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Article
Phytochemical Profiling and Biological Potential of Prunus dul-
cis Shell Extracts
Talel Ben khadher 1,2, Sameh Sassi-Aydi 2, Samir Aydi 2, Mohamed Mars 2 and Jalloul Bouajila 1,*
1. Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INP, UPS, Toulouse, France
2. Laboratory of Biodiversity and Valorization of Bioressources in Arid Zones, Faculty of Sciences at the Uni-
versity of Gabes, Zrig, 6072, Gabes, Tunisia.
* Correspondence: authors: Jalloul Bouajila (tel: + 33562256885; fax + 33562256885; e-mail
jalloul.bouajila@univ-tlse3.fr).
Abstract: Prunus dulcis is one of the most widely cultivated species in the world. Its fruit (almond)
is rich in various nutritious and bioactive compounds that exert several beneficial effects. The aim
of this study was to determine the chemical profile and evaluate the biological potential in vitro of
almond shell extracts. The chemical analysis of shell extracts led to the identification of 15 com-
pounds by HPLC-DAD of which 11 were first detected in the almond plant. Twenty-six volatile
compounds were identified by the GC-MS technique, among them; seven were firstly detected in
the studied plant. For the biological activities, the extracts demonstrated moderate inhibition poten-
tial against the antioxidant, antidiabetic, and cytotoxic activities. The methanol extract at 50 µg/mL
showed the highest antioxidant (45%) and antidiabetic activities (45% against alpha-glucosidase and
31% against alpha-amylase extracts), while the cyclohexane and dichloromethane at 50 µg/mL
showed the highest cytotoxic activity towards Hela (32.2% with cyclohexane), and RAW 264-7 (45%
with dichloromethane). Overall, these findings demonstrate the potential of almond shell extracts
as a source of bioactive compounds that could be applied in the pharmaceutical and medical fields.
Keywords: Prunus dulcis shell extracts; chemical composition; biological activities; GC-MS; HPLC-
DAD
1. Introduction
Nowadays, fruits and their by-products are widely studied as a promising source of
phytochemicals with various health benefits that have a high potential to be integrated
into the pharmaceutical and cosmetic industries given their various pharmacological and
pharmaceutical properties [1].
Nuts are a class of food rich with high lipids content. Its consumption provides us
with a multitude of bioactive compounds that present several pharmacological properties.
They are known for their distinguished flavor, their consumption provides us with wide
array of bioactive molecules, and nutrients [2,3].
Prunus dulcis (P.dulcis) is one of the most cultivated nuts species in the world. It be-
longs to the Rosaceae family. The almond fruit is a stone fruit comprising a kernel, skin,
shell, and hull.. It presents a high nutritional value given its richness with high quality
fats (monounsaturated fatty acids (MUFA, 60%) such as palmitic, stearic and palmitoleic
acid; polyunsaturated fatty acids (PUFA, 30%) such as linoleic acid), protein, carbohy-
drates fibers and vitamins [2,3] . Numerous bioactive compounds have been reported in
the fruit of P. dulcis such as phenolic acids ( hydroxycinnamic acid, hydro benzoic acid
sinapic acid, etc.), flavonoids (Catechin, epicatechin, cyanidin, etc.), lignans, stilbene, and
terpenoids [1,3,4]. Various biological proprieties have been reported in the almond such
as antimicrobial, antioxidant, anti-inflammatory, anticancer, laxative, hepatoprotective,
cardiometabolic protective, neotropic, sedative, hypnotic, anxiolytic, and neuroprotective
effects [4] .
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Interestingly, the almond shell, which accounts for 33% of the total fruit mass and
produces annually 0.8 to 1.7 million tons globally, has been traditionally utilized as animal
feed. However, it remains less characterized among the other parts, traditionally used as
animal food. It is rich in cellulose and lignin contents suggesting its potential for further
exploration [5].
