molecules

Article
Synergistic Interactions between Tocol and Phenolic Extracts
from Different Tree Nut Species against Human Cancer
Cell Lines
Jazmín C. Stevens-Barrón 1,2, *, Abraham Wall-Medrano 3 , Emilio Álvarez-Parrilla 1 ,
Imelda Olivas-Armendáriz 4 , Humberto Astiazaran-García 5 , Ramón E. Robles-Zepeda 6
and Laura A. De la Rosa 1, *

1 Department of Chemical-Biological Sciences, Institute of Biomedical Sciences,

Autonomous University of Ciudad Juarez, Ciudad Juárez 32310, Mexico; [email protected]
2 Department of Veterinary Sciences, Institute of Biomedical Sciences,
Autonomous University of Ciudad Juarez, Ciudad Juárez 32310, Mexico
3 Department of Health Sciences, Institute of Biomedical Sciences, Autonomous University of Ciudad Juarez,
Ciudad Juárez 32310, Mexico; [email protected]
4 Department of Physics and Mathematics, Institute of Engineering and Technology,
Autonomous University of Ciudad Juarez, Ciudad Juárez 32310, Mexico; [email protected]
5 Department of Nutrition, Food and Development Research Center A.C., Hermosillo 83304, Mexico;
[email protected]
6 Department of Chemical-Biological Sciences, University of Sonora, Hermosillo 83000, Mexico;
Citation: Stevens-Barrón, J.C.; [email protected]
Wall-Medrano, A.; Álvarez-Parrilla, * Correspondence: [email protected] (J.C.S.-B.); [email protected] (L.A.D.l.R.)
E.; Olivas-Armendáriz, I.;
Astiazaran-García, H.; Abstract: Tree nuts are rich in polar (phenolic compounds) and non-polar (tocols) antioxidants,
Robles-Zepeda, R.E.; De la Rosa, L.A. with recognized effects in the prevention of diseases such as cancer. These biomolecules possess
Synergistic Interactions between antiproliferative activity on cancer cells; however, the combined effect of both types of compounds
Tocol and Phenolic Extracts from has been scarcely studied, and this approach could give valuable information on the real anticancer
Different Tree Nut Species against potential of tree nuts. In the present study, the antiproliferative activity of pure tocols and phenolic
Human Cancer Cell Lines. Molecules
compounds, tocol- and phenolic-rich extracts (TRE and PRE, respectively) from tree nuts and the
2022, 27, 3154. https://doi.org/
extracts combinations, was evaluated in four cancer (HeLa, MCF7, PC3, A549) and one control
10.3390/molecules27103154
(ARPE) cell lines. The most sensible cell lines were HeLa and MCF7. TRE and PRE from nuts
Academic Editors: were chemically characterized; γ and δ tocopherols, total tocols, total tocopherols and total phenolic
M. Mizerska-Kowalska, Wojciech compounds were negatively correlated with cell viability in MCF7 cells. In HeLa cells, only δ and
Płaziński, Sylwia Sowa, Roman Paduch total tocopherols were negatively correlated with cell viability. TRE and PRE had a low effect in
and Alexandru Mihai Grumezescu
reducing cell viability of the cancer cell lines, the most effective extracts were those of emory oak acorn
Received: 12 April 2022 (EOA), pecan nut (PEC) and walnut (WAL), and these were further studied for their pharmacological
Accepted: 12 May 2022 interactions, using the combination index and the isobologram methods. Combinations of both
Published: 14 May 2022 extracts showed a synergistic and strongly synergistic behavior in the three nuts (EOA, PEC and WAL),
with combination indexes between 0.12 and 0.55. These results highlight the need to understand the
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
interactions among components found in complex natural extracts or food products in order to fully
published maps and institutional affil- understand their bioactivities.
iations.
Keywords: food antioxidants; antiproliferative; cell viability; pharmacological interactions; cancer
cell lines; bioactive compounds; polyphenols; tocopherols; tocotrienols

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and
Cancer is one of the leading causes of death worldwide with 10 million victims in
conditions of the Creative Commons
Attribution (CC BY) license (https://
2020, according to the World Health Organization (WHO) [1]. Resistance to chemotherapy
creativecommons.org/licenses/by/
is one of the main causes why cancer still has many victims, this occurs some of the cancer
4.0/).
cells develop a survival strategy and the therapy is ineffective. Scientific studies reveal that

Molecules 2022, 27, 3154. https://doi.org/10.3390/molecules27103154 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 3154 2 of 15

a good diet contributes to the effectiveness of chemotherapies. Certain foods such as grains,
fruits and vegetables have been considered as preventives or adjuvants in the treatment of
cancer patients, due to their content of bioactive compounds with anticancer activities [2].
Tree nuts are an example of this type of food [3–5]. Moreover, tree nuts are remarkable
because, since they are composed of an oily fraction and a polar fraction, they possess both
lipophilic and hydrophilic bioactive compounds. The oil fraction contains molecules from
the tocol family, tocopherols (Ts) and tocotrienols (T3s), whose difference is in the degree
of saturation of its side chain. Ts contain a saturated side chain and T3s an unsaturated
side chain with three double bonds. There are 4 forms of both T and T3: α, β, γ, and δ,
depending on the number and position of methyl groups in the chromanol [6]. Ts, mainly
αT, are the main forms of vitamin E; they are effective antioxidants, and the γ and δ forms
also possess anticancer effects [7,8]. They can modulate and inhibit estrogen receptors [7]
and cyclin D1 [8], thereby blocking the phases of cell growth and proliferation. T3s are even
more effective than Ts in modulating these antiproliferative targets and reducing cancer
cell survival [9]. δ and γ T3 are involved in the direct activation of the tumor suppressor
protein p53, which in turn modulates the pro apoptotic regulator BAX/BAK and inhibits
anti apoptotic Bcl-2 proteins [10–12].
In the polar fraction of nuts, the main phytochemicals are phenolic compounds whose
chemical structure consists of one or more benzene rings substituted by at least one hydroxyl
group. These molecules are mostly recognized for their antioxidant properties, and the
most common phenolic compounds in nuts are gallic, syringic, ellagic and chlorogenic
acids, catechin, epicatechin and related flavan-3-ols [13]. Their principal mechanism of
action in cancer cells is the generation of intracellular reactive oxygen species (ROS) by
autoxidation [14], cell cycle regulation [15] and activation of pro apoptotic pathways [16].
Although the antiproliferative activity of these molecules in cancer cells has been
proven, their effectiveness in complex mixtures, as they are found in nuts and other foods,
could be altered. A pharmacological interaction is the reciprocal action between two (or
more) drugs or active principles that may reduce (antagonism) or increase (synergism)
their effect when they are combined, so the combination should be considered as a different
drug [17]. Pharmacological interactions may occur between different components of a
natural extract or food product, but they are seldom studied. Combination studies that
aim to evaluate synergistic or antagonistic effects between tocols and phenolic compounds
should be carried according to the experimental and mathematical conditions of the Chou–
Talalay method [18,19]. Therefore, the objective of this work is to determine the type of
pharmacological interaction that may exist between tree nut tocols and phenolic compounds
with respect to their antiproliferative effect in cancer cells, following the Chou–Talalay
method. For this purpose, seven tree nut species were selected. Six of them are commercial
and commonly consumed as healthy snacks: almond (ALM), pecan (PEC), pine nuts from
two varieties (pink (PNP) and white (PNW)), pistachio (PIS) and walnut (WAL). The last
one is a wild emory oak acorn (EOA) species characteristic of Northern Mexico, which can
be used as an ingredient in baking products. Since each tree nut species possess a unique
profile of tocols and polyphenols, their antiproliferative potential will be compared among
them. This study will help to understand if the presence of both non-polar tocols and polar
and phenolic compounds in nuts could be one reason for the cancer-preventing properties
of nuts.

