olga Tzakou 1,
Vassilios Roussis 1and Efstathia Ioannou 1,*


Citation: Koutsaviti, A.; Toutoungy,
S.; Saliba, R.; Loupassaki, S.; Tzakou,
O.; Roussis, V.; Ioannou, E.
Antioxidant Potential of Pine Needles:
A Systematic Study on the Essential
Oils and Extracts of 46 Species of the
Genus Pinus.Foods 2021,10, 142.
https://doi.org/10.3390/foods10010
142
Received: 18 November 2020
Accepted: 8 January 2021
Published: 12 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and
Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece;
kkoutsaviti@pharm.uoa.gr (A.K.); tzakou@pharm.uoa.gr (O.T.); roussis@pharm.uoa.gr (V.R.)
2Department of Food Quality and Chemistry of Natural Products, Mediterranean Agronomic Institute of
Chania—Centre International de Hautes Etudes Agronomiques Méditerranéennes, 73100 Chania, Greece;
tutunji@hotmail.com (S.T.); rouba.saliba@hotmail.com (R.S.); sofia@maich.gr (S.L.)
*Correspondence: eioannou@pharm.uoa.gr; Tel.: +30-210-727-4913
† Dedicated to the memory of Dr. Panagiotis Kefalas.
Abstract:
The antioxidant activity of the essential oils, as well as of the organic and hydroethanolic
extracts, of the fresh needles of 54 pine taxa was evaluated using the peroxy-oxalate and luminol
chemiluminescence assays. Among all evaluated essential oils, P. canariensis and P. attenuata displayed
the highest levels of activity. P. contorta var. murrayana, followed by P. nigra var. caramanica, exhibited
the highest antioxidant capacity among the organic extracts, while the most active hydroethanolic
extract was that of P. nigra subsp. nigra. Based on the overall levels of activity, the latter taxon was
selected for phytochemical analysis targeting the isolation of the bioactive constituents. As such,
the organic extract of P. nigra subsp. nigra was subjected to chromatographic separations to yield 11
secondary metabolites (
1−11
) that were evaluated for their antioxidant activity. Nonetheless, the
isolated compounds were found to be less active than the crude extract, thus suggesting the potential
role of synergism.
Keywords:
Pinus; pine needles; antioxidant activity; chemiluminescence; secondary metabolites;
chromatographic separations
1. Introduction
In recent years, interest towards finding new antioxidant agents derived from natural
sources has increased, since synthetic antioxidant compounds currently in use, such as
butylated hydroxy-anisole (BHA) and tertiary butyl hydroquinone (TBHQ), may induce
serious side effects (e.g., carcinogenesis) [
1
,
2
]. Apart from preventing food deterioration by
militating against the activity of reactive oxygen species, natural antioxidant agents may
also add nutritional value as functional food ingredients [3].
Pines are monoecious woody plants, mostly seen as tall trees and rarely as shrubs,
with distinctive needle-shaped evergreen leaves, encountered in a variety of terrestrial
environments and climatic zones in both hemispheres, mainly distributed over the northern
hemisphere, while they also occur in subtropical and tropical areas of Central America and
Asia [
4
,
5
]. The genus Pinus, including approximately 110 species [
4
,
5
], is important from
an ecological point of view, since its representatives form extended forests either in pure
stands or mixed with other conifers. Furthermore, from an economic point of view, pines
are a valuable source of nuts and seeds, as well as resin, but also of pulp and paper, timber
and construction materials.
The genus Pinus is a well-known source of antioxidants, mainly phenolic compounds,
including procyanidins and other flavonoids and phenolic acids, already available in
the market as food supplements or phytochemical remedies, such as Pycnogenol
™
, a
Foods 2021,10, 142. https://doi.org/10.3390/foods10010142 https://www.mdpi.com/journal/foods

Foods 2021,10, 142 2 of 19
standardized bark extract from Pinus maritima, with a remarkable array of biological
activities, used also in the treatment of chronic inflammation and circulatory dysfunction [
6
].
In the last 25 years, various pine extracts and preparations have exhibited significant health-
promoting activities, e.g., protective activity against alcohol-induced liver disease or against
lipopolysaccharide-induced inflammation, hippocampal memory-enhancing activity, and
activity for the early management of dyslipidemia, that can be potentially useful in food,
functional food, and food supplement industries [7–15].
Besides the traditional use of pine seeds for human consumption either as edible raw
nuts or in cooked dishes due to their high nutritional value and enticing taste [
7
], the use of
pines cones, needles, bark and oil as food or food ingredients has already been established
and accepted in the EU [
16
]. Pine needles have been used as herb tea in Estonian folk
medicine [
17
], while pine needle-based food products, such as pine needle powder, wine
and herbal teas, have become quite popular in the Korean food market [
18
]. It is worth
noting that in recent years, the use of pine needles in herbal teas and as an ingredient in
various food recipes has gained significant interest [19,20].
The aim of the present study was the investigation of the antioxidant potential of the
essential oils and extracts of fresh needles from 46 pine species, including 37 and 17 taxa of
the subgenera Pinus and Strobus, respectively, using two methods based on the measure-
ment of chemiluminescence (CL), with the prospect of finding new natural antioxidant
agents for the nutraceutical, food and food supplement industries, thus capitalizing on the
renewable pine needle biomass as a sustainable and eco-friendly approach.
2. Materials and Methods
2.1. General Experimental Procedures
NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers
(Bruker BioSpin GmbH, Rheinstetten, Germany). Chemical shifts are given on a
δ
(ppm)
scale using TMS as internal standard. The 2D NMR experiments (HSQC, HMBC, COSY,
NOESY) were performed using standard Bruker pulse sequences. Optical rotations were
measured on a Perkin Elmer model 341 polarimeter (PerkinElmer Instruments, Norwalk,
CT, USA) with a 1 dm cell. UV spectra were obtained on a Shimadzu UV-160A spec-
trophotometer (Shimadzu Europa GmbH, Duisburg, Germany). IR spectra were obtained
on a Bruker Tensor 27 spectrometer (Bruker Optik GmbH, Ettlingen, Germany). High-
resolution ESI mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos
mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Low-resolution EI and
CI mass spectra were measured on a Thermo Electron Corporation DSQ mass spectrom-
eter a Direct-Exposure Probe (Thermo Fisher Scientific, Bremen, Germany), using CH
4
as reagent gas. Normal- and reversed-phase column chromatography separations were
performed with Kieselgel Si 60 (Merck, Darmstadt, Germany) and Kieselgel RP-18 (Merck,
Darmstadt, Germany), respectively. HPLC separations were conducted on an Agilent
1100 liquid chromatography system equipped with a refractive index detector (Agilent
Technologies, Waldbronn, Germany) or a CECIL 1100 Series liquid chromatography pump
(Cecil Instruments Ltd., Cambridge, UK) equipped with a GBC LC-1240 refractive in-
dex detector (GBC Scientific Equipment, Braeside, VIC, Australia), using the following
columns: Kromasil 100 SIL 5
µ
m (MZ-Analysentechnik GmbH, Mainz, Germany, 250 mm
×
8 mm i.d.), Econosphere 100 SIL 10u (Grace, Columbia, MD, USA, 250 mm
×
10 mm
i.d.), Nucleosil SIL 50-7 (Macherey-Nagel GmbH & Co. KG, Düren, Germany, 250 mm
×
10 mm i.d.), Kromasil 100 C
18
5
µ
m (MZ-Analysentechnik GmbH, Mainz, Germany,
250 mm
×
8 mm i.d.) or Econosphere C
18
10u (Grace, Columbia, MD, USA, 250 mm
×
10 mm i.d.). Thin layer chromatography (TLC) was performed with Kieselgel 60 F
254
aluminum plates (Merck, Darmstadt, Germany) and spots were detected after spraying
with H
2
SO
4
in MeOH (15% v/v) reagent and heating at 100
◦
C for 1 min. All solvents
used for preparative and analytical purposes (CH
2
Cl
2
, MeOH, cHex, EtOAc, EtOH, MeCN)
were of analytical or HPLC grade from LAB-SCAN Analytical Sciences (Gliwice, Poland),
whereas CDCl
3
and CD
3
OD used for NMR spectroscopic analyses were from Deutero