The present study aims to determine the chemical composition of P. dulcis shell ex-
tracts using HPLC-DAD and GC-MS methods as well as evaluate their biological potential
in vitro, mainly the antioxidant (DPPH), antidiabetic (alpha-amylase, alpha-glucosidase),
and cytotoxic (Hela, RAW 264.7 )
2. Results
2.1. Extraction yield:
In this study, maceration using an increasing polarity organic solvent (cyclohexane,
dichloromethane, ethyl acetate, methanol, and H2O) was used in the extraction of the
phytochemicals compounds. As presented in Table 1, the polar fractions (MeOH, H2O)
presented higher yield values than the nonpolar fractions, with the highest value recorded
with the H2O extract (2.22%), while the EtOAc extract presented the lowest yield value
(0.03%). These findings suggest that the shell part is relatively richer in polar compounds
such as polyphenols and polysaccharides. Conversely, the nonpolar fraction, which in-
cluded dichloromethane and cyclohexane extracts, presented low yield values ranging
from 0.09% to 0.14% respectively, suggesting the presence of nonpolar compounds, such
as fatty acids, in the shell material. In this context, Sarwar et al. [6] studied the effect of
different solvents in the yield extraction of two varieties of P. dulcis shell, using 80 or 100%
MeOH and 80 or 100% EtOH. The results showed significant variation in the yield be-
tween the different solvents, with the highest recorded with the 80% MeOH (18.2% for the
thick shell and 57.9% for the thin shell). In another study, Queirós et al. (2020) obtained an
extraction yield value similar to our findings (3.4% with the ethanol-water extract).
Table 1: Extraction yields of shell extracts of P. dulcis (DW %) (Cyclohexane: CHYA; Dichloro- 1
methane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH) 2
Fractional extraction
CHYA DCM EtOAc MeOH H2O
Maceration 0.14 0.09 0.03 0.63 2.22
3
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2.2 Total Polyphenol Content (TPC):
The total phenolic content of the almond shell extracts was deter-
mined using the Folin-Ciocalteu method. The results are shown in figure
1. Statistically, we note a significant difference between the extracts. As
shown in the figure below, the polyphenol content of the obtained extracts
is relatively low. The polar extracts have a higher TPC content than the
apolar ones. The highest TPC content was recorded with the H2O extract
(31.77 mg GAE / g of DW), while the lowest value was obtained with the
CHYA extract (3.56 mg GAE / g of DW). Compared to the literature, the
obtained TPC values are higher than that found by Sarwar et al. (2012)
with the methanolic and the ethanolic shell extracts of the two almond
varieties (values range from 1.4 to 2.3 mg GAE/g DW with the thick shell
and from 2.3 to 7.21 mg GAE/g DW with the thin shell). In another study,
Queirós et al. (2020) obtained a higher TPC content (188.6 mg GAE/g DW)
with the almond shell ethanol-water extract.
Figure 1. Total phenolic content (TPC) of P. dulcis shell extracts (Cyclohexane: CHYA; Di-
chloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH). Mean values ± SD (n = 3);
Different letters on the histograms mean a significant difference (p <0.05).
2.3. Antioxidant Activity (DPPH):
The antioxidant activity of the almond shell extracts was assessed by the DPPH assay. The
extracts concentration was adjusted to 50 µg/mL, the ascorbic acid was used as reference at 4
µg/mL. The results are presented in figure 2. Statistically, there is a significant difference between
the different extraction solvents. As shown in Figure 2, only three extracts have shown antioxidant
activity against the DPPH radical (DCM, MeOH, H2O). The highest inhibition values was recorded
with the polar extracts: 37.6% with the MeOH extract and 16.6% with the H2O extract. This suggests
that polar molecules seem to be responsible for the present effect. In fact, several polyphenol com-
pounds identified in the almond, such as Catechin, epicatechin are known for their antioxidant
properties. These compounds could interact synergistically with molecules of other classes, such
as fatty acids, vitamins such as A, B (thiamine B1, riboflavin B2, niacin B3 and pyridoxine B6), and
E present in almonds [8], and polysaccharides (glycosides) that also present as well according to
recent studies [8]. Compared to the literature, a study conducted by Queirós et al. (2020), evaluating
the antioxidant potential of almond shell extracts using the DPPH test, showed that the different
extracts presented a moderate inhibition activity range of 12.1 to 18.22% for the thick shell and 15
to 57.9% for the thin shell. In another study, the DPPH assay was used to evaluate the antioxidant
potential of almond shell extract. The results showed that the ethanol-water extract exerted a high
inhibition activity with IC50 equal to 7.9 µg/mL [1].