2. Results and Discussion

2.1. Antiproliferative Activity of Pure Tocopherols and Phenolic Compounds in Cancer Cell Lines
Tocopherols and phenolic compounds have shown antiproliferative and pro-apoptotic
properties in many cancers cell lines [20,21]. In accordance with this premise, all compounds
tested in the present work showed antiproliferative activity in all cell lines (Table 1). Gallic
acid, a phenolic acid, was the most effective and showed the best effect in PC3 prostate
cancer cell line (EC50: 98.6 µM), and these values are higher than those reported by
Heidarian et al. [22] and Saffari-Chaleshtori et al. [23], where they establish an EC50 of
Molecules 2022, 27, 3154 3 of 15

50 µM and 35 µM, respectively. It is known that gallic acid can promote apoptosis in prostate
cancer and other cancer cell lines by inducing oxidative stress [14,24] and activating the
intrinsic and extrinsic apoptosis pathways [16]. Of the three tocopherols studied, δT was
more effective and its effect stronger in the MCF7 breast cancer cell line (EC50: 319.9 µM),
followed by γT, most effective in PC3 cells (EC50: 436.9 µM). This was expected, since
it is generally admitted that αT has lower antiproliferative, proapoptotic and, in general,
anticancer activity than γ and δ isoforms [25].

Table 1. Effect of pure compounds on the viability of cancer cells.

HeLa MCF7 A549 PC3

EC50 µM EC50 µM EC50 µM EC50 µM
αT 764.9 ± 19.2 c i 631.5 ± 12.3 e i 969.8 ± 78.8 c i 866.3 ± 106.2 c i
γT 659.2 ± 16.9 c ii 438.3 ± 8.0 d i 463.6 ± 14.7 b i 437.1 ± 0.3 b i
δT 386.7 ± 6.0 b ii 319.9 ± 9.8 c i,ii 491.9 ± 21.0 b iii 436.9 ± 11.6 b ii,iii
Gallic acid 281.4 ± 44.0 b i 151.3 ± 23.4 b i 421.9 ± 42.7 ab i 98.6 ± 32.6 ab i
Doxorubicin 2.36 ± 0.08 a i 2.30 ± 0.03 a i 4.7 ± 0.05 a ii 7.00 ± 0.5 a iii
α (αT), γ (γT), δ (δT), gallic acid and doxorubicin (Mean ± SEM). Each EC50 was calculated using a linear equation
by log (dose) response curves. Different roman numerals in the same row indicate different significant values
between cancer cell lines for each compound (p < 0.05). Different letters in the same column indicate different
significant values between compounds for each cancer cell line (p < 0.05).

To our best knowledge, there are no studies that indicate the EC50 values of γT and δT
in MCF7, there is an estimate reported by Figueroa, Asaduzzaman, and Young [26] where
the mean effective concentration for γT was between 50 to 75 µM. As well as the studies
by Hong et al. [27], where γT and δT presented a > 50% inhibition of cell growth at doses
between 60 to 100 µM. No studies were found that determined the EC50 of tocopherols in
HeLa cells, while the study by Jiang et al. [28] in PC3 and A549 cells also observed a dose
response effect of γT, with EC50 values of 50 and 40 µM, respectively.
The mechanisms of action of tocopherols in cell lines include upregulation (increased
expression) of PPARγ [29,30], cell cycle arrest by decreasing protein levels of cyclins D1
and E [30,31] and apoptosis induction by activation of the p53 tumor suppressor protein
and caspases 9 and 3, which in turn activate apoptotic proteins such as BAX and inhibit
anti-apoptotic proteins such as Bcl-2 [12,30]. Nevertheless, none of the natural compounds
was as effective as the chemotherapeutic agent doxorubicin, whose mechanism of action
involves the generation of free radicals that damage DNA, cell membrane and proteins [32].

2.2. Antiproliferative Activity of Tree Nut TRE and PRE

The profile of tocols (T and T3) in the TRE and content of total phenolic compounds
in PRE are shown in Tables 2 and 3, respectively. γT was the most abundant isoform in
all TRE and was highest in EOA; EOA, PNP and WAL extracts were also rich in δT, while
αT showed the lowest recovery in all TRE and was only moderately high in ALM. High
levels of T3 were present in both pine nut varieties (PNP and PNW), and moderate levels
in EOA and PIS (Table 2). The presence of T3 in pink and white pine nuts and in EOA has
been recently reported [33] and is relevant since tree nuts are not usually considered good
sources of these vitamin E forms, and only PIS has been previously recognized for its T3
content [34,35]. The highest content of phenolic compounds was found in PEC and WAL
extracts, while PIS showed the lowest content (Table 3). Next, the effect of TRE and PRE
obtained from six species of tree nuts on the viability of the same cancer cell lines studied
with pure compounds, plus a non-cancer retinal epithelial cell line (ARPE) was examined.
Antiproliferative activity of TRE against all cell lines is shown in Figure 1. In general, HeLa
cells were more sensible to all extracts, followed by MCF7 and PC3; A549 lung cancer cells
and the non-cancer line ARPE were not affected by the TRE. In the HeLa cell line, EOA
and WAL were statistically the most effective extracts, in MCF7 cells, EOA, WAL and PNP
were the most effective and in PC3, PNP, WAL and EOA were the most effective (p < 0.05,
statistical analysis in Table S1, Supplemental Materials). Since ARPE cells were not affected
Molecules 2022, 27, 3154 4 of 15