Foods 2021,10, 142 3 of 19
GmbH (Kastellaun, Germany). Hydrogen peroxide (H
2
O
2
, 35%),
β
-carotene, quercetin and
CoCl
2.
6H
2
O were from Merck (Darmstadt, Germany), while 9,10-diphenylanthracene (9,10-
DPA), bis(2,3,6-trichlorophenyl) oxalate (TCPO), ethylenediaminetetraacetic acid (EDTA),
imidazole, luminol, NaOH and H3BO3were from Sigma-Aldrich (St. Louis, MI, USA).
2.2. Plant Material
Fresh needles of 54 taxa of genus Pinus, namely 37 taxa of subgenus Pinus and 17
taxa of subgenus Strobus, were collected from either well-documented wild localities or
from botanical gardens, as previously described [
21
]. Voucher specimens of the taxa
have been deposited at the Herbarium of the Section of Pharmacognosy and Chemistry
of Natural Products, Department of Pharmacy, National and Kapodistrian University of
Athens. The moisture content of the fresh needles ranged between 49–57%, as determined
after incubation in an oven at 80 ◦C for 6 h.
2.3. Isolation of Essential Oils
Fresh needles of each sample (30–50 g) were cut into small pieces (0.5–1 cm) and
separately subjected to hydro-distillation for 3 h using a modified Clevenger-type apparatus
with a water-cooled receiver, in order to reduce overheating artifacts. The isolated essential
oils were taken up in pentane, dried over anhydrous sodium sulfate and stored at 4
◦
C
until analyzed.
2.4. Preparation of Extracts
Fresh needles of each sample were cut into small pieces (0.5–1 cm) and divided into
two parts (A and B), of approx. 0.5 g each. Part A was macerated with 5 mL CH
2
Cl
2
/EtOH
(2:1) to prepare the organic extract, while part B was macerated with 5 mL EtOH/H
2
O (1:2)
to prepare the hydroethanolic extract. In both cases, extraction was repeated twice for 24 h
each time at 25
◦
C. After evaporation of the solvents in vacuo, the extracts were weighted
and stored at 4 ◦C until assayed.
2.5. Extraction and Isolation of Secondary Metabolites from P. nigra subsp. nigra
Fresh needles of P. nigra subsp. nigra (60.0 g) were exhaustively extracted with
CH
2
Cl
2
/EtOH (2:1) (three times with fresh volume of solvents; no additional amount of
residue was obtained afterwards) at 25
◦
C. After evaporation of the solvents in vacuo, the
crude extract (1.4 g) was subjected to gravity column chromatography on silica gel, using
cHex with increasing amounts of EtOAc, followed by EtOAc with increasing amounts
of MeOH as the mobile phase, to afford 16 fractions (1–16). Fractions 3 (28.8 mg), 4
(47.6 mg), 5 (24.9 mg) and 6 (49.5 mg) were separately purified by normal-phase HPLC,
using cHex/EtOAc (85:15) as eluent, to yield
1
(9.2 mg),
2
(0.5 mg),
7
(3.2 mg),
8
(6.8 mg),
9
(15.2 mg) and
β
-sitosterol (4.7 mg). Fractions 7 (60.8 mg) and 8 (34.7 mg) were separately
purified by normal-phase HPLC, using cHex/EtOAc (75:25) as eluent, to yield
3
(3.0 mg),
4
(6.9 mg),
6
(11.2 mg),
9
(6.8 mg) and
10
(0.7 mg). Fraction 10 (22.2 mg) was purified by
normal-phase HPLC, using cHex/EtOAc (70:30) as eluent, to yield
5
(4.7 mg). Fraction 14
(40.1 mg) was subjected to reversed-phase HPLC, using MeOH/H
2
O (50:50) as eluent, to
yield 11 (3.5 mg).
5,4
0
-Dihydroxy-3,6,7-trimethoxy-8-C-methylflavone (
10
): Yellow solid;
1
H NMR (CD
3
OD,
400 MHz)
δ
12.47 (s, 1H, 5-OH), 8.11 (dd, J= 8.8, 2.1, 2H, H-2
0
and H-6
0
), 6.97 (dd, J= 8.8,
2.1, 2H, H-3
0
and H-5
0
), 3.99 (s, 3H, 7-OMe), 3.92 (s, 3H, 6-OMe), 3.86 (s, 3H, 3-OMe), 2.15
(s, 3H, 8-Me);
13
C NMR (CD
3
OD, 50.3 MHz)
δ
157.8 (C-4
0
), 157.2 (C-7), 155.4 (C-2), 153.9
(C-8a), 138.9 (C-3), 132.6 (C-6), 130.4 (C-2
0
and C-6
0
), 123.3 (C-1
0
), 115.6 (C-3
0
and C-5
0
), 113.4
(C-8), 61.7 (6-OMe), 60.9 (7-OMe), 60.0 (3-OMe), 7.9 (8-Me).
2.6. Evaluation of Antioxidant Activity Using the Peroxy-Oxalate Chemiluminescence Assay
The antioxidant activity of the essential oils was evaluated using the peroxy-oxalate
chemiluminescence (POCL) assay, based on the measurement of CL as a result of the