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Figure 2. Antioxidant Activity of P.dulcis shell extracts (Cyclohexane: CHYA; Dichloro-
methane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH; ascorbic acid: VIT C: used as a refer-
ence at 4 µg/mL). Extracts was tested at 50 µg/mL. Mean values ± SD (n = 3); Different letters
on the histograms mean a significant difference (p <0.05).
2.4. Chromatographic analysis:
2.4.1. Identification of compounds in P. dulcis shell extracts by HPLC-DAD:
The identification of compounds in the almond shell extracts was performed using the
HPLC-DAD technique. The retention time and the maximum wavelength (λmax) of each peak
were compared to those of standards compounds, which were injected under identical condi-
tions as the shell extracts. Following this identification, each recognized compound was quan-
tified by referencing the calibration curve tailored to each corresponding standard. The anal-
ysis of the chemical composition of P. dulcis shell extracts led to the identification of 15 com-
pounds as presented in Table 2. Of these, only four compounds were previously identified in
the almond plant (Catechin, epicatechin, , rutin, and trans-cinnamic acid), while all the com-
pounds were firstly identified in the shell part. The majority of the identified compounds were
detected in the CHYA and the EtOAc extracts. The EtOAc extract was particularly rich in ru-
tin, with a concentration of 13.06 mg per gram of extract. The Methanol (MeOH) extract had a
high content of epicatechin, at 4.42 mg/g extract, while the water (H2O) extract was notable for
its high Synephrine content, at 1.62 mg/g extract. According to the literature, few studies have
been interested in the chemical composition of the almond shell, a study conducted by Moure
et al. (2007), identified vanillic, syringic, and p-coumaric acids in the P. dulcis shell part. In
another study, Kahlaoui et al. (2019) identified several polyphenolic compounds such as quer-
cetin-3-glucoside, p-coumaric acid, caffeic acid, and protocatechuic acid in the hulls of several
varieties of almonds.
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Table 2. Identification of compounds by HPLC-DAD of almond shell extracts Cyclohexane: CHYA;
Dichloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH).
mg/g extract
compounds Rt (min)
Ref
Extracts
CHYO
DCM
EtOAc
MeOH
H
2
O
Catechin 0.87 0.39 [10]
Rutin 0.93 13.06 [11]
Synephrine 1.09 1.62 [12]
Epicatechin 1.9 4.42 [13]
trans-3-hydroxycinnamic acid
3.5 0.08 [14]
trans-cinnamic acid
10.8 0.02 [15]
7,8-dihydroxy-2,2-dimethylchromane-6-carbox-
ylic acid
10.93 0.31 [16]
5,7-dihydroxy-3',4',5'-trimethoxyflavone
19.51 0.31 [17]
3-tert-butyl-4-hydroxybenzoic acid 19.55 0.02 [16]
7-hydroxyflavone 19.71 0.59 [18]
Shikonin 20.72 0.19 [19]
3,3′-dimethoxyflavone 21.59 0.23 [20]
3,6,3′-trimethoxyflavone 21.74 0.33 0.11 [21]
Isobutyl 4-hydroxybenzoate 21.02. 0.25 [16]
5-hydroxy-3'-methoxyflavone 21.95 0.33 [22]
2.4.2. Identification of volatile compounds in P. dulcis shell extracts by GC-MS (before and after
derivatization):
The volatile composition of the different almond shell extracts was determined by the GC-MS
technique. The identified compounds were presented in tables 3 and 4. As shown in the tables below.
The analysis by GC-MS method has led to the identification of 26 compounds before and after deri-
vatization. Seven compounds were detected for the first time in the almond plant (2,3-dimethyl-
decane,undecane, 1,1'-bicyclohexyl, ben-zaldehyde, 3-hydroxy-4-methoxy , 2,4-Di-tert-butylphenol,
dodecanoic acid, 1-methylethyl ester, and hydracrylic acid). Whereas only 11 compounds were previ-
ously reported in almond shell part: dodecane; tridecane, hexanoic acid; glycolic acid; glyceric acid;
butanedioic acid; malic acid, Dodecanoic acid, Pentadecanoic acid; 9-octadecenoic acid,(E)-, and stea-
ric acid. As shown in Tables 3 and 4 most of the identified compounds have been detected in the apolar
extracts. Various classes of compounds are present in the almond shell extract, such as alkanes (un-
decane; dodecane, tridecane…); organic acids (hexanoic acid, glycolic acid, butanedioic acid…); poly-
phenols (2,4-Di-tert-butylphenol). Some compounds were present in two or three extracts, such as 2,4-
Di-tert-butylphenol, benzaldehyde, 3-hydroxy-4-methoxy- and palmitic acid. According to the litera-
ture, only few studies have investigated the volatile profile of the almond shell part. In this context,
Moure et al. (2007) identified some compounds in the almond shell part by the GC-MS method: fatty
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acids (butanedioic, octadecanoic and trans-9 octadecenoic); phenolic acids (vanillic, syringic, and p-
coumaric acids). Furthermore, in another study several volatile compounds were identified in the P.