by TRE, all extracts showed selectivity indexes (SI) greater than one in HeLa, MCF7 and
PC3 cells (Table S2, Supplementary Materials); however, all SI values were lower than 3;
the highest value was for EOA TRE in HeLa cells (SI = 2.33), which indicates that these
compounds do not show high selectivity toward cancer cells [36]. Moreover, cell viabilities
at the highest extract concentrations were all above 50%, except for EOA extract in HeLa
cells, indicating a low antiproliferative effect of all TRE. This was most probably due to the
relatively low tocol concentrations in the extracts; the highest total tocol content was found
in EOA extract, followed by PNP, and WAL (see Table 2), which were also the most active
extracts. Correlation analysis of tocol content in extracts (Table 4) and cell viability showed
a significant negative correlation for γT (MCF7 and PC3 cells), δT (HeLa and MCF7), total T
(HeLa, MCF7 and PC3) and total tocols (T + T3 in MCF7 and PC3 cells). This indicates that
γT and δT were the most active tocol forms, which is consistent with the results obtained
from pure compounds (Table 1) and other published studies [25].

Table 2. Content of individual tocoferols (T) and tocotrienols (T3) in TRE extracts from nuts.

Nuts αT γT δT αT3 γT3 TT

Almond 43.4 ± 0.5 a 318.3 ± 11.4 c 97.38 ± 0.0 b ND ND 459.1 ± 4.5 b
Emory oak acorn 1.52 ± 0.1 c 7170.5 ± 1227.1 a 1695 ± 0.3 a UQL 219.7 ± 26.7 c 9086.8 ± 250.0 a
Pecan 0.05 ± 0.0 c 564.0 ± 20.5 bc 204.2 ± 0.2 b 0.3 ± 0.0 b UQL 768.3 ± 93.0 b
Pine nut, pink 7.79 ± 2.3 b 3235.5 ± 807.1 abc 818.2 ± 0.2 ab 1740.5 ± 445.1 a 793.4 ± 193.0 b 6595 ± 164.0 ab
Pine nut, white 0.18 ± 0.0 c 4538.0 ± 38.9 abc UQL 136.3 ± 4.7 b 1303.9 ± 1.2 a 5978 ± 12.3 ab
Pistachio 0.02 ± 0.0 c 429.5 ± 2.4 c 97.1 ± 0.0 b 44.2 ± 3.4 b 93.5 ± 0.0 c 664.3 ± 4.6 b
Walnut ND 5225.1 ± 542.1 ab 827.1 ± 0.3 ab ND ND 6052.4 ± 207.1 ab
UQL: Under quantification limit, ND: not detected. Values are presented as mean ± SEM (n = 3). Different letters
in the same column indicates values significantly different between nuts (p < 0.05). Tocol rich extract (TRE),
Tocopherols (T), tocotrienols (T3) and total tocols (TT) were expressed as micrograms of tocols per gram of extract
(µg tocols/g of TRE).

Table 3. Content of total phenolic compounds in PRE from nuts.

Nuts µg GAE/g PCE

Almond 512.5 ± 31.0 b
Emory oak acorn 475.1 ± 16.3 b
Pecan 823.4 ± 31.1 a
Pine nut, pink 369.7 ± 0.8 b
Pine nut, white 383.6 ± 11.3 b
Pistachio 141.6 ± 2.91 c
Walnut 717.9 ± 72.2 ab
Values are presented as mean ± SEM (n = 3). Different letters in the same column indicates values significantly
different (p < 0.05). Phenolic compounds rich extract (PCE) was expressed as micrograms of gallic acid equivalents
(GAE) per grams of extract (µg GAE).

Table 4. Pearson correlation coefficients between content of bioactive compounds and cell viability in
TRE- and PRE-treated cell cultures.

Correlation Coefficient ®with % Cell Viability of Cell Lines

Compound in Extract
HeLa MCF7 A549 PC3 ARPE
αT 0.1995 0.5733 0.4529 0.5563 0.5060
γT −0.7481 −0.9071 * −0.6761 −0.7696 * −0.5662
δT −0.8644 * −0.8515 * −0.2293 −0.6346 −0.6466
Total T −0.7975 * −0.9277 * −0.6135 −0.7699 * −0.6004
αT3 0.2925 −0.2807 −0.2215 −0.4657 −0.0666
γT3 0.2229 −0.2377 −0.8048 * −0.3382 −0.2324
Total T3 0.3063 −0.3061 −0.5591 −0.4790 −0.1631
Total T + T3 −0.6426 −0.9336 * −0.7176 −0.8380 * −0.5944
Total phenols −0.5172 −0.9715 * −0.6034 −0.1582 0.9778
Individual T and T3 were quantified in the TRE; total phenols were quantified in the PRE. Bold * indicates
significant correlation (p < 0.05).
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Molecules 2022, 27, 3154 Individual T and T3 were quantified in the TRE; total phenols were quantified in the PRE. 5 of
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PT 5050
(WN).
(WN 50 WN
).).WN
),),Pistachio
Pistachio (PIS Walnut (WN).
%CELL
VIABILITY
VIABILITY

Pistachio (PIS
(PIS
(PIS PT
), PT
Walnut
PTWalnut
Walnut
Walnut (WN(WN
(WN
(WN).50 ). WN
VIABILITY
VIABILITY
VIABILITY

Pistachio (PIS PT
), Walnut ). WNWPN PPN
PPN
WN (WN
CELLVIABILITY

WN WN
WN
WN WPN
WPN
WPN
WPN PT
PT PPN PPN
PPN
PPN
WN WPN PT
PT PT WN
WN
%
%

PT WN
%

WN
WNWN
CELL

50
50
50 PN WN
CELL

5050 PN
PN
CELL

50 PNPN
PN
PN
PNon cell viability PN PPNPPN
PPN PT
PT
%

The effect of PRE PN is shown in Figure 2. It is noticeable that the firstPT

PTPT
%
%

PPN
PPN PT
%

PPN PN
PN
PN
PN
PN PN
WPN
WPN
WPN WPN
WPN
WPNof live cells with respect to control (DMSO-treated PN
50
50 50
doses of all extracts increased
WPN
WPN
WPN the number
WPN
50 PT
PT
PT
CELL
CELL

WPN
WPN
%

00
50000 5050200 200200
200 400
400
400 600
600
600 800
800
800 PT PT WPN
%CELL

50
%

50000 200
200 400 400
400 600
600
600 cells) 800800
800in all cell 50 lines. For PT WPN
WPNWPNof
000000 PPN the four cancer PPNcell lines, viability was maximal at 200 µg/mL
%