Foods 2021,10, 142 4 of 19
oxidation of an aryl oxalate ester by H
2
O
2
in the presence of 9,10-DPA as a fluorophore
(activator) and developed for assessing the hydrogen peroxide scavenging activity of low
polarity hydrophobic samples [
22
]. Briefly, 0.2 mL of TCPO solution (0.45 mM) and 0.05 mL
of the sample solution (at least three different concentrations were tested) or solvent
(EtOAc) in the case of blank measurements were placed in a cuvette and immediately
1.8 mL 9,10-DPA solution (0.5 mM), 0.2 mL imidazole solution (4.5 mM) and 0.025 mL
H
2
O
2
solution (2.25 mM) were added and mixed well for 5 s. All solutions were prepared
in EtOAc/MeCN (9:1), with the exception of the sample solutions which were prepared
in EtOAc. CL was continuously monitored in a JENWAY 6200 fluorimeter (Jenway Ltd.,
Essex, UK), keeping the lamp off and using only the photomultiplier of the apparatus, until
the reaction reached a plateau and CL intensity was recorded.
2.7. Evaluation of Antioxidant Activity Using the Luminol Chemiluminescence Assay
The antioxidant activity of the organic and hydroethanolic extracts, as well as of the
isolated metabolites was evaluated using the luminol chemiluminescence (LCL) assay,
based on the measurement of CL as a result of the oxidation of luminol by H
2
O
2
in the
presence of cobalt (II) as a transition metal and EDTA as a metal chelator, and developed
for assessing the hydroxyl free radical scavenging activity of medium and high polarity
samples [
23
,
24
]. Briefly, 1 mL of Co(II)/EDTA solution (8.4 mM CoCl
2.
6H
2
O and 34.25 mM
EDTA) and 0.1 mL of luminol solution (0.56 mM) were placed in a cuvette and mixed well
for 15 s and subsequently 0.025 mL H
2
O
2
solution (5.4 mM) and 0.025 mL of the sample
solution (at least three different concentrations were tested) or solvent (MeOH) in the case
of blank measurements were added and mixed well for 15 s. The Co(II)/EDTA and luminol
solutions were prepared in borate buffer (H
3
BO
3
0.05 M, adjusted to pH 9 using NaOH 1 M),
the H
2
O
2
solution was prepared in H
2
O and the sample solutions were prepared in MeOH.
CL was continuously monitored in a LS-55 fluorescence spectrometer (PerkinElmer, Inc.,
Waltham, MA, USA), until the reaction reached a plateau and CL intensity was recorded.
2.8. Determination of Antioxidant Activity and Statistical Analysis
For both assays, an equation in the form I
0
/I = a
×
C
±
b was obtained by plotting
I
0
/I against C, where I
0
is the initial CL intensity recorded for the blank, I is the reduced
CL intensity recorded after the addition of the sample and C is the concentration of the
sample (in
µ
g/mL). Correlations were established using linear regression analysis (with a
coefficient R
2
> 0.98), employing Microsoft Office Excel 2007 software. Assignments a and
b represent the gradient and the intercept of the equation, respectively. The concentration
necessary to decrease the CL intensity by 50% (IC
50
) was calculated by setting I
0
/I = 2. All
measurements were performed at least in three independent experiments and data are
presented as mean ±SEM (standard error of the mean).
3. Results and Discussion
3.1. Evaluation of the Antioxidant Activity of Essential Oils
The antioxidant activity of the essential oils obtained from the fresh needles of 46
pine species, including 37 and 17 taxa of the subgenera Pinus and Strobus, respectively,
was evaluated using the POCL assay. According to the results of the evaluation (
Table 1,
Figure 1a)
, the IC
50
values of the pine needle essential oils ranged from 1.00
±
0.08 (P.
canariensis) to 20.03
±
2.77 (P. cembroides var. monophylla). Besides P. canariensis oil which ex-
hibited the most significant antioxidant activity, high levels of activity were also displayed
by the essential oils of P. attenuata (1.30
±
0.02), P. muricata (1.60
±
0.09), P. sylvestris var.
scotica (1.67
±
0.05), P. halepensis (1.78
±
0.17), P. mugo var. prostrata (1.79
±
0.21), P.mugo
(1.89
±
0.16) and P. monticola (1.94
±
0.09). As can be observed, with the exception of the
latter needle oil derived from a species belonging to the subgenus Strobus, the most active
essential oils were obtained from taxa of the subgenus Pinus.

Foods 2021,10, 142 5 of 19
Table 1.
Antioxidant activity (expressed as IC
50
in
µ
g/mL) of the essential oils, the organic (CH
2
Cl
2
/EtOH 2:1) and the
hydroethanolic (EtOH/H2O 1:2) extracts of the fresh needles of 54 Pinus taxa.
Taxon
IC50 (µg/mL)
Essential Oil Organic Extract Hydroethanolic Extract
Subgenus Pinus
Section Pinus
Subsection Pinaster
1P. brutia 4.67 ±0.14 0.27 ±0.01 0.20 ±0.02
2P. canariensis 1.00 ±0.08 0.29 ±0.05 0.20 ±0.01
3P. halepensis 1.78 ±0.17 0.30 ±0.03 0.90 ±0.02
4P. heldreichii 7.26 ±0.54 0.33 ±0.02 0.66 ±0.08
5P. pinaster 7.03±1.12 0.21 ±0.01 0.25 ±0.02
6P. pinea 4.40 ±0.37 0.30 ±0.03 0.40 ±0.02
7P. roxburghii 15.96±1.45 0.30 ±0.01 0.34 ±0.01
Subsection Pinus
8P. densiflora 4.45 ±0.40 0.17 ±0.01 0.28 ±0.01
9P. massoniana 8.29 ±0.41 0.20 ±0.02 0.41 ±0.02
10 P. mugo 1.89 ±0.16 0.19 ±0.01 0.34 ±0.01
11 P. mugo var.
prostrata
1.79 ±0.21 0.15 ±0.01 0.33 ±0.01
12 P. mugo var.
pumilio
3.42 ±0.06 0.27 ±0.01 0.24 ±0.01
13
P. nigra subsp.
caramanica
3.28 ±0.27 0.08 ±0.01 0.27 ±0.01
14
P. nigra subsp.
laricio
5.25 ±0.19 0.30 ±0.02 0.35 ±0.02
15
P. nigra subsp.
nigra
2.05 ±0.20 0.17 ±0.01 0.14 ±0.02
16
P. nigra subsp.
salzmannii
4.05 ±0.12 0.12 ±0.03 0.66 ±0.03
17 P. sylvestris 4.86 ±0.48 0.17 ±0.04 0.37 ±0.01
18 P. sylvestris
subsp. scotica
1.67 ±0.05 0.15 ±0.01 0.30 ±0.01
19 P. tabuliformis 3.97 ±0.62 0.30 ±0.01 0.22 ±0.04
20 P. taiwanensis 9.31 ±0.29 0.19 ±0.02 0.42 ±0.02
21 P. thunbergii 2.68 ±0.12 0.38 ±0.04 0.41 ±0.02
Section Trifoliae
Subsection Australes
22 P. attenuata 1.30 ±0.02 0.20 ±0.01 0.28 ±0.02
23 P. elliottii 3.97 ±0.35 0.32 ±0.01 0.52 ±0.03
24 P. muricata 1.60 ±0.09 0.24 ±0.01 0.40 ±0.03
25 P. patula 5.63 ±0.02 0.36 ±0.02 0.43 ±0.02
26 P. radiata 5.65 ±0.10 0.35 ±0.04 0.41 ±0.03
27 P. rigida 2.09 ±0.12 0.16 ±0.01 0.59 ±0.02
28 P. teocote 5.36±1.23 0.29 ±0.03 0.56 ±0.06
Subsection Contortae
29 P. banksiana 3.60 ±0.14 0.31 ±0.03 0.44 ±0.02
30
P. contorta var.
contorta
5.11 ±0.40 0.22 ±0.00 1.30 ±0.10
31
P. contorta var.
latifolia
9.57 ±0.64 0.31 ±0.02 0.22 ±0.01
32
P. contorta var.
murrayana
3.51 ±0.16 0.06 ±0.00 0.41 ±0.02
Subsection Ponderosae
33 P. coulteri 2.64 ±0.08 0.24 ±0.01 0.35 ±0.01
34 P. jeffreyi 3.72 ±0.39 0.20 ±0.00 0.57 ±0.03