dulcis shell, such as syringol, 1-(4-hydroxy-3-methoxyphenyl)-propyne; hexadecanoic acid, and syrin-
galdehyde [6].
Table 3. Identification of volatile compounds by GC-MS of almond shell extracts (before derivatization)
Cyclohexane: CHYA; Dichloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH).
N RI Compounds
Area (
E+07)
Ref
Extracts
CHYA
DCM
EtOAc
MeOH
H
2
O
1 1075 2,3-Dimethyldecane
12.6 [23]
2 1100 Undecane
0.0922 [24]
3 1205 Dodecane
6.76 [25]
4 1306 Tridecane
1.63 [26]
5 1377 1,1'-Bicyclohexyl
25300 4.06 [26]
6 1535 Benzaldehyde, 3-hydroxy-4-methoxy-
370 4.99 [25]
7 1560 2,4-Di-tert-butylphenol
6.24 3.55 7.868 [27]
8 1566 Dodecanoic acid, 1-methylethyl ester
3.69 [28]
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Table 4. Identification of volatile compounds by GC-MS of almond shell extracts (after derivatization) (CHYA: Cyclohexane; DCM:
Dichloromethane; EtOAc: Ethyl acetate; MeOH: Methanol).
N RI Compounds
Area(E+07)
Ref
Extracts
CHYA
DCM
EtOAc
MeOH
H
2
O
1 1025 Cyclohexanol 15.4 4.02 [29]
2 1038 Furfuryl alcohol 3.13 14.3 39.1 23.4 [29]
3 1075 Lactic Acid 45.9 58.2 86.5 [30]
4 1094 Hexanoic acid 15.1 [30]
5 1094 Glycolic acid 121 [30]
6 1162 Hydracrylic acid 13.8 32.9 [31]
7 1266 Glycerol 88.9 1130 621 [26]
8 1338 Glyceric acid 41.7 109 [28]
9 1351 Butanedioic acid 97.9 0.691 [32]
10 1384 Nonanoic acid 51.6 [26]
11 1500 Malic acid 1760 1310 [33]
12 1591 2,4-Di-tert-butylphenol 22.2 [27]
13 1672 Dodecanoic acid 23.8 [34]
14 1865 Myristic acid 12.8 [34]
15 1959 Pentadecanoic acid 10.6 [33]
16 2054 Palmitic acid 511 4.60 2.84 [35]
17 2074 Myo-Inositol 428 1150 [36]
18 2241 9-octadecenoic acid, (E)- 278 [37]
19 2248 Stearic acid 165 121 392 [38]
2.5. Biological activities:
2.5.1. Antidiabetic activity:
• The evaluation of the antidiabetic potential of almond shell extracts was performed in vitro using alpha-
glucosidase and alpha-amylase assays.
• Alpha-glucosidase activity:
To the best of our knowledge, no previous studies were conducted to assess the an-
tidiabetic activity of the P. dulcis shell. The shell extracts were adjusted at 50 µg/mL. Acar-
bose was used as a reference at the same concentration. The results of the present activity
are shown in Figure 3. As presented in the figure below, only three of the tested extracts
have shown inhibition activity against the alpha-glucosidase enzyme (DCM, EtOAc, and
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MeOH). The highest inhibition value was recorded with the MeOH extract (45%), while
the ethyl acetate extract presented the lowest one (7%). The variation in inhibition activity
between the extracts could be correlated with their chemical composition. Indeed, several
bioactive compounds, especially the polar compounds, could be responsible for this ac-
tivity mainly the phenolic and polysaccharides. In the same context, a study conducted
on the antidiabetic potential of almond kernel using the protein tyrosine phosphatase (1B
PTP1B) inhibitory assay showed that seven alcoholic extracts exerted strong antidiabetic
activity with an IC50 = 0.46 µg/mL with ETOH extract [1].
