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Chemical Composition, Antioxidant, Antimicrobial and Antiproliferative

Activities of Wastes from Pecan Nut [Carya illinoinensis (Wagenh) K. Koch]

Article · April 2019

DOI: 10.1007/s12649-019-00681-2

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Waste and Biomass Valorization
https://doi.org/10.1007/s12649-019-00681-2

ORIGINAL PAPER

Chemical Composition, Antioxidant, Antimicrobial

and Antiproliferative Activities of Wastes from Pecan Nut [Carya
illinoinensis (Wagenh) K. Koch]
Ramón A. Flores‑Estrada1 · Nohemí Gámez‑Meza1   · Luis A. Medina‑Juárez1 · Lucía G. Castillón‑Campaña2 ·
Claudia C. Molina‑Domínguez1 · Luisa A. Rascón‑Valenzuela2 · Alfonso García‑Galaz3

Received: 29 August 2018 / Accepted: 15 April 2019

© Springer Nature B.V. 2019

Abstract
This study was carried out to determine the antioxidant, antiproliferative, and antimicrobial activities of shell and husk
extracts from two pecan nut cultivars (Wichita and Western). The methanolic extracts by mass spectrometry (MS/MS) were
analyzed. The antioxidant activity by ABTS and DPPH methods was evaluated. Antiproliferative activity was evaluated
against cervical (HeLa), lung (A549), prostate (PC-3), colon (LS180), cancer cell lines and normal retinal cell (ARPE-19)
by MTT assay. Antibacterial activity against Staphylococcus aureus and Escherichia coli by disk diffusion and macrodilu-
tion was tested. The results indicated that the phenolic compounds content and antioxidant capacity in nutshell was greater
than in husk. Also, an antiproliferative effect of the nutshell extracts from both cultivars in the HeLa gynecological cell line
was found. Furthermore, nutshell extracts of both varieties showed antibacterial activity against Staphylococcus aureus. The
agroindustrial waste of pecan nutshell to obtain bioactive compounds could be used.

Keywords  Nut wastes · Carya illinoinensis · Nutshell · Husk · Antibacterial activity · Antiproliferative activity

Abbreviations CFU Colony-forming unit

ABTS 2,2-Azino-bis (3-ethylbenzthiazoline-6-sul- DMEM Dulbecco’s modified Eagle’s medium
fonic acid) DMSO Dimethyl sulphoxide
ARPE-19 Human retinal pigment epithelial cell line DPPH 2,2-Diphenyl-1-picrylhydrazyl
ATCC​ American type culture collection ELISA Enzyme-linked immunosorbent assay
A549 Human alveolar epithelial cell line FBS Fetal bovine serum
BCL-2 Apoptosis regulator [Homo sapiens (Human)] GAE Gallic acid equivalent
BIRC5 Gen baculoviral inhibitor of apoptosis repeat- HeLa Cervical cell line
containing 5 HepG2 Human liver cancer cell line
CaCo-2 Human epithelial colorectal adenocarcinoma HHDP Hexahydroxydiphenoyl
cells HTB4 Bladder cancer cell line
CE Catechin equivalent IC50 Concentration of a substrate needed to reduce
50 percent of the cell population
LS180 Intestinal human colon adenocarcinoma cell
* Nohemí Gámez‑Meza
nohemi.gamez@unison.mx line
LLC-PKI Kidney epithelial cells
1
Departamento de Investigaciones Científicas y Tecnológicas, MBC Minimum bactericidal concentration
Universidad de Sonora, Rosales y Blvd. Luis Encinas, MIC Minimum inhibitory concentration
CP 83000 Hermosillo, Sonora, Mexico
2
MS Mass spectrometry
Departamento de Ciencias Químico Biológicas, MTT Colorimetric assay for assessing cell meta-
Universidad de Sonora, Rosales y Blvd. Luis Encinas,
CP 83000 Hermosillo, Sonora, Mexico bolic activity by reduction of 3-(4,5-dimethyl-
3 thiazol-2-yl)-2,5-diphenyltetrazolium bromide
Ciencia de los Alimentos, Centro de Investigación en
Alimentación y Desarrollo AC, 83304 Hermosillo, Sonora, NF-κB Nuclear factor kappa-light-chain-enhancer of
Mexico activated B cells