Foods 2021,10, 142 6 of 19
Table 1. Cont.
Taxon
IC50 (µg/mL)
Essential Oil Organic Extract Hydroethanolic Extract
35 P. ponderosa 2.86 ±0.09 0.19 ±0.01 0.25 ±0.01
36 P. sabineana 9.05±1.25 0.31 ±0.04 0.36 ±0.05
37 P. torreyana 9.58 ±0.40 0.28 ±0.01 0.37 ±0.03
Subgenus Strobus
Section Parrya
Subsection Balfourianae
38 P. aristata 16.39±1.52 0.27 ±0.01 0.28 ±0.02
Subsection Cembroides
39 P. cembroides 2.38 ±0.16 0.41 ±0.02 1.43 ±0.11
40 P. culminicola 11.71±2.17 0.17 ±0.01 1.00 ±0.05
41 P. monophylla 20.03±2.77 0.39 ±0.01 0.66 ±0.04
Section Quinquefoliae
Subsection Gerardianae
42 P. bungeana 4.99 ±0.14 0.28 ±0.02 0.40 ±0.02
43 P. gerardiana 11.35±2.03 0.44 ±0.05 0.46 ±0.01
Subsection Strobus
44 P. armandii 4.95 ±0.17 0.58 ±0.03 0.48 ±0.07
45 P. cembra 2.36 ±0.05 0.40 ±0.02 0.67 ±0.02
46 P. flexilis 3.62 ±0.57 0.27 ±0.01 0.36 ±0.01
47 P. koraiensis 2.73 ±0.07 0.25 ±0.02 0.55 ±0.03
48 P. monticola 1.94 ±0.09 0.14 ±0.03 0.45 ±0.02
49 P. parviflora 7.04 ±0.44 1.34 ±0.01 0.38 ±0.03
50 P. peuce 4.04 ±0.26 0.35 ±0.01 0.67 ±0.03
51 P. pumila 4.24 ±0.27 0.19 ±0.01 0.75 ±0.06
52 P. strobiformis 2.68 ±0.15 0.16 ±0.02 0.47 ±0.10
53 P. strobus 11.54±3.27 0.22 ±0.04 0.35 ±0.02
54 P. wallichiana 2.23 ±0.12 0.43 ±0.04 0.49 ±0.04
β-carotene 0.23 ±0.01
quercetin 0.15 ±0.00
Analysis of the chemical composition of the essential oils evaluated for their antiox-
idant activity in the present study has shown that mono- and sesquiterpene derivatives
characterize the majority of the essential oils [
21
]. In most cases,
α
- and
β
-pinene were the
major representatives of the monoterpene fraction. However, occasionally
β
-phellandrene
and/or
δ
-3-carene were also present in high percentages. The sesquiterpene group was
characterized by germacrene D, while the levels of diterpenes varied notably. Germacrene
D was one of the common main metabolites among the three most active samples (P.
canariensis 44.0%, P. attenuata 29.0%, and P. muricata 41.5%), and while it was detected in
notably lower amounts in the essential oils of the following in activity order P. sylvestris
var. scotica (5.1%) and P. mugo var. prostrata (2.8%), its oxygenated derivative germacrene
D-4-ol reached a relatively higher percentage (10.0% and 6.0%, respectively). Instead, the
major metabolite in P. halepensis needle oil was
β
-caryophyllene (19.0%). It should be noted
though that no clear pattern correlating the antioxidant effect and the chemical composition
of the investigated essential oils can be observed overall. Thus, according to our results
and in agreement with the literature data [
25
], it can be deduced that the antioxidant
activity exhibited by our samples may be a result of synergism, since pinenes, ubiquitous
constituents of pine essential oils often appearing as major components, do not possess
antioxidant properties [
26
]. On the other hand, terpene derivatives such as germacrene D,
β-caryophyllene, and γ-terpinene have been reported to exert antioxidant activity [27].

Foods 2021,10, 142 7 of 19
Foods 2021, 10, x FOR PEER REVIEW 8 of 19
P. rigida, P. teocote, P. banksiana, P. contorta var. contorta, P. contorta var. latifolia, P. contorta
var. murrayana, P. coulteri, P. jeffreyi, P. ponderosa, P. sabineana, P. torreyana, P. aristata, P.
cembroides, P. culminicola, P. monophylla, P. bungeana, P. gerardiana, P. armandii, P. flexilis, P.
koraiensis, P. monticola, P. pumila, P. strobiformis, and P. strobus.
3.2. Evaluation of the Antioxidant Activity of Extracts
In the framework of the present study, two extracts of different polarity, namely an
organic extract resulting from maceration of the needles in CH2Cl2/EtOH (2:1) containing
less polar constituents and a hydroethanolic extract resulting from maceration of the nee-
dles in EtOH/H2O (1:2) containing more polar constituents, were prepared from the fresh
needles of 54 pine taxa and evaluated for their antioxidant potential using the LCL assay.
An overall comparison of the IC50 values of the investigated organic extracts (Table
1, Figure 1b) revealed the superiority of P. contorta var. murrayana of section Trifoliae (sub-
genus Pinus), followed by P. nigra subsp. caramanica and P. nigra subsp. salzmanii of section
Pinus (subgenus Pinus), along with P. monticola of section Quinquefoliae (subgenus Strobus),
with the organic extracts of the four taxa exhibiting stronger antioxidant activity than
quercetin.
Figure 1. Graphical representation of the antioxidant activity (expressed as IC50 in μg/mL) exerted by (a) the essential oils,
(b) the organic (CH2Cl2/EtOH 2:1) and (c) the hydroethanolic (EtOH/H2O 1:2) extracts of the fresh needles of 54 Pinus taxa,
in comparison to that of the positive control (CTR: β-carotene for (a) and quercetin for (b) and (c)). The various boxes
represent the following subsections: (i) Pinaster, (ii) Pinus, (iii) Australes, (iv) Contortae, (v) Ponderosae, (vi) Balfourianae, (vii)
Cembroides, (viii) Gerardianae, (ix) Strobus.
Figure 1.
Graphical representation of the antioxidant activity (expressed as IC
50
in
µ
g/mL) exerted by (
a
) the essential oils,
(
b
) the organic (CH
2
Cl
2
/EtOH 2:1) and (
c
) the hydroethanolic (EtOH/H
2
O 1:2) extracts of the fresh needles of 54 Pinus
taxa, in comparison to that of the positive control (CTR:
β
-carotene for (
a
) and quercetin for (
b
) and (
c
)). The various boxes
represent the following subsections: (i) Pinaster, (ii) Pinus, (iii) Australes, (iv) Contortae, (v) Ponderosae, (vi) Balfourianae, (vii)
Cembroides, (viii) Gerardianae, (ix) Strobus.
The antioxidant activity of the Aleppo pine (P. halepensis) needle oils from Algeria
was studied using four different assays, namely 2,2-diphenyl-1-picrylhydrazyl radical
scavenging (DPPH),
β
-carotene bleaching (BCB), iron (II) chelating ability employing the
Fe
2+
-ferrozine system (FICA) and potassium ferricyanide reducing power (PFRAP) assays,
and high levels of activity, especially for a specific chemotype rich in caryophyllene oxide,
were also observed, as in our case [
28
,
29
]. The high antioxidant potential of P. halepensis
essential oil was further verified by Postu et al. who observed remarkable activity in the
DPPH and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) radical cation scavenging
(or Trolox equivalent antioxidant capacity, ABTS/TEAC) assays [
30
]. The antioxidant
potential of P. mugo essential oil has been evaluated by Grassmann et al., employing
a variety of biochemical tests in both aqueous (e.g., Fenton system, xanthine oxidase-
induced superoxide radical formation) and more lipophilic environments (e.g., ACC-
cleavage by activated neutrophils in whole blood, copper-induced oxidation of low-density
lipoprotein), observing good antioxidant activity in more lipophilic rather than in aqueous
environments [
31
]. In contrast, Kurti et al. observed a rather low to moderate DPPH
radical scavenging activity for the needle essential oil of P. mugo from Kosovo [
32
]. High
to moderate activity, as observed in the present study, for the needle oil of the Himalayan
blue pine (P. wallichiana), was also noted by Dar et al. using the DPPH assay [
26
]. On