Figure 3. Antidiabetic Activity (alpha-glucosidase) of different extracts of P. dulcis shell Cyclohex-
ane: CHYA; Dichloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH; Acarbose: was used
as reference at 50 µg/mL). Extracts was tested at 50 µg/mL. Mean values ± SD (n = 3); Different letters
on the histograms mean a significant difference (p <0.05).
• Alpha-amylase activity:
Evaluation of the antidiabetic potential of almond shell extracts was performed using
the alpha-amylase assay. The shell extracts and the reference concentration were adjusted
at 50 µg/mL. The antidiabetic activity was illustrated in figure 4. As shown in the extracts
figure below, all the tested had an inhibition activity against the alpha-amylase enzyme
with some variation. The highest inhibition value was recorded with the CHYA extract
(34.34%), while the lowest value was recorded with the H2O extract (20.71%). In compar-
ison with the alpha-glucosidase inhibition activity, we deduce that the polar shell extracts
are more effective with the alpha-glucosidase assay while the apolar ones are more active
with the alpha-amylase assay. These results could be explained by the difference in the
inhibition mechanism between the two enzymes as well as the compounds responsible
for the inhibition activity. In the same context, Nowicka et al. (2016) have investigated the
antidiabetic potential of several Prunus fruit smoothies and puree in vitro using the alpha
glucosidase and the alpha amylase assays. The obtained results showed that the 100%
apricot puree, 50% sour cherry juice 50% apricot puree, and 20% sour cherry juice 80%
apricot puree exhibited the highest inhibition activity toward the alpha-amylase enzyme
with an IC50 < 1mg/mL
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Figure 4. Antidiabetic activity (alpha-amylase) of different extracts of P. dulcis shell cyclohexane:
CHYA; Dichloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH; Acarbose: was used as
reference at 50 µg/mL). Extracts was tested at 50 µg/mL. Mean values ± SD (n = 3); Different letters
on the histograms mean a significant difference (p <0.05)..
2.5.2. Cytotoxic activity:
The determination of the cytotoxic potential of the obtained shell extracts was con-
ducted using the MTT test. Two cancer cell lines (HELA and Raw 264-7). The extracts were
adjusted at 50 µg/mL the tamoxifen was used as a reference at 100µM. The results of the
inhibition activity are presented in Figure 5. As shown in the figure below, for the first cell
line (HELA), we note that a significant difference was recorded between extracts with the
highest inhibition activity with the apolar extracts (CHYA, DCM), while the MeOH extract
has shown the lowest. For the second cell line (RAW 264-7), the apolar fractions have pre-
sented also the highest cytotoxic activity. These results suggest that apolar bioactive com-
pounds could be responsible for this inhibition effect. Moreover, the shell extracts have
demonstrated cytotoxic potential toward the two cell line. This could be explained by the
presence of bioactive substances in the shell fractions capable of inhibiting the Hela and
Raw 264-7 cells. In the same context, a study by Mericli et al. (2017) investigating the anti-
cancer potential of P. dulcis seed oil against Colo-320 and Colo-741 cells showed that the
almond seed oil from northern cyprus and turkey varieties was an effective antiprolifera-
tive agent against the two cancer cell lines. In another study, Amico et al. (2006) investi-
gated the anticancer activity of terpenoids compounds obtained by bioguided fractiona-
tion assay of Sicilian almond hulls, the results showed that betulinic acid, a compound
isolated from almond hulls in this study, exhibited anticancer potential against the MCF-
7 cell line with an antiproliferative activity GI50 = 0.27 µM.
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Figure 5. Cytotoxic activity of P. dulcis shell extracts against two cell types (HELA; RAW 264-7 ). Cyclohexane: CHYA;
Dichloromethane: DCM; Ethyl acetate: EtOAc; Methanol: MeOH; Tamoxifen: was used as a reference at 100 µM). Ex-
tracts was tested at 50 µg/mL Mean values ± SD (n = 3); Different letters on the histograms mean a significant differ-
ence (p <0.05).