13
Vol.:(0123456789)

PC-3 Human prostate cancer cell line
UV–Vis Ultraviolet–Visible spectral region
We Nut Western
Wi Nut Wichita
Statement of Novelty
This work shows the presence of phenolic substances in
pecan nut wastes, which are capable of preventing the bac-
terial growth, multiplication of cancer cells, and the produc-
tion of oxidant substances.
Introduction
Some agroindustrial processes generate byproducts or resi-
dues that are not handled properly, are generating diverse
environmental problems. Some wastes are burned or dumped
into landfills producing large amounts of carbon dioxide.
However, these materials are attractive sources for their
chemical (sugars, pigments, dietary fiber, protein, lignin,
and polyphenols) contents that can be used in antimicrobial
and antiproliferative studies [1–4]. Therefore is important
discover bioactive compounds from natural sources that
inhibit bacterial growth [5].
The plants contain an extensive variety of bioactive com-
pounds with great interest in food, cosmetic and the phar-
maceutical industries. These compounds behave directly
or indirectly as antioxidants, anticancer and antimicrobial
agents. Approximately 60–80% of the world population,
especially in unindustrialized countries, still rely on tradi-
tional medicine for treating more than 87% of diseases [6].
In the last several years, more than half of the products that
were used as pharmaceuticals were derived from natural
sources [7].
Pecan nut cultivation in Mexico is a growing activity,
especially in northern states, due to the climatic and edapho-
logical adaptation of pecan trees, as well as market condi-
tions and the attractive returns from selling pecan nuts to
the USA [8]. In Mexico in 2015 according to the report of
the Servicio de Información Agroalimentaria y Pesquera
there was a production of around 122,714 tons of walnut
of which 50% of waste was produced [9, 10]. Separate and
independent studies have shown that the pecan nut (Carya
illinoinensis) could be a source of compounds with potential
to inhibit oxidation, cell proliferation, and bacterial growth
[11–15]. Therefore, the objetive was to study the chemical
composition, antioxidant, antiproliferative and antibacterial
potential of extracts husk and shell from two cultivars pecan
nut Wichita and Western (Carya illinoinensis (Wangenh.)
K. Koch).
13
Waste and Biomass Valorization
Materials and Methods
Chemicals and Reagents
Müeller Hinton broth (Difco), Müller Hinton agar
(Bioxon), gentamicin (PubChem CID: 3467), dime-
thyl sulfoxide (DMSO, PubChem CID: 679), gallic acid
(PubChem CID: 370), catechin hydrate (PubChem CID:
107957), 6-Hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid (Trolox, PubChem CID:40634), 2,2-Azino-
bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS,
PubChem CID: 5815211), 2,2-Diphenyl-1-picrylhydra-
zyl (DPPH, PubChem CID:2735032), 2-(3,5-diphenyl
tetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole; bromide
(MTT, PubChem CID: 64965), DMEM culture medium,
doxorubicin (PubChem CID:31703) were obtained from
Sigma-Aldrich. Methanol was acquired from JT Baker
(Phillipsburg, USA). Cell lines: cervical (HeLa), lung
(A549), prostate (PC-3), colon (LS180) and human retinal
pigment epithelium (ARPE-19, ATCC CRL-2302) were
bought in the American Type Culture Collection (ATCC,
Rockville, MD, USA). The Department of Chemical Bio-
logical Sciences-University of Sonora provided bacterial
strains: Staphylococcus aureus ATCC 6538P, Escherichia
coli ATCC 25922.
Raw Material
The samples of pecan nut Western (We) and Wichita (Wi)
[Carya illinoinensis (Wagenh) K. Koch], were obtained
from Grupo Alta S.A. de C.V. The samples were collected
in México (28°59′19.69″N 111°32′29.86″W) and were
stored in absence of light to − 20 °C until analysis.
Quantification of Phenolic Compounds
The samples of nutshell and husk were powdered using an
Osterizer mixer (Oster model 4122, Mexico City) and sieved
with a 30-mesh screen. Methanolic extracts were obtained
according to the Molina [16] methodology and lyophilized.
The total phenols content in the extracts was determined by
the Folin-Ciocalteu method [17]. After 30 min, the absorb-
ance at 765 nm was measured using a UV–Vis spectropho-
tometer (Cary 100 Bio, Varian, Victoria Australia). Values
were expressed as milligrams of gallic acid equivalent/g dry
weight (mg GAE/g dw) of sample. The flavonoid contents
(mg/g) were measured spectrophotometrically at 415 nm
using a UV–Vis spectrophotometer (Cary 100 Bio, Varian,
Victoria Australia). Values were expressed as milligrams of
catechin equivalent/g dry weight (mg CE/g dw) [16, 18].
Waste and Biomass Valorization
MS/MS Analysis
A Varian 500-MS IT mass spectrometer (Walnut Creek, CA,
USA) equipped with an electrospray ionization source and
an ion trap mass analyzer was used, which were controlled
by MS WorkStation software v.16 (Agilent Technologies,
Santa Clara, CA, USA). The mass spectrometer was oper-