Foods 2021,10, 142 8 of 19
the other hand, the essential oil of the Swiss stone pine (P. cembra), which showed high
to moderate activity in our study, has previously exhibited rather weak DPPH radical
scavenging activity [
33
]. The needle essential oil of the Japanese black pine (P. thunbergii)
exerted a strong DPPH radical scavenging potential, as in our study, but insignificant
nitrite radical scavenging activity [
34
]. P. tabuliformis, which exhibited moderate levels of
activity in the present study, has also previously displayed moderate antioxidant activity
when evaluated using the DPPH, ABTS/TEAC and ferric reducing antioxidant power
(FRAP) assays [
35
]. In the study of Yener et al., the essential oil of P. brutia exhibited
strong iron (II) chelating ability and relatively lower levels of activity in the DPPH and
PFRAP assays, whereas the foliage essential oil of umbrella pine (P. pinea) displayed weak
iron (II) chelating ability, as well as weak reducing power [
36
]. Moreover, in the study
of Ustun et al., the essential oil of P. brutia exhibited weak activity in the PFRAP assay,
while P. sylvestris essential oil showed moderate iron (II) chelating ability [
37
]. P. sylvestris
essential oil and its fractions from Kosovo were also tested as DPPH radical scavenging
agents, displaying a weak to moderate potential [
32
]. In the same study, the needle oils
of P. nigra,P. peuce and P. heldreichii and their fractions were evaluated for their DPPH
radical scavenging activity, which was proven rather weak [
32
]. Similarly, P. heldreichii var.
leucodermis needle oil from central Herzegovina exhibited weak DPPH radical scavenging
activity [
38
]. The red pine needle oil (P. densiflora) has exerted a rather weak DPPH radical
scavenging potential, as well as nitrite radical scavenging ability [
34
]. The needle oil of
the maritime pine (P. pinaster) has been evaluated by Tümen et al. for its antioxidant
potential using the DPPH, ABTS/TEAC and FRAP assays, as well as for its hydroxyl
radical scavenging activity, displaying a rather moderate potential [
39
]. The Monterey pine
(P. radiata) needle oil, evaluated for its antioxidant capacity using the DPPH, BCB and LCL
assays, exhibited a rather moderate to weak activity in all three tests [
40
], similarly to our
results. The Japanese white pine (P. parviflora) needle oil has demonstrated weaker DPPH
scavenging activity compared to thymol, but strong hydroxyl radical scavenging activity in
reference to mannitol [
27
]. P. massoniana needle oil has exerted low to moderate antioxidant
potential, as determined using the DPPH, ABTS/TEAC and FRAP assays [
35
], while the
Chir pine (P. roxburghii) needle oil has showed weak DPPH radical scavenging activity [
41
].
To the best of our knowledge, this is the first report on the evaluation of the antioxidant
potential of the essential oils of P. canariensis,P. mugo var. prostrata,P. mugo var. pumilio,
P. nigra var. caramanica,P. nigra var. laricio,P. nigra subsp. nigra,P. nigra var. salzmanii,
P. sylvestris subsp. scotica,P. taiwanensis,P. attenuata, P. elliottii, P. muricata, P. patula, P.
rigida, P. teocote, P. banksiana, P. contorta var. contorta,P. contorta var. latifolia,P. contorta var.
murrayana, P. coulteri, P. jeffreyi, P. ponderosa, P. sabineana, P. torreyana, P. aristata,P. cembroides,
P. culminicola,P. monophylla,P. bungeana,P. gerardiana,P. armandii,P. flexilis,P. koraiensis,P.
monticola,P. pumila,P. strobiformis, and P. strobus.
3.2. Evaluation of the Antioxidant Activity of Extracts
In the framework of the present study, two extracts of different polarity, namely an
organic extract resulting from maceration of the needles in CH
2
Cl
2
/EtOH (2:1) containing
less polar constituents and a hydroethanolic extract resulting from maceration of the needles
in EtOH/H
2
O (1:2) containing more polar constituents, were prepared from the fresh
needles of 54 pine taxa and evaluated for their antioxidant potential using the LCL assay.
An overall comparison of the IC
50
values of the investigated organic extracts (Table 1,
Figure 1b) revealed the superiority of P. contorta var. murrayana of section Trifoliae (subgenus
Pinus), followed by P. nigra subsp. caramanica and P. nigra subsp. salzmanii of section Pinus
(subgenus Pinus), along with P. monticola of section Quinquefoliae (subgenus Strobus), with
the organic extracts of the four taxa exhibiting stronger antioxidant activity than quercetin.
The antioxidant activity evaluation of the hydroethanolic extracts (Table 1,
Figure 1c
)
showed that only P. nigra subsp. nigra exhibited a lower IC
50
value than quercetin. However,
significant levels of activity were also observed for the hydroethanolic extracts of P. brutia,

Foods 2021,10, 142 9 of 19
P. canariensis,P. tabuliformis,P. contorta var. latifolia,P. mugo var. pumilio,P. pinaster, and P.
ponderosa. All aforementioned taxa belong to the subgenus Pinus.
A number of studies employing different assays for the evaluation of the antioxi-
dant activity of various pine needle extracts have been undertaken and their results are
summarized in Table 2. Nonetheless, due to the different extraction protocols used in
these investigations, direct comparison of the results obtained in the current study is
not straightforward.
Table 2. Antioxidant activity of various needle extracts of Pinus taxa previously reported in the literature.
Species Extract Assay Activity Reference
P. brutia Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1165.8 ±0.06 µg/mL 9
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 131.89 ±0.02 µg/mL 9
Dry needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1327.5 ±0.08 µg/mL 9
Dry needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 118.38 ±0.06 µg/mL 9
Dry needles, Me2CO extract
DPPH 210.36 ±0.13–16.00 ±0.26% (at
250–1000 µg/mL) 10
[37]
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.316 ±0.042–0.889 ±0.011 (at
250–1000 µg/mL) 11
Dry needles, EtOAc extract
DPPH 214.14 ±0.45–28.27 ±0.26% (at
250–1000 µg/mL) 10
DMPD 32.15 ±0.56–12.66 ±2.14% (at
250–1000 µg/mL) 10
PFRAP 40.311 ±0.013–0.792 ±0.033 (at
250–1000 µg/mL) 11
Dry needles, EtOH extract
DPPH 213.41 ±0.19–25.59 ±0.19% (at
250–1000 µg/mL) 10
DMPD 3
inactive–7.72
±
1.24% (at 250–1000
µ
g/mL)
10
PFRAP 40.229 ±0.042–0.630 ±0.037 (at
250–1000 µg/mL) 11
Dry needles, MeOH extract
DPPH 227.5 ±0.4–85.0 ±0.8% (at 0.2-1.0 mg/mL) 10
[36]
PFRAP 40.119 ±0.009–0.438 ±0.008 (at
0.2–0.8 mg/mL) 11
FICA 521.5 ±0.4% (at 1.0 mg/mL) 12
P. halepensis Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1175.0 ±0.03 µg/mL 9
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 1inactive
Dry needles, Me2CO extract
DPPH 27.61 ±0.20–31.18 ±1.02% (at
250–1000 µg/mL) 10
[37]
DMPD 313.32 ±0.98–17.66 ±1.65% (at
250–1000 µg/mL) 10
PFRAP 40.330 ±0.008–0.941 ±0.018 (at
250–1000 µg/mL) 11