200 400 600 800 PPN

PPN
PPN
PPN PPN
PPN WPN
CELL

50
50 PPN PPN
CELL
CELL

 
CELL

 50
50 50
%CELL

ggg
gggTRE TRE
TRE
TRE 00 200
200
50 400
400 0 600
600
00cell line,600 800
800
gTRE
TRE
TRE most 0000PRE, and 200
200for
200
200 thePT
PT
ARPE 400
400400
4000 0
0 viability
600
600
600 continued
800
800
800
800 to increase as the extract
PPNdose
PPN
PPN
PPN
PPNPPN
PPN
PT
PT PT  g TRE 000000 PT PT 200
200 400
400 600
600 800
800
increased, reaching values PT 
ggofgTRE
g 120.9
TRE (PIS) PT 200
200200 400
400400 600
600
600
PT to 157.2 (PEC) % from control. Phenolic compounds 800
800
800
%

200 400 600 800

%

g TRE
%

TRE
TRE
gg
ggg TRE
TRE PT
PTPT
%

TRE
gTRE
TRE PT
PT PT
%

000000
%
%

are well known for their antioxidant and cell-protective TREactivities; nonetheless, they PT may
%

000000 200
200
200
200200
200
400
400
400
400
behave
400
400
600
600
600
600
as pro-oxidant
600
600 800
and 800
800
800
800 cytotoxic at high concentrations (above 50 µM according to
800
000000
000000 0000 200
200
200
200
200
200
400
400
400
400
400400 
600
600
600
600600
600
gggTRE
TRE
TRE
gseveral
ggTRETRE 800
TRE studies)
800
800
800
800
800
and in the presence of metals; their prooxidant activity and ability to induce
0000 

g
200
200g
g
200200
g
gTRETRE
TRE
TRE
gTRE
TRE
400
400
400400 mitochondrial 600
600600
600 0000000 800
dysfunction 800
800 and consequently apoptosis has been suggested as one of their
800
 g possible
TRE anticancer 0000
mechanisms
00 200
200
200
[21].
200200 Lower 400
200 400
400
400
doses
400400 of phenolic 600
600
600
600
600 600 compounds 800
800
800
800 usually increase
800
800
gggTRE
TRETRE 0 200 400 600 800
the viability of cells subjected to oxidativegstress gg TRE
ggTRE
g TRE
TRE
TRE
g TRE
TRE but have no effect in non-stressed cells;
however, one study has demonstrated stimulation of proliferation in an osteoblastic cell
line by pure phenolic compounds used at low micromolar concentrations and by phenolic
extracts from different olive oil varieties [37]. It is not clear if, and how, phenolic compounds
could possess mitogenic activity, but the possibility should be further investigated.
 gested
gestedasasone oneofoftheir
theirpossible
possibleanticancer
anticancermechanisms
mechanisms[21]. [21].Lower
Lowerdosesdosesofofphenolicphenoliccom- com-
Total
Total
TotalT3T3
T3 0.3063
pounds
0.3063
pounds0.3063 usuallyincrease
usually increase −0.3061
theviability
−0.3061
−0.3061 viabilityof −0.5591
ofcells
cells subjectedto
−0.5591
−0.5591 −0.4790
tooxidative
oxidative
−0.4790 stressbut
−0.4790stress −0.1631
but−0.1631
haveno
−0.1631 noef-
ef-
pounds
pounds usuallyincrease
usually increase the
theviability
the viabilityof ofcells subjected
subjectedto
subjected
cells tooxidative
oxidative stress
stressbut buthave have
haveno noef-
ef-
Total
Total T + T3
TotalT T+ +T3
T3 fect
fect −0.6426
fect in
in
−0.6426 non-stressed
non-stressed
−0.6426
in non-stressed −0.9336
cells;
cells;
−0.9336
cells;−0.9336 *
however,*
however,
however,* one
one
one −0.7176
study
study
−0.7176
study has
has
−0.7176
has −0.8380
demonstrated
demonstrated
−0.8380
demonstrated −0.8380 *
stimulation
stimulation
* *
stimulation −0.5944
of
of
of prolifera-
prolifera-
−0.5944
−0.5944
prolifera-
fect in non-stressed cells; however, one study has demonstrated stimulation of prolifera-
Total phenols tion−0.5172
tion inan
in anosteoblastic −0.9715
osteoblastic−0.9715cell line *by
*by purephenolic−0.6034
phenolic compounds −0.1582
usedat atlow
lowmicromolar
micromolar 0.9778 con-
Total
Totalphenols
phenols tion
tion inan
−0.5172
−0.5172
in anosteoblastic
osteoblasticcell cell line
−0.9715
cellline *by
line pure
purephenolic
bypure −0.6034
−0.6034
phenolic compounds
compounds
compounds used
usedatatlow
−0.1582
−0.1582
used lowmicromolar
micromolar 0.9778 con-
0.9778con-
con-
Individual
Individual
centrations
centrations T and
Tand
and
and by T3
T3
by were
were
phenolic
phenolic quantified
quantified
extracts
extracts in
inin the
the
from
from TRE;
TRE; total
total
different
different phenols
phenols
oliveoil
olive were
were
oil quantified
quantified
varieties in
in
[37].in Itthe the
the
is PRE.
PRE.
not Bold
Bold
clear if,****
Individual
IndividualTand
centrations
centrations Tand
and
and T3
by
byT3were
werequantified
phenolic
phenolic quantified
extracts
extracts the
in
fromtheTRE;
from TRE;total
different
different oliveoil
totalphenols
phenols
olive werevarieties
were
oil quantified[37].
[37].ItItin
quantified
varieties
varieties [37]. Itisisisnot
thePRE.
not clear
PRE.
not Bold
clear if,
Bold
clearif,
if,
indicates
indicates
and
and how,
how, significant
significant
phenolic
phenolic correlation
correlation
compounds
compounds (p
(p <
< 0.05).
0.05).
could possess
could possess mitogenic
mitogenic activity,
activity, butbut the the possibility
possibility
Molecules 2022, 27, 3154 indicates
and indicates
and how,
how,significant
significant
phenolic
phenolic correlation
correlation
compounds
compounds could possess
(p(p< <0.05).
0.05).
could possess mitogenic
mitogenic activity,
activity, butbut the the possibility 6 of 15
possibility
shouldbe
should befurther
furtherinvestigated.
investigated.
shouldbe
should befurther
furtherinvestigated.
investigated.

HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa ALM
ALM
ALM
ALM ALM
ALM
ALM
ALM
ALM
ALM ALM
ALM
ALM
ALM
100
100
100
100
100
100 EOA
EOA
EOA
EOA
EOA
EOA EOA
EOA
EOA EOA
EOA
EOA
HeLa
HeLa
HeLa EOA EOA ALM
ALM
ALM
ALMALM
ALM
VIABILITY

ALM
VIABILITY
VIABILITY
CELLVIABILITY

100
100
100
100100 ALM
ALM
100
100
100 HeLa
HeLa
HeLa
HeLa WN WN
WN
WN WN
WN
WNWN
100100 HeLa
HeLa
HeLa
HeLa
ALM
ALM
HeLa
HeLa ALM
ALM WN
WN WN
WNWN
WN EOA EOA
EOA
EOA
EOA
EOAEOA
VIABILITY

100
VIABILITY

100
VIABILITY
VIABILITY

100 PN
PN PN
CELLVIABILITY

PN PN
PNPN EOA
EOA
EOA
PN EOA EOA
EOA PN PN
PN
PNPN WN
WN
WN
PN WNWNWN
VIABILITY

WN
VIABILITY
VIABILITY

ALM
ALM
CELL VIABILITY

WPN WPN
WPNWPN
WPN
WPN
WPN ALM
ALM
100
100 WPN ALM WN
WN
WN WPN
WPN PN
PN
PN
100
100 Figure
Figure ALM
ALM WN WN
WN WPN WPN
WPN
WPN ALM
ALM
ALM PNPNPN
000
00
Figure
Figure
Figure
Figure
50
50 Figure
Figure 2.2.2.
1.1.
1.
2.Effect
Effect
Effect
Effect
1. of
Effect
of
Effect
2.Effect
Effect of of
of phenols-rich
of
of
of tocol-rich
phenols-rich
of
ALM
tocol-rich
phenols-rich
phenols-rich ALM
PPNALM extracts
tocol-rich
tocol-rich
phenols-rich
PPN
PPN
PPN
extracts
extracts
extracts
extracts
extracts
extracts (PRE)
(TRE)
(TRE)
extracts
(PRE)
(TRE)
extracts (TRE)
(PRE) (PRE)
(PRE)EOA
EOA on
on on
on
onon
on
on
viability
viability
viability cancer
cancer
viability cancer
cancer
viabilitycancer
viability
viability cancer
cancer
cells.
cells.
cells.
cells.
Almond
cells.
cells. Almond
Almond
Almond
Almond
cells. Almond
Almond
Almond
Almond
(ALM
(ALM
(ALM
(ALM
(ALM
(ALM (ALM ),),
(ALM
(ALM ),),),),
),Emory
ALM
ALM
ALM
EmoryEmory
Emory
Emory
ALM PN
Emory
), Emory
ALM
Emory
CELL

00 EOAEOA
CELL

50
50 50 PPN PPN
PPN
%CELL

100
00 50 100
100
100
100
100 PPN WPN
WPN
oak
oak acorn (EOA
(EOA 100 ),),EOA
Pecans ),),),),),PNPine nut pink (PPN ),),),),PPN
Pine nut white
white (PNW ),),WPN
WPN
VIABILITY

oak oak acorn

acorn
acorn (EOA
(EOA 100
(EOA100 ),),),EOA Pecans
Pecans
EOA
EOA (PEC
(PEC
(PEC Pine
PN
PN
PN
Pine nut
nut pink (PPN
(PPN PPN
Pine
PPN nut
nut white (PNW
(PNW WPN
VIABILITY

oak acorn (EOA ),Pecans

Pecans(PEC ),Pine
Pinenutnutpink
pink(PPN Pine
Pinenutnutwhite (PNW EOA), ), WPN
CELL VIABILITY

oak oakoak acorn

acorn
acorn (EOA
(EOA EOA
Pecans
EOA (PEC
(PEC ), PN
Pine
PN nutpink
pink(PPN(PPN
(PPN ),
), Pine
Pine
PPN
PPN
PPN nut
nut white
white
white (PNW
(PNW EOA EOA
EOA
EOA
EOA
EOA
WPN
CELL

50
50
50 EOA
CELL
CELL

),Pistachio
Pistachio
Pistachio
Pistachio (PIS ),),),
(PIS
(PIS PTPT
PT
),PT
),),), Walnut
Walnut
Walnut
PT 5050
(WN).
(WN
(WN 50 WN
).).WN
). WN
),),Pistachio
Pistachio (PIS Walnut (WN).
%CELL
VIABILITY
VIABILITY

Pistachio (PIS
(PIS
(PIS PT
), PT
Walnut
Walnut
Walnut
Walnut (WN(WN
(WN
(WN). 50 ). WN
VIABILITY
VIABILITY
VIABILITY

), Pistachio (PIS PT
), Walnut (WN). PPN
PPN
PPN
CELLVIABILITY

WNWNWN
WN
WN WPN WPN
WPN
WPN
WPN PT
PT PPN PPN
PPN
PPN
WN WPN PT
PTPT WN
WN
%

WN
%

PT WN
%

WN WN WN
CELL

50
50
50 PN
PN WN
CELL

5050 PN PN
%CELL

50 PN PN
PN
PNPNof PRE used in the PPN PT
PT
PT
%

The highest doses PN PPN

PPNpresent work were capable to reduce cell viability PTPT
PT
%
%

PPN PPN PT
%

PPN PN PN
PN
PN PN
PN
PN
WPN
WPN
WPN WPN
WPN
WPNWPN PN
50
in MCF7 and HeLaWPN cells
WPN (except PNW extract in HeLa). The PEC extract was the most
WPN
050
50
50 50 PT
PT
PT
CELL
%CELL

WPN
WPN
%

0000000
50 5050200200
200
200
200 400
400
400
400 600
600
600
600 800
800
800
800 WPN
WPN
inPT PT
%

050 200
200 400 400
400 600
600
600
600 00000 800
800
800
effective 800 in the MCF7 line and WAL PT
HeLa cells, but differences were not statistically WPN
WPN WPN
%

200 400 PPN PPN

PPN
PPN
PPN PPN
PPN
PPNPPN WPN
0
CELL

5050
50
50 PPN
CELL
CELL

 
CELL

 g g TRE
TRE 50
50 50
%CELL

g g TRE
ggTRETRE
ggTRE
TRE 0
0 200
50
200 400
400 00
0 600600 800
800
TRE significant
00 0
0 with200 the other
200
200
200 PT
PT
active
400
400 00 00 extracts.
400
400 600
600 600Nevertheless,
600 800
800
800
800 a significant negative correlation PPN
PPN
PPN
PPN PPN
PPN
PPN
PPN
PT PT
PT  g TRE 000000 PT PT
PT 200
200
200 400
400
400 600
600
600 800
800
800
was observed in MCF7 PT 
gg
cells between PT
the 200200
200
extracts, total 400
400400
phenolic 600
600
600
content, 800
and 800
800
cell viability
%
%

g TRE 0 200 400 600 800

gTRE
g TRE
TRE
TRE
gg
 ggg TRE
TRE
TRE PT
PTPT
PT
%

TRE
g TRE PTPTPT
%

000000
%

gTRE
%

(Table 4). Previous studies that have found antiproliferative TRE activity of tree nut phenolic PT
%