ated in negative mode in the range of 100–2000 m/z using
a flow of 10 mL/min, a voltage of − 17 kV, a capillary tem-
perature of 350 °C and helium at a flow of 0.8 mL/min. The
identification of several metabolites was realized by second
order fragmentation (MS/MS).
Antioxidant Activity Evaluation
Antioxidant activity of husk and nutshell extracts was evalu-
ated by measuring the free radical scavenging activity using
two different radicals ­(ABTS·+ and ­DPPH·). ABTS method
is based on the reduction of the green/blue color produced
by the ­ABTS· radical reaction with the antioxidants. To gen-
erate the radical cation, 19.2 mg of ­ABTS·+ were dissolved
in 5 mL of distilled water. Subsequently 88 µL of a potas-
sium persulfate solution (0.0378 mg/mL) were added, and
the solution was homogenized and incubated in darkness
at room temperature for 16 h. The ­ABTS·+ radical formed
was diluted in methanol to an absorbance of 0.70 ± 0.1
at 734 nm. The filtered samples (0.1 mL of extract) were
placed in a cell and mixed with 3.9 mL of the radical. Initial
absorbance ­(Absi) and after 7 min of reaction (­ Absf) was
measured using a UV–Vis spectrophotometer (Cary 100 Bio,
Varian, Victoria Australia).
DPPH method is based on the color changes caused by
antioxidants. An alicuot (0.1 mL) of each methanolic extract
was mixed with 3.9 mL of the D ­ PPH· radical (0.025 mg/
mL methanol). The mixture was shaken in a vortex mister
and kept 30 min in darkness. The absorbance was read at
515 nm using a UV–Vis spectrophotometer (Cary 100 Bio,
Varian, Victoria Australia) [19]. The antioxidant activity
was expressed in μmol of Trolox equivalent (μmol TE/g). A
calibration curve (0.00–0.89 μmol/mL) was used.
Antiproliferative activity evaluation
Antiproliferative activity was measured using MTT reduc-
tion assay with modifications [20]. Cells (1 × 104 per well,
50 μL) in each well of a 96-well plate were placed and
incubated for 24 h at 37 °C in 5% ­CO2 atmosphere. Lyo-
philized extracts at different concentrations (0–200 μg/
mL) were dissolved in DMSO, and 50 μL of medium
(DMEM 5% FBS) were added to cells, and incubated for
48 h. The maximum concentration of DMSO used was
2% (v/v). Doxorubicin was used as positive control, due
to its broad spectrum in the cancer treatment. After 4 h of
incubation, the cells were washed with PBS, and then 100
μL of culture medium and 10 μL of MTT solution (5 mg/
mL) were added to each sample. The cells were measured
by the ability to reduce the salt of tetrazolium (yellow)
to formazan (violet). The formazan crystals with 100 μL
of acid isopropanol were dissolved. The absorbance was
measured with an ELISA microplate reader (BioMarBrad),
at 570 nm and a reference wavelength of 630 nm. The
absorbance of the cells treated with DMSO was consid-
ered as 100% of proliferation. The calculations were made
using a GraphPad Prism 5 software (GraphPad Software,
Inc., CA, USA).
Antibacterial Activity Evaluation
The evaluation of antibacterial activity of lyophilized
extract from husk and nutshell was determined by the
disc diffusion method [21]. All samples were dissolved in
DMSO (5% v/v) in order to obtain serial dilution concen-
trations of 250 to 31.25 mg/mL. S. aureus and E. coli ­(108
CFU/mL) were inoculated using a sterile swab on the Petri
dish (90 mm) with 20 mL of Mueller–Hinton agar. Sterile
disks of 6 mm of diameter were impregnated with 10 µL
of extract. Gentamicin (10 μL/disc) was used as positive
control, and DMSO (10 μL/disc) as negative control. The
bacteria were incubated at 36 °C for 24 h.
The concentrations that presented an inhibition zone
≥ 13 mm were determined as the minimum inhibitory con-
centration (MIC) using the technique of macrodilution of
agar [22]. The determination was performed in tubes with
different concentrations of extracts (0.00, 0.58, 1.17, 2.34,
4.68, 9.37, 18.75, 37.5, 75.0, 150.0, and 300.0 mg/mL).
A final volume of 3 mL with Mueller–Hinton agar in each
tube was used. An aliquot (100 μL) of the bacterial sus-
pension was added to each tube, similar to 0.5 MacFarland
standard (1.5 × 108 CFU/mL). After of a gently homog-
enization, the tubes were incubated at 37 °C for 24 h. The
DMSO was used as negative control, and the gentamycin
as positive control. It was considered as MIC to tube with
the lower concentration where no presented a visible bac-
terial development.
To determine the minimum bactericidal concentration
(MBC) of extracts, an inoculum with a sterile inoculating
loop was taken, and was subcultured into Petri dishes with
Mueller–Hinton agar. The plates were incubated at 37 °C
for 18–24 h, after which the presence or absence of bacte-
rial colonies was evaluated. The MBC was determined as
the minimum concentration of the compound that does not
allow the visible growth of colonies in the Petri dish. If
growth was observed, it was concluded that this concen-
tration of the extract produces a bacteriostatic effect [23].
13
 Waste and Biomass Valorization