Foods 2021,10, 142 10 of 19
Table 2. Cont.
Species Extract Assay Activity Reference
Dry needles, EtOAc extract
DPPH 2inactive–21.05 ±0.71% (at
250–1000 µg/mL) 10
DMPD 35.43 ±1.44–9.96 ±0.57% (at
250–1000 µg/mL) 10
PFRAP 40.264 ±0.012–0.849 ±0.010 (at
250–1000 µg/mL) 11
Dry needles, EtOH extract
DPPH 28.98 ±0.79–18.39 ±1.22% (at
250–1000 µg/mL) 10
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.412 ±0.042–1.250 ±0.022 (at
250–1000 µg/mL) 11
Dry needles, MeOH extract DPPH 243.1 ±3.1–93.9 ±0.1% (at 0.2-1.0 mg/mL) 10
[36]
PFRAP 40.236 ±0.010–0.914 ±0.008 (at
0.2–0.8 mg/mL) 11
FICA 55.5 ±0.8% (at 1.0 mg/mL) 12
P. pinaster Dry needles, Me2CO (80%)
extract, filtrate ORAC 6478.8 ±32.8 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 6128.0 ±9.6 µM TE/g 13
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 660.2±7.1 µM TE/g 13
Fresh needles, n-Hex extract
DPPH 2203.28 µg/mL 9
[39]
ABTS/TEAC 7170.92 µg/mL 9
FRAP 816.28% (concentration not specified) 14
hydroxyl
radical
scavenging
158.26 µg/mL 9
Fresh needles, Me2CO extract
(sequentially)
DPPH 2171.12 µg/mL 9
ABTS/TEAC 7163.45 µg/mL 9
FRAP 819.74% (concentration not specified) 14
hydroxyl
radical
scavenging
192.35 µg/mL 9
P. pinea Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1161.8 ±0.07 µg/mL 9
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 1129.6 ±0.04 µg/mL 9
Dry needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 142.1 ±0.01 µg/mL 9
Dry needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 179.2 ±0.03 µg/mL 9
Dry needles, Me2CO (80%)
extract, filtrate ORAC 6901.5 ±35.2 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 670.9 ±0.9 µM TE/g 13

Foods 2021,10, 142 11 of 19
Table 2. Cont.
Species Extract Assay Activity Reference
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 639.7±5.5 µM TE/g 13
Dry needles, MeOH extract
DPPH 227.9 ±0.8–91.4 ±0.5% (at 0.2-1.0 mg/mL) 10
[36]
PFRAP 40.154 ±0.016–0.542 ±0.031 (at
0.2–0.8 mg/mL) 11
FICA 51.2 ±0.4% (at 1.0 mg/mL) 12
P. roxburghii Dry needles, n-Hex fraction of
MeOH extract DPPH 2inactive
[44]
Dry needles, CH2Cl2fraction
of MeOH extract DPPH 2163.45 µg/mL 9
Dry needles, EtOAc fraction of
MeOH extract DPPH 211.62 µg/mL 9
Dry needles, n-BuOH fraction
of MeOH extract DPPH 23.283 µg/mL 9
Dry needles, H2O fraction of
MeOH extract DPPH 2120.0 µg/mL 9
Dry needles, EtOH (95%)
extract ABTS/TEAC 70.57 mM (maximum TEAC content at
12.5 µg/mL)
[45]
Dry needles, n-Hex fraction of
EtOH (95%) extract ABTS/TEAC 7inactive
Dry needles, CHCl
3
fraction of
EtOH (95%) extract ABTS/TEAC 70.14 mM (maximum TEAC content at
12.5 µg/mL)
Dry needles, n-BuOH fraction
of EtOH (95%) extract ABTS/TEAC 70.38 mM (maximum TEAC content at
12.5 µg/mL)
Dry needles,
n-BuOH-insoluble fraction of
EtOH (95%) extract
ABTS/TEAC 70.57 mM (maximum TEAC content at
12.5 µg/mL)
P. densiflora
Dry needles, MeOH extract
DPPH 232.5 µg/mL 9
[46]
nitrite radical
scavenging 80.38 ±1.44% (at 10 µg/mL) 10
hydroxyl
radical
scavenging
−29.79 ±5.18% (at 40 µg/mL) 10
reactive oxygen
species (ROS)
scavenging
−392.80 ±21.3% (at 40 µg/mL) 10
Dry needles, CH2Cl2fraction
of MeOH extract
DPPH 245.4 µg/mL 9
nitrite radical
scavenging 21.36 ±1.04% (at 10 µg/mL) 10
hydroxyl
radical
scavenging
−357.45 ±10.4% (at 40 µg/mL) 10
reactive oxygen
species (ROS)
scavenging
−907.36 ±50.0% (at 40 µg/mL) 10
Dry needles, EtOAc fraction of
MeOH extract
DPPH 213.2 µg/mL 9
nitrite radical
scavenging 95.60 ±0.09% (at 10 µg/mL) 10

Foods 2021,10, 142 12 of 19
Table 2. Cont.
Species Extract Assay Activity Reference
hydroxyl
radical
scavenging
82.13 ±5.31% (at 40 µg/mL) 10
reactive oxygen
species (ROS)
scavenging
59.15 ±3.4% (at 40 µg/mL) 10
Dry needles, n-BuOH fraction
of MeOH extract
DPPH 224.3 µg/mL 9
nitrite radical
scavenging 82.28 ±1.89% (at 10 µg/mL) 10
hydroxyl
radical
scavenging
61.70 ±4.42% (at 40 µg/mL) 10
reactive oxygen
species (ROS)
scavenging
50.55 ±3.7% (at 40 µg/mL) 10
Dry needles, H2O fraction of
MeOH extract
DPPH 225.1 µg/mL 9
nitrite radical
scavenging 69.02 ±1.29% (at 10 µg/mL) 10
hydroxyl
radical
scavenging
27.66 ±0.43% (at 40 µg/mL) 10
reactive oxygen
species (ROS)
scavenging
40.38 ±3.20% (at 40 µg/mL) 10
Dry needles, Me2CO (80%)
extract, filtrate ORAC 6466.1±27.3 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 661.4±3.7 µM TE/g 13
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 655.3±2.8 µM TE/g 13
Dry needles, EtOH (95%)
extract
inhibition of
lipid
peroxidation
53.48 µg/mL 9
[47]
DPPH 295.12 µg/mL 9
Dry needles, H2O extract DPPH 2176.37±29.84 µg/mL 9
[48]
ABTS/TEAC 714.90 ±0.37 µg/mL 9
Dry needles, EtOH (20%)
extract
DPPH 283.70 ±6.22 µg/mL 9
ABTS/TEAC 79.02 ±0.55 µg/mL 9
Dry needles, EtOH (40%)
extract
DPPH 275.96 ±11.60 µg/mL 9
ABTS/TEAC 78.56 ±0.51 µg/mL 9
Dry needles, EtOH (60%)
extract
DPPH 278.46 ±7.99 µg/mL 9
ABTS/TEAC 79.12 ±0.43 µg/mL 9
Dry needles, EtOH (80%)
extract
DPPH 2126.47 ±4.38 µg/mL 9
ABTS/TEAC 711.80 ±0.08 µg/mL 9
Dry needles, EtOH (100%)
extract
DPPH 2373.70 ±60.67 µg/mL 9
ABTS/TEAC 719.76 ±1.32 µg/mL 9