000000 200
200
200
200
200
200
400
400
400
400
extracts
400
400 have 600
600
600
600
used extract
600
600
800
800
800
800 concentrations in the order of mg/mL [38], so it is reasonable
800
800
000000


gg
to
g TRE
think
TRE
TRE that PRE doses used in the present work were too low to exert a robust antipro-
000000 0000 200
200
200
200
200
200
400
400
400
400
400
400
600
600
600
600600
600 g TRE 800
g TRE
g TRE 800
800
800
800
800
0000 200
200

g
200 200
gTRE
gg TRE
TRE
gTRE
TRE
400
400
400400 600
600
liferative 600
effect.
600 000 0000 800
0
800
800
Comparison800 of PRE and TRE antiproliferative effects indicated that TRE
 g TRE 0000 200
200 400
400 600
600 800
800

 g
g were
TRE
TRE more effective, 00although
0 200
200
reduction
200
200 200 in400 400
400
cell
400400viability 600
600
600
600 was 800
800
600 still under
800
800 800 50% for all TRE
 g g
TRETRE 0 200 400 600 800
treatments but one. Therefore, we decided to 

g

ggg
g TRE
TRE
TRE
combine
 TRE both extracts and analyze the antipro-
 gTRE
g TRE
g TRE
TRE
liferative activity of the mixtures to determine if any kind of pharmacological interaction
could exist between both types of bioactive compounds. For this, we selected the most
active extracts, EOA (most active TRE), PEC (most active PRE) and WAL (good activity of
TRE and PRE), and the most sensible cell lines, MCF7 and HeLa.
Molecules 2022, 27, 3154 7 of 15

2.3. Analysis of TRE and PCE Combinations on HeLa and MCF7 Cell Viability
Combinations of TRE and PRE were assayed by adding a sub-optimal concentration
(EC20) of each extract to different concentrations of the other extract. The effect of all
combinations in HeLa cells are shown in Figure 3, and their effect in MCF7 cells are shown
in Figure 4. Calculated EC20 and EC50 values for all treatments and combinations are
shown in Table S3 in Supplementary Materials. Extracts from all nuts (EOA, PEC and WAL)
showed the same tendency in both cell lines. TRE + PREEC20 had a greater effect in reducing
cell viability than the TRE alone, EC50 values of all TRE in combination with PRE were
lower than 800 µg/mL, unlike when tested alone on cells, this means that combined extracts
are more effective than extracts alone. For the combinations PRE + TREEC20 the effect was
more remarkable in HeLa and MCF7, the EC50 of the combinations always remained below
500 µg (Table S3 in Supplementary Data), and the combinations were more effective than
PRE alone [EC50 above 2.0 mg (Table S3 in Supplementary Data); moreover, combination
treatments did not induce an increase in viability at low PRE concentrations (200 µg), as
observed when PRE were used alone (Figures 3 and 4). These data suggested that PRE
and TRE had a synergic effect on cell viability. To confirm the synergic interaction, the
combination index (CI) was calculated for each experiment and data were also analyzed by
the isobologram method. Figure 5 shows the isobolograms obtained from the combinations
of EOA extracts in HeLa an MCF7 cell lines. In all cases, the combination appeared in the
lower part of the isobologram, indicating a synergistic effect. The other extracts showed
the same tendencies, which can be also observed in the CI values (Table 5).

Table 5. Combination index (CI) to evaluate the interaction between TRE and PRE in cancer cell lines.

CI for Combination TRE + PREEC20 CI for Combination PRE + TREEC20

Nuts
HeLa MCF7 HeLa MCF7
EOA 0.53 (synergy) 0.55 (synergy) 0.12 (strong synergy) 0.30 (synergy)
PEC 0.44 (synergy) 0.37 (synergy) 0.18 (strong synergy) 0.28 (strong synergy)
WAL 0.52 (synergy) 0.43 (synergy) 0.27 (strong synergy) 0.22 (strong synergy)

The values of the CI were classified following the parameters of the Chou-Talalay
method [19], where a CI between 0.1 and 0.29 indicates strong synergy and a CI of 0.3 to
0.69 indicates synergy. The combination PRE + TREEC20 of all nuts presented values below
0.29 in both cells, which indicates strong synergy. In the combination TRE + PREEC20 all CI
values showed synergy between extracts (values between 0.37 and 0.55) in both cell lines.
The results consistently showed a synergy between both extracts; moreover, when a
suboptimal concentration of TRE was added to PRE (PRE + TREEC20 ) the interaction was
strongly synergic for all nut extracts in HeLa cells and PEC and WAL extracts in MCF7
cells. A synergic interaction between tocols and phenolic compounds has been documented
using pure compounds. In two studies, the antiproliferative activity of δT3 was potentiated
by ferulic acid [39] and the lignan sesamin [40]. In both works, the authors concluded that
the mechanism for the synergy was that the phenolic compound decreased the intracellular
degradation of δT3 by inhibiting the activity of cytochrome P450 (CYP450), and thus the
intracellular concentration of active δT3 was effectively increased [39,40]. CYP450 4F2 is
involved in tocol side chain degradation, which is initiated by hydroxylation catalyzed by
CYP450 families in the cell membrane [41]. Therefore, the synergistic interaction between
our extracts could be mediated by the inhibition of CYP4F2 by the polyphenols in PRE and
a decrease in the degradation of the tocols in TRE, which increases tocol bioavailability and
its capacity to reduce cell viability.
Furthermore, a direct antiproliferative action of the PRE cannot be excluded. It
is possible that tocols and phenols from the extracts modulate in synchrony different
antiproliferative or cytotoxic pathways, such as the inhibition of mitogenic proteins (cyclins
D1 and cMyc) and activation of caspases (3 and 9) by tocols [8], and a pro-oxidant activity
and inhibition of mitogenic signaling (NF-kβ, MAPK, Cdk2-4) by phenols [42].
Molecules 2022, 27, x FOR PEER REVIEW 8 of 15

Molecules 2022, 27, 3154 8 of 15

Figure
Figure 3. HeLa
3. HeLa cellviability
cell viability alone
aloneand in combination
and of EC20PRE:
in combination TRE (left)TRE
of EC20PRE: and (left)
EC20TRE:
andPRE (Right). PRE
EC20TRE:
(Right).
Molecules 2022, 27, x FOR PEER REVIEW 9 of 15

Molecules 2022, 27, 3154 9 of 15

Figure 4. MCF7 cell viability alone and in combination of EC20PRE: TRE (left) and EC20TRE: PRE (Right).
Figure 4. MCF7 cell viability alone and in combination of EC20PRE: TRE (left) and EC20TRE: PRE
(Right).
Molecules 2022, 27, x FOR PEER REVIEW 10 of 15

Molecules 2022, 27, 3154 10 of 15

Figure 5.5.Isobologram
Figure ofEC20PRE:
Isobologram of EC20PRE: TRE
TRE (left)
(left) and and EC20TRE:
EC20TRE: PRE (Right)
PRE (Right) from
from EOA EOA(up)
in HeLa in HeLa
and (up)
and MCF7
MCF7 cells
cells (down).
(down).