Statistical Analysis extracts, vanillic, gallic, ellagic, ferulic, methyl-ellagic and

Statistical analysis (p < 0.05) of the data was done with SPSS
Statistics 17.0 (Chicago: SPSS Inc). Data were presented as
mean value ± standard deviation (SD), which were calcu-
lated from triplicate analysis.
Results and Discussion
Phenols and Total Flavonoids Content
The nutshell extract showed higher content of phenolic
compounds than husk extracts. Phenols and flavonoids
contents in husk extracts did not show any significant dif-
ferences (p < 0.05) between the two varieties (Table 1).
Fernández-Agulló et al. [24] reported higher levels of phe-
nols (81.50 ± 2.55 mg/g) for husk of Juglans regia than
found in this study. De La Rosa et al. [12] reported values
in the range of 24.73–54.29 mg GAE/g for husk from pecan
nut. Comparing the levels of total phenols and flavonoids in
shell, there were significant differences (p < 0.05) between
the varieties, with the highest content the Western variety.
Akbari [25] reported flavonoids contents ranging from 5.34
to 10.64 mg CE/g for husk of Juglans regia. De La Rosa
et al. [11] reported levels of flavonoids in methanolic extract
of pecan nutshell four times lower than the results obtained
in this study. The content of phenols and flavonoids can be
an important parameter that establishes both the quality of
the material and its biological potential.
MS/MS Analysis
MS/MS analysis of shell and husk extracts from pecan nut
revealed the presence of around 30 compounds. Comparing
the primary ion fragments present in the husk and shell sam-
ples with information in literature was made the identifica-
tion (Tables 2, 3, 4, and 5). These substances were classified
into the following groups according to their nature: phenolic
acids, flavonoids, glycosylated phenolic acids, glycosylated
flavonoids and (epi) catechin dimers and trimers. In nutshell
ellagic-pentose acids were found (Fig. 1) as well as [epi]
catechin, protocatechuic aldehyde, [epi]afzelekine-hexose,
myricetin-pentose, myricetin-3-O-rhamnose, HHDP-hexose,
[epi]gallocatechin-[epi]catechin, [epi]gallocatechin-O-gal-
late, [epi]gallocatechin dimer, HHDP-galloyl glucose, [epi]
catechin trimer, [epi]catechin-[epi]gallocatechin-[epi]gal-
locatechin, [epi]gallocatechin-O-gallate-[epi]gallocatechin-
O-gallate (Fig. 2).
Because samples were not treated with acid hydrolysis,
it was possible to identify substances in the form of glyco-
sides. Catechin dimers and trimers were found in nutshell of
both cultivars (Tables 2 and 4) as [epi]catechin dimer, [epi]
gallocatechin dimer, [epi]gallocatechin-[epi]catechin and
trimer of [epi]catechin, [epi]catechin-[epi]gallocatechin-
[epi] gallocatechin, [epi] gallocatechin-O-gallate- [epi]
gallocatechin-O-gallate.
Antioxidant Activity
The values of the antioxidant activities found in husk
extracts were lower than those found in nutshell extracts
(Table 1). The antioxidant activities (ABTS method) of husk
and nutshell extracts were higher for Western variety than
those of Wichita. De La Rosa et al. [11] reported similar
antioxidant activities (518.4–644.2 μmol TE/g) in nutshell
from a different region of Mexico to that found in the present
study. Prado et al. [13] reported antioxidant activities higher
(1562.51 μmol TE/g) for pecan nut Barton variety, than
those obtained in this study. Villarreal-Lozoya et al. [15]
carried out a study with nutshell of cultivars belonging to the
species Carya illinoensis species, finding higher antioxidant
activities by DPPH method (1322.46–2697.41 μmol TE/g)
than present study. Also, Prado et al. [14] reported inhibition
values ranging from 1220 to 1950 μmol TEAC/g. Studies
conducted by De La Rosa et al. [11] showed lower inhibi-
tions values (537.8–720.3 μmol TE/g) by DPPH method,
compared to the present study. Therefore, results suggest
that the nutshell can serve as raw material for obtains natural
antioxidants.