Foods 2021,10, 142 13 of 19
Table 2. Cont.
Species Extract Assay Activity Reference
P. nigra Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1inactive
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 1174.6 ±0.15 µg/mL 9
Dry needles, Me2CO extract
DPPH 210.14 ±0.58–17.14 ±1.09% (at
250–1000 µg/mL) 10
[37]
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.273 ±0.022–0.893 ±0.078 (at
250–1000 µg/mL) 11
Dry needles, EtOAc extract
DPPH 212.91 ±0.26–24.36 ±1.80% (at
250–1000 µg/mL) 10
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.346 ±0.001–0.969 ±0.041 (at
250–1000 µg/mL) 11
Dry needles, EtOH extract
DPPH 214.41 ±1.09–28.36 ±0.77% (at
250–1000 µg/mL) 10
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.360 ±0.024–0.965 ±0.029 (at
250–1000 µg/mL) 11
Dry needles, MeOH extract
DPPH 234.0 ±2.1–92.5 ±0.4% (at 0.2-1.0 mg/mL) 10
[36]
PFRAP 40.163 ±0.002–0.586 ±0.008 (at
0.2–0.8 mg/mL) 11
FICA 521.3±2.1% (at 1.0 mg/mL) 12
P. sylvestris Dry needles, Me2CO (80%)
extract, filtrate ORAC 6560.0±36.3 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 691.7±3.2 µM TE/g 13
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 659.3±4.0 µM TE/g 13
Dry needles, Me2CO extract
DPPH 215.77 ±1.74–31.41 ±0.84% (at
250–1000 µg/mL) 10
[37]
DMPD 3inactive–4.22 ±0.11 (at 250–1000 µg/mL) 10
PFRAP 40.327 ±0.048–1.015 ±0.066 (at
250–1000 µg/mL) 11
Dry needles, EtOAc extract DPPH 28.32 ±0.19–13.55 ±0.01% (at
250–1000 µg/mL) 10
DMPD 3inactive (at 250–1000 µg/mL) 10
PFRAP 40.230 ±0.013–0.627 ±0.011 (at
250–1000 µg/mL) 11
Dry needles, EtOH extract
DPPH 222.64±1.41–45.86±1.35% (at
250–1000 µg/mL) 10
DMPD 33.03 ±0.45–14.57 ±1.91% (at
250–1000 µg/mL) 10
PFRAP 40.515 ±0.005–1.343 ±0.013 (at
250–1000 µg/mL) 11
P. attenuata Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1inactive
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 1144.1 ±0.01 µg/mL 9

Foods 2021,10, 142 14 of 19
Table 2. Cont.
Species Extract Assay Activity Reference
P. radiata Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F1 LCL 1228.1 ±0.02 µg/mL 9
[42]
Fresh needles, CHCl3/MeOH
(3:1) extract, organic phase F2 LCL 1inactive
P. cembra
Dry needles, MeOH (80%)
extract
DPPH 2186.1 ±1.7 µg/mL 9
[49]
ABTS/TEAC 724.0 ±0.2 µg/mL 9
PFRAP 4104 ±2µg/mL 9
FICA 51755 ±22 µg/mL 9
P. koraiensis Dry needles, Me2CO (80%)
extract, filtrate ORAC 6402.0±7.5 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 6111.6±6.2 µM TE/g 13
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 632.0±4.5 µM TE/g 13
P. strobus Dry needles, Me2CO (80%)
extract, filtrate ORAC 61223.3±12.6 µM TE/g 13
[43]
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/EtOAc-soluble
fraction
ORAC 682.3±3.1 µM TE/g 13
Dry needles, Me2CO (80%)
extract, alkaline hydrolysis of
the residue/H2O-soluble
fraction
ORAC 681.3±2.4 µM TE/g 13
P. wallichiana
Dry needles, n-Hex fraction of
MeOH extract DPPH 2inactive
[44]
Dry needles, CH2Cl2fraction
of MeOH extract DPPH 2inactive
Dry needles, EtOAc fraction of
MeOH extract DPPH 28.403 µg/mL 9
Dry needles, n-BuOH fraction
of MeOH extract DPPH 285.90 µg/mL 9
Dry needles, H2O fraction of
MeOH extract DPPH 2inactive
1
luminol chemiluminescence,
2
2,2-diphenyl-1-picrylhydrazyl radical scavenging,
3
N,N-dimethyl-p-phenylene diamine radical scavenging,
4
potassium ferricyanide reducing power,
5
iron (II) chelating ability employing the Fe
2+
-ferrozine system,
6
oxygen radical absorbance
capacity,
7
2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) radical cation scavenging or Trolox equivalent antioxidant capacity,
8
ferric
reducing antioxidant power,
9
expressed as IC
50
/ EC
50
in
µ
g/mL,
10
expressed as % of scavenging activity (at a given concentration),
11
expressed as absorbance at 700 nm (at a given concentration),
12
expressed as % of chelating ability (at a given concentration),
13
expressed
as µM Trolox equivalents (TE) per g dry weight, 14 expressed as % of reducing capacity (at a given concentration).
3.3. Phytochemical Analysis of P. nigra subsp. nigra and Evaluation of the Antioxidant Activity of
the Isolated Metabolites
In the current study, both organic and hydroethanolic extracts, as well as the essential
oil of the black pine (P. nigra subsp. nigra), were constantly among the most active samples
tested, with IC
50
values of 0.17
±
0.01, 0.14
±
0.02, and 2.05
±
0.20, respectively. Therefore,
phytochemical analysis of the black pine needle extract was undertaken, aiming at the
isolation of the metabolites responsible for the observed antioxidant activity.