3. Materials and Methods

The values of the CI were classified following the parameters of the Chou-Talalay
3.1. Samples and Reagents
method [19], where a CI between 0.1 and 0.29 indicates strong synergy and a CI of 0.3 to
Raw tree nuts of 6 species (5 commercial and 1 wild) were used in this study. Commer-
0.69 indicates synergy. The combination PRE + TREEC20 of all nuts presented values below
cial species were almonds (ALM; Prunus dulcis), pecans (PEC; Carya illinoinensis), pine nuts
0.29 in both
(Pinnus cells, which
cembroides) indicates
of pink (PNP) and strong
whitesynergy. In the combination
(PNW) varieties, pistachios (PIS; TRE + PRE
Pistacia EC20 all CI
vera),
values showed(WAL;
and walnuts synergy between
Juglans regia).extracts
They were (values between from
all purchased 0.37 andlocal0.55) in both
retailers. Emorycell lines.
oakThe results
acorns (EOA; consistently
Quercus emory) showed a synergy
were collected frombetween both
wild trees extracts;
located moreover,
in Cuitaca, Sonora when a
suboptimal ◦ 0 00
Mexico (31concentration
00 17 N, 110 29 ◦ 0
of 33 00
TREW)was added
in the toSeptember–October
period PRE (PRE + TREEC20 ) the interaction was
2018.
strongly Pure (≥93%)for
synergic standards
all nut (gallic
extractsacid,in α–,
HeLa and δ–tocopherol);
γ–, cells and PEC anddoxorrubicin,
WAL extracts Folin–
in MCF7
Ciocalteau phenol reagent, dimethyl sulfoxide (DMSO), Dulbecco’s
cells. A synergic interaction between tocols and phenolic compounds has been docu- modified Eagle’s
medium,
mented fetalpure
using bovine serum (FBS),In3-two
compounds. (4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium
studies, the antiproliferative activity of δT3 was
bromide (MTT), sodium chloride (NaCl), sodium hydroxide (NaOH), trypan blue, trypsin-
potentiated by ferulic acid [39] and the lignan sesamin [40]. In both works, the authors
EDTA, sodium phosphate salts were purchased from Sigma-Aldrich (St. Louis, MO, USA).
concluded that the
Non-essential aminomechanism for the synergy
acids, L-Glutamine was penicillin-streptomycin
(200 mM), that the phenolic compound decreased
from Gibco
the(Gibco,
intracellular degradation of δT3
Austria). High-performance liquidby inhibiting the(HPLC)
chromatography activityandof cytochrome P450
analytical-grade
(CYP450), and thus the intracellular concentration of active δT3 was
solvents (hexane, methanol, acetone, and isopropyl and isobutyl alcohol) were obtained effectively increased
[39,40]. CYP450
from JT- 4F2 is involved
Baker (Avantar Performance in tocol side chain
Materials SA de degradation,
CV, Mexico). which is initiated by hy-
droxylation catalyzed by CYP450 families in the cell membrane [41]. Therefore, the syn-
ergistic interaction between our extracts could be mediated by the inhibition of CYP4F2
by the polyphenols in PRE and a decrease in the degradation of the tocols in TRE, which
increases tocol bioavailability and its capacity to reduce cell viability.
Molecules 2022, 27, 3154 11 of 15

3.2. Preparation and Characterization of Tocol- and Phenolic-Rich Extracts (TRE, PRE)
All nuts were ground, oil was extracted by cold extraction method [43], and 10 g of
grounded nut were mixed with 100 mL of hexane at 8000 rpm for 3 min. The mixture was
filtered, the residue was re-extracted twice, the solvent portions were combined, and the
hexane was removed by rotary evaporation (BUCHI Series-114, BUCHI Labortechnik AG,
Flawil, Switzerland) at 40 ◦ C. Oil was weighed and transferred to an amber bottle, sealed
with nitrogen, and stored at −80 ◦ C until analysis.
Tocols were then extracted from the oil fraction, and phenolic compounds from the
defatted flour. The tocol-rich extract (TRE) was prepared according to Miraliakbari and
Shahidi [43], with minor modifications. An amount of 20 g of oil was mixed with 200 mL
hexane in a separatory funnel, then 100 mL of MeOH was added and the separating funnel
was sealed and stirred for 15 min, and the methanolic fraction was recovered in a flask. The
extraction process was repeated four times, all the methanolic fractions were mixed and
the solvent was removed by rotary evaporation.
The quantification of individual tocols (α, γ and δT, α and γT3) in TRE was performed
by normal-phase HPLC (Perkin Elmer model 200) according to the chromatographic condi-
tions and methodology of Stevens et al. [33] and the results were expressed in µg tocols per
gram of extract (µg tocols/g of TRE). For the phenolic rich extract (PRE), 1 g of defatted
flour was mixed with 10 mL of 80% aqueous acetone in an ultrasound at room temperature
for 10 min, and the mixture was centrifuged for 10 min at 3000 rpm at 4 ◦ C. Next, the
supernatant was recovered, and the sample was subjected to a further extraction process
under the same conditions. The supernatants were combined, the solvent was removed by
rotary evaporation, and water removed by lyophilization (LABCONCO Freezone 6, Lab-
conco, Kansas City, MO, USA). Total phenolic compounds were quantified in the extracts
according to De la Rosa et al. [44] and the results were expressed in µg equivalents of gallic
acid/g of extract (µg GAE/g of PRE).

3.3. Cell Cultures

Cancer cell lines MCF7 (breast cancer), HeLa (human cervical carcinoma), A549 (hu-
man alveolar carcinoma), PC-3 (prostate adenocarcinoma), and noncancerous ARPE (en-
dothelial retinal) were purchased from the American Type Culture Collection (ATCC,
Rockville, MD, USA). Cells were cultured in Dulbecco Modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum, non-essential amino acids (7.5 mL), 10%
L-Glutamine (200 mM), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco, Austria)
under 5% CO2 and 95% air atmosphere at 37 ◦ C. Cells were grown in 25 cm2 plastic flasks.