Table 1  Total phenols, Husk Nutshell

flavonoids and antioxidant
activity of shell and husk Western Wichita Western Wichita
extracts from pecan nut
Phenols (mg GAE/g) 27.36 ± 1.91a 28.42 ± 1.73a 102.78 ± 4.33a 87.611 ± 2.04b
Flavonoids (mg CE/g) 23.37 ± 1.12a 21.57 ± 1.02a 94.89 ± 1.17a 79.03 ± 1.25b
ABTS (μmol TE/g) 152.59 ± 13.34b 115.54 ± 2.94a 631.09 ± 17.48b 502.74 ± 19.73a
DPPH (μmol TE/g) 334.86 ± 13.60a 310.54 ± 15.48a 955.69 ± 7.49a 945.78 ± 7.49a

Values represent the mean ± standard deviation of the triplicate. Values in rows with different superscript
(a, b) are significantly different (p < 0.05)
GAE gallic acid equivalent, CE catechin equivalent, ABTS method, DPPH method, TE trolox equivalents

13
Waste and Biomass Valorization

Table 2  Phenolic substances in the methanolic extract of shell from Wichita nut

Substances Precursor ion Ion products ESI-MSn (m/z)a
[M–H] (m/z)

1 Protocatechuic aldehyde [43] 137 109 (61)

2 Protocatechuic acid [44] 153 137 (100)
3 Coumaric acid [44] 163 119 (69)
4 Vanillic acid [43] 167 109 (43), 123 (93)
5 Gallic acid [44] 169 125 (100)
6 Caffeic acid [44] 179 135 (100), 179 (21)
7 Ferulic acid [44] 193 111 (64), 121 (12), 137 (20), 175 (42)
8 Trihydroxyflavone [45] 269 225 (51)
9 [Epi]afzelechin [46] 273 167 (100), 229 (59)
10 [Epi]catechin [43] 289 203 (64), 205 (64), 227 (24), 245 (100)
11 Brevifoline carboxylic acid [47] 291 203 (34), 247 (100)
12 Ellagic acid [43] 301 185 (67), 229 (62), 257 (100)
13 Dihydroquercetin [48] 303 285 (100)
14 [Epi]gallocatechin [43] 305 179 (48), 219 (16), 261 (21)
15 Methyl-ellagic acid [11] 315 185 (100), 300 (64)
16 Myricetine [49] 317 229 (12), 271 (100), 287 (44)
17 Laricitrin [50] 331 169 (100), 287 (75), 315 (15)
18 Dihydrocaffeic-hexose acid 377 179 (14), 341 (100)
19 Elagic-pentose acid [47] 433 300(100), 301 (59)
20 [Epi]afzelechin-3-O-glucoside [51] 435 273 (31), 300 (100), 301 (58)
21 Methyl-galloyl-ellagic acid [47] 447 300 (50), 301 (86), 315 (100)
22 Myricetin-3-O-pentoside [52] 449 316 (100), 317 (52)
23 Myricetin 3-O-rhamnoside [43] 463 300 (38), 301 (63), 316 (100)
24 Valoneic acid bilactone [11] 469 301 (60), 426 (43)
25 Miricetine-hexose [50] 479 300 (88), 315 (100), 316 (22)
26 HHDP-hexose [45] 481 275 (7), 301 (100)
27 Proanthocyanidin Type B [Epi]catechin dimer [53] 577 289(87), 407 (100), 425 (58), 451 (97)
28 Ellagic-galoyl-pentose acid [47] 585 301 (100), 433 (65), 541 (58)
29 [Epi]gallocatechin-[epi]catechin [53] 593 289 (100), 303 (50), 305 (92), 423 (73)
30 [Epi]gallocatechin dimer [53] 609 305 (100), 423 (32), 441 (45), 483 (69)
31 HHDP-galloyl-glucose [45] 633 301 (100)
32 [Epi]catechin trimer [53] 865 543 (100), 575 (52), 713 (78), 738 (46)
33 [Epi]catechin-[epi]gallocatechin-[Epi]gallocatechin [54] 897 289 (57), 305 (43), 413 (16), 606 (100), 711 (92)
34 [Epi]gallocatechin-O-gallate-[epi]gallocatechin-O-gallate [53] 913 305 (12), 423 (15), 607 (10), 727 (100), 787 (42)
a
 Relative abundance of ion fragments is in parentheses