Foods 2021,10, 142 15 of 19
A series of chromatographic separations of the organic extract of the fresh needles
of P. nigra subsp. nigra led to the isolation of compounds
1−11
(Figure 2), which were
identified as dehydroabietic acid (
1
) [
50
], 15-hydroxy-dehydroabietic methyl ester (
2
) [
50
],
8,12
α
-epidioxy-abiet-13-en-18-oic acid (
3
) [
50
], 15-hydroxy-8,12
α
-epidioxy-abiet-13-en-18-
oic methyl ester (
4
) [
50
], 15-hydroperoxy-8,12
α
-epidioxy-abiet-13-en-18-oic acid (
5
) [
50
],
15-hydroxy-8(17)-labden-18-oic acid (
6
) [
51
], 15-hydroxy-8(17)-labden-18-oic methyl ester
(
7
) [
51
], 15-oxo-8(17)-labden-18-oic acid (
8
) [
51
], 8(17)-labden-15,18-dioic acid 18-methyl
ester (
9
) [
51
], 5,4
0
-dihydroxy-3,6,7-trimethoxy-8-C-methylflavone (
10
) [
51
,
52
], (-)-catechin
(
11
) [
51
], a rare stereoisomer of catechin, and
β
-sitosterol [
51
] by comparison of their
spectroscopic and physical characteristics with those reported in the literature. Among
them, compounds
2
–
4
,
6
,
11
and
β
-sitosterol are reported for the first time from black pine,
whereas metabolite
10
is reported for the first time in Gymnospermae. It is worth noting
that the chemical structure of
10
, which has been reported from the leaves of three Vellozia
species and the fungus Colletotrichum dematium f.sp. epilobii, has been so far only tentatively
assigned [52,53].
Foods 2021, 10, x FOR PEER REVIEW 15 of 19
that the chemical structure of 10, which has been reported from the leaves of three Vellozia
species and the fungus Colletotrichum dematium f.sp. epilobii, has been so far only tenta-
tively assigned [52,53].
Figure 2. Structures of compounds 1–11 isolated from the organic extract of the fresh needles of P. nigra subsp. nigra.
The structure of compound 10 was elucidated after thorough analysis of its spectro-
scopic data. Specifically, according to the NMR and MS spectra, metabolite 10 was identi-
fied as a flavonol with a para-substituted ring B, bearing one aromatic methyl, two hy-
droxy and three methoxy groups. The positions of the functional groups were determined
after analysis of a standard set of six UV spectra [54]. In particular, in the presence of
NaOMe, band Ib exhibited a bathochromic shift of 56 nm with no decrease in intensity,
typical of the presence of a free hydroxy group at C-4′. Moreover, no small additional peak
or shoulder at 330 nm was observed, indicating the absence of a free hydroxy group at C-
7. With AlCl3 and AlCl3-HCl, bathochromic shifts of 25 nm and 24 nm, respectively, were
observed, diagnostic for the presence of 5-OH and 6-OMe in 3-O-substituted flavonols.
No shift was observed in band II in the presence of NaOAc, verifying the presence of 6-
OMe, as well as of a methyl group at C-8, also confirming a 7-O-substitution. The presence
of 7-OMe was confirmed by the fact that no shift was observed in the presence of NaOAc
and H3BO3. The proposed structure was further supported by the heteronuclear correla-
tions observed in the HMBC spectrum of metabolite 10. The 1H and 13C NMR chemical
shifts for compound 10 are reported herein for the first time, complementing the relevant
literature.
Metabolites 1–11 were subjected to evaluation of their antioxidant potential using the
LCL assay (Table 3). Phenolic compounds 10 and 11 displayed significant levels of activity
with IC50 values of 1.95 ± 0.21 and 1.34 ± 0.16 μg/mL, respectively, whereas the isolated
diterpenes showed moderate levels of activity (1 and 3) or were proven inactive (2 and 4–
9). The fact that both extracts of the black pine needles showed higher antioxidant activity
compared to that of the isolated compounds indicates that the higher antioxidant potential
of the extracts may be the result of synergism.
Figure 2. Structures of compounds 1–11 isolated from the organic extract of the fresh needles of P. nigra subsp. nigra.
The structure of compound
10
was elucidated after thorough analysis of its spec-
troscopic data. Specifically, according to the NMR and MS spectra, metabolite
10
was
identified as a flavonol with a para-substituted ring B, bearing one aromatic methyl, two
hydroxy and three methoxy groups. The positions of the functional groups were deter-
mined after analysis of a standard set of six UV spectra [
54
]. In particular, in the presence
of NaOMe, band Ib exhibited a bathochromic shift of 56 nm with no decrease in intensity,
typical of the presence of a free hydroxy group at C-4
0
. Moreover, no small additional peak
or shoulder at 330 nm was observed, indicating the absence of a free hydroxy group at C-7.
With AlCl
3
and AlCl
3
-HCl, bathochromic shifts of 25 nm and 24 nm, respectively, were
observed, diagnostic for the presence of 5-OH and 6-OMe in 3-O-substituted flavonols. No
shift was observed in band II in the presence of NaOAc, verifying the presence of 6-OMe,
as well as of a methyl group at C-8, also confirming a 7-O-substitution. The presence of
7-OMe was confirmed by the fact that no shift was observed in the presence of NaOAc and
H
3
BO
3
. The proposed structure was further supported by the heteronuclear correlations

Foods 2021,10, 142 16 of 19
observed in the HMBC spectrum of metabolite
10
. The
1
H and
13
C NMR chemical shifts for
compound
10
are reported herein for the first time, complementing the relevant literature.
Metabolites
1
–
11
were subjected to evaluation of their antioxidant potential using the
LCL assay (Table 3). Phenolic compounds
10
and
11
displayed significant levels of activity
with IC
50
values of 1.95
±
0.21 and 1.34
±
0.16
µ
g/mL, respectively, whereas the isolated
diterpenes showed moderate levels of activity (
1
and
3
) or were proven inactive (
2
and
4
–
9
).
The fact that both extracts of the black pine needles showed higher antioxidant activity
compared to that of the isolated compounds indicates that the higher antioxidant potential
of the extracts may be the result of synergism.
Table 3.
Antioxidant activity (expressed as IC
50
in
µ
g/mL) of compounds
1
–
11
isolated from the
organic extract of the fresh needles of Pinus nigra subsp. nigra.
Compound IC50 (µg/mL)
135.52 ±0.65
2>100
325.91 ±4.95
4>100
592.45 ±13.19
6>100
7>100
8>100
9>100
10 1.95 ±0.21
11 1.34 ±0.16
4. Conclusions
The antioxidant activity of the essential oils, as well as of the organic (CH
2
Cl
2
/EtOH
2:1) and hydroethanolic (EtOH/H
2
O 1:2) extracts of the fresh needles, from 54 pine taxa
was evaluated using the POCL and LCL assays. The extracts showed overall higher I
0
inhibition in comparison to the essential oils. Two samples from subgenus Pinus were
proven to be the most potent among the investigated essential oils, namely P. canariensis
(section Pinus) followed by P. attenuata oil (section Trifoliae), albeit with observed IC
50
values higher than that of the reference (
β
-carotene). The organic extracts of P. contorta var.
murrayana (section Trifoliae), followed by P. nigra subsp. caramanica (section Pinus), P. nigra
subsp. salzmanii (section Pinus), P. monticola (section Quinquefoliae), P. mugo var. prostrata
(section Pinus) and P. sylvestris subsp. scotica (section Pinus), exhibited the same or higher
levels of activity compared to the reference (quercetin). Among the hydroethanolic extracts,
however, only P. nigra subsp. nigra (section Pinus) demonstrated stronger antioxidant
activity than that of the reference (quercetin), albeit with several other taxa of subgenus
Pinus displaying significant levels of activity.
Based on the overall levels of activity, P. nigra subsp. nigra was selected for phytochem-
ical analysis targeting the isolation of the bioactive constituents. Among the secondary
metabolites isolated from the organic extract of the black pine needles, the abietane and
labdane diterpenes
1
–
9
were not active, whereas the two phenolic compounds
10
and
11
showed noteworthy levels of antioxidant activity. To the best of our knowledge, this is
the first report on the evaluation of the antioxidant activity of the needle essential oils and
extracts from 37 and 41 pine taxa, respectively.
Author Contributions:
Conceptualization, V.R. and E.I.; methodology, V.R. and E.I.; formal analysis,
A.K., S.T., R.S., S.L., O.T., V.R. and E.I.; investigation, A.K., S.T. and R.S.; resources, S.L., V.R. and E.I.;
writing—original draft preparation, A.K.; writing—review and editing, V.R. and E.I.; visualization,
A.K. and E.I.; supervision, O.T., V.R. and E.I.; project administration, V.R. and E.I.; funding acquisition,
V.R. and E.I. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was partially funded by the research projects MARINOVA (grant number
70/3/14684) and BioNP (grant number 70/3/14685).

Foods 2021,10, 142 17 of 19
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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