3.6. Principal component analysis (PCA):
Principal component analysis was performed to determine the correlation between
the TPC and the different biological assays. The inertia axes was withheld from this anal-
ysis. As shown in figure 6, the total variation percentage recorded at 81.58%. The axes PC1
and PC2 expressed, respectively, 50.63% and 30.94% of variability. The “loading plot”
(Figure 6) express the correlation between the tested activities, the TPC, and the correla-
tion between the PC and the original variables. As presented in table 5, PC1 is positively
correlated with DPPH, alpha-glucosidase, Raw276-4, and HELA with loading respec-
tively 0.903, 0.547, 0.711, and 0.753. The PC2 is positively correlated with TPC and alpha-
amylase with loading, respectively, 0.818, and 0.917. Based on the correlation matrix (Ta-
ble 6) and Figure 8, we note the presence of moderate correlation between the TPC and
the DPPH (0.388) which suggests the presence of other compounds classes such as fatty
acids or polysaccharides contributing to the antioxidant activity detected in the present
study. Moreover, the TPC present negative correlation with (alpha-glucosidase, alpha-
amylase, and cytotoxic activity against RAW 276-4 and HELA) which means that phenolic
compounds present in the extracts could be not responsible for the obtained results and
then, we can suggest the presence of other classes of compounds responsible for these
effects like the fatty acids, alkaloids, and polysaccharides. Furthermore, DPPH presents
high correlation with alpha-glucosidase activity (0.680), suggesting that the substances
exerting these effects could be the same. In addition, alpha-glucosidase present a low cor-
relation with alpha-amylase, which could indicate that these activities were exerted by
different bioactive compounds, and let us suggest the presence of a different class of bio-
active molecules in the shell extracts. For the cytotoxic activity, the correlation between
the two-cell line is high (0.679). As shown in figure 7, we can class the different extracts
into three groups: C1 (MeOH), C2 (H2O), and C3 (CHYA, DCM, and EtOAc). The correla-
tion between observations and variables presented in figure 8 shows that the MeOH ex-
tract is near to the DPPH and the alpha-glucosidase. This suggests that polar components
such as polyphenols and polysaccharides may have a synergistic influence on the ob-
served effects. In addition, the apolar fractions (CHYA, DCM, and EtOAc) are near to the
alpha-amylase and the cytotoxic activities against RAW 276-4 and HELA, which suggest
the presence of bioactive molecules in the mentioned fractions responsible for the present
effects. In conclusion, based on the results of the biological assays and the statistical anal-
ysis mainly the PCA, we can deduct that the shell extracts are rich in several bioactive
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compounds of different classes and polarity such as Catechin, epicatechin, and
Synephrine exerting moderate cytotoxic, antidiabetic and antioxidant effects.
Figure 6. Principal Components analysis (PCA) “loading plot” of (TPC: total phenolic content),
(DPPH: Antioxidant activity) and biological activities (Alpha-amylase, Alpha-glucosidase: Antidi-
abetic activity); (HELA and RAW 276-4: cytotoxic activity) of shell extracts of P. dulcis.
Table 5. Correlations between variables and factors.
F1
F2
TPC
0.121
0.818
DPPH
0.903
0.000
ALPHA
-GLUCOSIDASE
0.547
0.100
ALPHA
-AMYLASE
0.003
0.917
RAW 276
-4
0.711
0.021
HELA
0.753
0.000
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Figure 7. Principal component analysis (PCA) “score plot” of TPC: total phenolic content), (DPPH:
antioxidant activity), and biological activities (Alpha-amylase, Alpha-glucosidase: Antidiabetic ac-
tivity); (HELA and RAW 276-4: cytotoxic activity) of shell extracts of P. dulcis.
Table 6. Correlation matrix (Pearson (n)).
Variables TPC DPPH ALPHA-GLUCO-
SIDASE
ALPHA-AMYL-
ASE
RAW 276
-4
HELA
TPC 1 0.388 -0.018 -0.828 -0.150 -0.209
DPPH 0.388 1 0.680 0.011 -0.757 -0.762
ALPHA-GLUCOSIDASE -0.018 0.680 1 0.185 -0.490 -0.535
ALPHA-AMYLASE -0.828 0.011 0.185 1 -0.099 0.063
RAW 276-4 -0.150 -0.757 -0.490 -0.099 1 0.679
HELA -0.209 -0.762 -0.535 0.063 0.679 1
.