3.4. Antiproliferative Activity of Pure Compounds and Extracts

Cells were removed with trypsin-EDTA, seeded into 96-well plates (100 µL/well) at a
density 1 × 104 cells/mL and incubated for 24 h. Pure compounds (α, γ and δT, gallic acid,
and doxorubicin), TRE and PCE extracts were initially dissolved in DMSO to prepare a stock
solution and further diluted with DMEM to obtain the appropriate final concentrations in
the microplate (1–1000 µM for pure compounds, 100–800 µg/mL for the extracts). DMSO
was used as control with a final concentration of 800 µg/mL. The cell viability assay with
the MTT reagent was performed according to Meneses-Sagrero et al. [45], 50 µL of DMEM
with treatments or DMSO were added and incubated for 48 h. After the incubation period,
cells were washed with 100 µL phosphate-buffered solution (PBS), then 100 µL of DMEM
with 10 µL of MTT (5 mg/mL) was added to each well and later it was left incubating
for 4 h more. The formazan crystals were dissolved with acidic isopropyl alcohol (where
337 µL of HCL are dissolved on 100 mL of isopropyl alcohol), and the absorbance of the
samples was read at 570 nm in a UV-Vis microplate reader (BioRad Benchmark Plus) and a
wavelength reference of 630 nm. Cell viability was expressed in terms of percentage, where
the optical density of cells treated with only DMSO was considered as 100% viability.
EC50 (half effective concentration) and EC20 (20% effective concentration) values were
determined by linear regression between % viability vs. log concentration [19]. A modified
Molecules 2022, 27, 3154 12 of 15

selectivity index (SI, Equation (1)) was determined for the extracts using the viability at the
maximum concentration of extract (800 µg/mL):

% cell viability noncancerous cell line ( ARPE)

SI = (1)
% cell viability cancer cell line

where SI > 1 means that cytotoxicity for cancer cells exceeds cytotoxicity in normal cells,
and a value greater than 3 is considered as a high selectivity and safe for normal cells [36].

3.5. Experimental Design for Combinations and Analysis of the Pharmacological Interaction
between TRE and PRE
TRE and PRE were combined and its antiproliferative effect determined as previously
described. Two types of combinations were carried out: TRE + PREEC20 and PRE + TREEC20.
In both, a sub-effective (EC20) concentration of each extract (extract 2 in Equation (2)) was
mixed with different concentrations (100–800 µg/mL) of the other extract (extract 1 in
Equation (2)), both from the same nut. Once the combinations were calculated and the
extracts mixed and diluted with DMEM to the appropriate concentrations, the cell viability
was determined with MTT as described in the previous section.
The combination index (CI) was calculated according to the Chou-Talalay method to
determine the kind of interaction between TRE and PRE [19]:

D1 D2
CI = + (2)
E1 E2
where D1 and E1 are the doses of extract 1 necessary to elicit the same effect and D2 and
E2 are the doses of extract 2 necessary to elicit the same effect. E1 and E2 were calculated
with each extract used alone in a different experiment, and D1 and D2 were calculated
for each extract in the combination experiment. Considering the experiment design, D1
and E1 were EC50 values of extract 1 alone or in the combination, respectively; D2 was the
suboptimal concentration (EC20) of extract 2 (since in the combination experiment, this
extract concentration was able to elicit a 50% viability when combined with the appropriate
concentration of extract 1) and E2 the EC50 of extract 2 alone. A CI of 0.90 or lower
indicates synergy between extracts, 0.91 to 1.10 an additive effect, and 1.11 or greater means
an antagonistic effect.
Interactions were also evaluated by the isobologram method, which is related to
Loewe’s additivity [17]. In this graphical procedure, the doses of one drug (extract 1) are
shown along an Y axis, and the doses of a second drug (extract 2, the one that potentiates the
effect of extract 1), are shown along the X axis. Then, on each axis, the EC50 value of each
extract alone is plotted (E1 and E2) and both points are joined by means of a line (isobole).
Finally, a point is drawn in which x corresponds to the concentration of extract 2 in the
combination that elicits a 50% viability (D2, which according to our experimental design
will be the EC20 of extract 2) and y is the concentration of extract 1 in the combination that
elicits a 50% viability (D1). The kind of interaction is deduced from the position that the
point (D2, D1) occupies with respect to the isobole: if the point is below, the interaction is
synergistic, close to the isobole is additive interaction (or no interaction), and above the
isobole it indicates antagonistic interactions.

3.6. Statistical Analysis

All analyses were carried out three times in triplicate, and assays data were expressed
as mean ± standard error of the mean (SEM). The data were subjected to a one- or two-way
ANOVA, followed by a post hoc Tukey test to evaluate difference between samples (tree
nut species) and between effects in different cell lines. A value of p < 0.05 was considered
statistically significant.
Molecules 2022, 27, 3154 13 of 15

4. Conclusions
This study indicates that although pure tocols and phenolic compounds can decrease
the viability of cancer cells, phenol- and tocol-rich extracts (PRE and TRE) from tree nuts
(known for their abundance in both types of antioxidants) had low antiproliferative activity,
especially PRE. However, combination of both extracts (PRE + TRE) had greater effective-
ness, showing strongly synergistic interactions. This finding highlights the importance
of studying and understanding the interactions that occur among the multiple chemical
components of complex natural extracts and food matrixes in order to fully understand
their biological activities and health-promoting potential. More studies are still required to
determine the mechanism of the synergy between tocols and polar phenolic compounds in
the modulation of the pathways responsible for cell viability, proliferation, or apoptosis.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules27103154/s1, Table S1: Effect of TRE and PCE on the
viability of cancer cells title; Table S2. Selectivity of the cytotoxicity of tocol and phenol extracts in
cancer cell lines; Table S3: EC20 and EC50 of extracts alone and in combination.
Author Contributions: J.C.S.-B. and L.A.D.l.R. contributed equally to this work, on methodology,
investigation, and writing the original version. I.O.-A., E.Á.-P., A.W.-M., H.A.-G. and R.E.R.-Z. on
investigation, writing—review and editing. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported by CONACYT (Mexican Council of Science and Technology)
Projects CB-2015-1/254063, CB-2016-286449.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study is openly available in Mendeley Data,
Stevens Barron, Jazmin (2022), “Synergistic interactions between tocol and phenolic extracts from
different tree nut species against human cancer cell lines”, doi: 10.17632/4n4rjp9pdk.1.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available.

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