Figure 8. Principal Components Analysis (PCA) “Biplot” of TPC: total phenolic content), (DPPH:
antioxidant activity), and biological activities (Alpha-amylase, Alpha-glucosidase: Antidiabetic ac-
tivity); (HELA and RAW 276-4: cytotoxic activity) of shell extracts of P. dulcis.
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3. Materials and Methods
3.1. Plant Material:
In this study, the shell material of P. dulcis (Achaak variety) was collected from the
south-Est of Tunisia (Zarzis). After air-drying, the collected plant material was manually
ground using mortar (LGC France) and stored at room temperature.
3.2. Extraction:
• Maceration:
Fifty grams of dried shell material was successively extracted, using organic solvents
of increasing polarity cyclohexane (CHYA); dichloromethane (DCM); ethyl acetate
(EtOAc); methanol (MeOH); and ultra-pure water (H2O) for 2 h with medium agitation
at ambient temperature and pressure. The extraction mixture was subsequently filtered
with Wattman paper .The solvents were then, evaporated under a vacuum at 35 ° C using
a rotary evaporator (IKA; RV 10 auto V; Staufen Germany).
3.3. Total Phenolic Content (TPC):
The total polyphenolic content of P. dulcis was evaluated using the Folin-Ciocalteu
method described by Ben khadher et al..
3.4. Determination of Radical Scavenging Activity:
The antioxidant scavenging activity was assessed using 1,1-diphenyl-2-picrylhydra-
zyl free radical (DPPH); as reported by Rahmani et al [42] with some modifications. In a
96-well microplate (Micro Well; Thermo Fisher Scientific; Illkirch France); 20 µL of each
diluted plant extract (0.5 mg/mL) was mixed with 180 µL of methanolic DPPH solution
(0.2N). The reaction mixture was then incubated at 25 °C for 25 min. Subsequently, the
absorbance was measured at 524 nm using a microplate reader (Multiskan Go F1-01620;
Thermo Fisher Scientific; Vantaa, Finland). The ascorbic acid was used at 4 µg/mL as the
reference for this activity.
DPPH inhibition was calculated as follows: % Inhibition = 100 × (Ablank −Asam-
ple)/Ablank.
3.5. Biological Activity:
3.5.1. Antidiabetic activity:
• Anti-α-Amylase Activity:
The anti-α-amylase activity of the shell extracts was performed as described by Ben
khadher et al.[43].
• Anti-α-glucosidase Activity:
The anti-α-glucosidase activity of the different shell extracts was performed using
the PNP-G method as described by Rahmani et al[42]. In the reaction mixture, 50 µL of
phosphate buffer (0.1M, pH 6.9) was mixed with 100 µL of the alpha-glucosidase enzyme
(1 U / ml) and 50µL of each extract (0.5 mg/mL), after 10 min of incubation at 25°C, 50µL
of PNP-G (5 mM) was added to the mixture. After 5 min of incubation, the absorbance
was measured at 405 nm. Inhibition of enzyme activity was calculated as follows:
% inhibition = 100 × (Ablank − Asample)/Ablank.
3.5.2. Cytotoxic Activity:
The Cytotoxic activity of the shell extracts was determined on the cell line RAW 264.7
(mouse macrophage cell line) and Hela (human cervical cancer cell line), as described by
Ben Khadher et al. (2022)[43] with slight modification. In a 96-well microplate, cells have
been distributed at 13 × 103 cells/well for Hela and 12 × 103 cells/well for RAW 264-7 in
100 µL. After 24 h of incubation at 37 °C, 100 µL of each extract diluted in the medium
after being solubilised in DMSO was added to 100 µL of the corresponding culture me-
dium; DMEM (Advanced DMEM, Thermo Fisher Scientific). The plate was then incu-
bated for 48 h at 37°C. The cytotoxic potential of the samples was evaluated using the
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MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. After re-
moving the supernatant, cells were treated with 50 µL of MTT solution then, the plate
was incubated at 37°C for 40 min. then MTT was then eliminated and 80 µL of DMSO
was added. The absorbance was determined at 605 nm using the microplate reader
(Mullikan Go, F1-01620, Thermo Fisher Scientific, Vantaa, Fin-land). The Tamoxifen at
100 µM was used as a reference.
3.6. Chromatographic analysis:
3.6.1. High-Performance Liquid Chromatography Analysis (HPLC-DAD):
Analysis of the different shell extracts by HPLC-DAD was carried out in a Thermo Scien-
tific Accela pump equipped with Accela PDA detector (280 nm wavelength was used for
the detection of compounds). Separation has been performed using an RP-C18 column
(Phenomenex; Le Pecq; France). The dimensions of the column are 25 cm × 4.6 mm and
particle size 5 µm. Elution was carried out with flow rate of 0.5 mL/min.The mobile phase
made up of acidified water (pH = 2.65) as solvent A and acidified water/ACN (20:80 v/v)
as solvent B. The samples were eluted by the linear gradient as follows: from 12% B to
30% B for 15 min; from 30% B to 50% B for 2 min; from 50% B to 99.9% B for 3 min; and
finally from 99.9% B to 12% B for 7 min. Extracts were prepared at 10 mg/mL using acidi-
fied water/ACN (80:20 v/v) and filtered using a filter (Sigma Aldrich, Millex-HA filter 0.45
µm, Saint-Quentin-Fallavier, France). The identification and the quantification of the mol-
ecules was determined by comparison them with the retention time and lambda max of
known standards. Additionally, the quantity of each compound present in the shell ex-
tracts was determined using their specific calibration curves.
3.6.2. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:
The volatile composition of almond shell extracts was determined using the method
mentioned by Rahmani et al. (2020)[42] with some modifications. Shell extracts was solu-
bilized in their extraction solvents (except for water extract, where methanol was used) at
3 mg/mL. the analysis was carried out on Saturn 2000 gas chromatography (Les Ulis,
France), equipped with a fused silica capillary DB-5MS column (5% phenylmethylpoly-
syloxane, 30 × 0.25 mm, film thickness 0.25 µm). H2 was used as a carrier gas in this anal-
ysis. Chromatographic conditions were: 60 ° C for 1 min, then up to 150°C, at a gradient
of 10°C/min, then hold for one minute. A second gradient was applied to reach 260 at
12°C/min, and then was held for 10 min. The trap temperature was 220 °C and that of the
transfer line was 240 °C. Mass scanning was performed from 70 to 650 m/z. 5 µL of each
extract was injected.
• Derivatization method:
The derivatization method was conducted as described by ben khadher et al (2022).
• Compounds identification:
Identification of volatile compounds by GC-MS of P. dulcis shell extracts has been
performed using the Xcalibur software version 3.0.63 by comparison of their mass spectra
and linear retention index (LRI) with those recorded in the NIST MS library version 2.4
built on 25 March 2020.
3.7. Statistical Analysis:
All data were expressed as means ± standard deviations from triplicate measure-
ments. Confidence limits were set at p < 0.05 and calculated according to the ANOVA test
using the Statistical Package for the Social Sciences (SPSS) 22 (version IBM. 22.0. 2013, San
Francisco, CA, USA). The difference between the extraction solvents was determined us-
ing the Tukey’s test. Principal component analysis (PCA) was performed using XLSTAT
(version 2021.3.1, Addinsoft, Pearson edition, Waltham, MA, USA)
4. Conclusions
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The present study asses the chemical profile as well as the biological potential of P. dulcis
shell extracts. The chemical analysis led to the identification of 15 compounds by HPLC-
DAD of which 11 were firstly identified in the almond plant, and 26 volatile compounds
by GC-MS among them seven were identified for the first time in the P. dulcis plant. For
the biological activities, shell extracts displayed moderate antioxidant, antidiabetic, and
cytotoxic potential. Further studies are required to confirm the obtained results and to
isolate the active compounds responsible for those activities.
Author Contributions: Talel ben Khadher carried out the practical part and drafting of
the manuscript. Jalloul Bouajila, Mohamed Mars., Samir Aydi, and Sameh Sassi-Aydi.
were involved in the correction of the manuscript, the orientation of work, and the coor-
dination of the project.
Funding: This work has been supported financially by the ERASMUS PLUS program and
the Tunisian Minister of Higher Education and Scientific Research, which provided a PhD
scholarship for the student Talel ben khadher.
Conflicts of Interest: The authors declare that they have no conflict of interests
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