Antiproliferative Activity nutshell of Western and Wichita, respectively. The husk

The antiproliferative activity of lyophilized extracts of
shell and husk from Western and Wichita pecan nut were
evaluated at concentrations established for the screen-
ing of extracts with activity (6.25–200 μg/mL) [26, 27].
The graphs obtained for the triplicates of three independ-
ent assays of effect of the various extracts on differents
cell lines are shown in Figs. 3 and 4. Nutshell extracts of
both cultivars showed an antiproliferative effect on HeLa
gynecological cell line at the highest concentrations, with
­IC50 values of 153.72 ± 2.03 and 174.19 ± 1.05 µg/mL for
extracts did no show antiprolifetative activity in any cell
lines. This differential effect may be due to the specific
components of each extract. Nutshell extracts were rich in
resveratrol, phenolic acids, and derivatives of gallic acid,
and especially rich in proanthocyanidins of high molecular
weight (Tables 2 and 4); whereas the extracts of husk pre-
sent mainly phenolic acids and flavonoids. Also, it can be
seen that the extracts show no significant effect on human
cell lines A549, LS 180 and ARPE-19 since even the ­IC50
value could not be determined at the highest concentra-
tion tested.

13
 Waste and Biomass Valorization

Table 3  Phenolic substances in Substances Precursor ion Ion products ESI-MSn (m/z)a

the methanolic extract of husk [M–H] (m/z)
from Wichita nut
1 Protocatechuic aldehyde [43] 137 109 (34)
2 Cinnamic acid 147 119 (42)
3 Protocatechuic acid [44] 153 109 (100)
4 Vanillic acid [43] 167 109 (100), 123 (41)
5 Gallic acid [43] 169 125 (100)
6 Caffeic acid [44] 179 135 (29)
7 Syringaldehyde [45] 181 166 (66)
8 Phloretin [45] 273 151 (100)
9 [Epi]afzelechin [46] 273 167 (100), 229 (23)
10 [Epi]catechin [43] 289 203 (16),205 (100), 229 (93), 245 (89)
11 Methyl-ellagic acid [11] 315 185 (62), 245 (44), 300 (30)
12 Laricitrin [50] 331 287 (100), 315 (25)
13 Elagic-pentose acid [47] 433 301 (36)
14 Quercetin 3-O-rhamnose [55] 447 301 (47), 387 (100)
15 Diosmetin-8-C-glucoside [56] 461 281 (100), 371 (52)
16 Quercetin 3-O-hexoside [43] 463 300 (64), 301 (44)
17 Quercetin 3-O-glucuronide [52] 477 301 (13)
18 HHDP-hexoside [45] 481 301 (61)
19 [Epi]catechin dimer [53] 577 289 (43)
a
 Relative abundance of ion fragments is in parentheses

In this regard, numerous in vitro studies have demon-
strated the antiproliferative effect of substances such as res-
veratrol, ellagic acid and proanthocyanidins, demonstrating
that these metabolites are capable of inducing cell death.
They reduce the proliferative capacity and decrease metas-
tasis due to the control of NF-κB signaling pathways and
the inactivation of cancer-promoting genes such as BCL-2
and BIRC5 [28]. Kim et al. [30] showed that the levels of
BAX and CASPASE-3 increased in SNU-C4 cells treated
with oligomeric proanthocyanidins. Also, the level of BCL-2
mRNA expression was significantly decreased compared
with control.
In previous studies the antiproliferative activity of ace-
tone soluble extracts of the edible part of 10 different types
of walnuts was evaluated, it was determined that the nuts had
­IC50 values of 9.7 ± 0.5 and 2.5 ± 0.1 mg/mL in cells HepG2
and CaCo-2, respectively [29]. The antiproliferative activity
of extracts rich in polyphenols against cancer cell lines has
been used as an indicator of chemopreventive properties.
De la Rosa et al. [12] measured the antiproliferative activity
of walnut paste extracts rich in polyphenolic compounds in
the cancer cell line HTB4 (bladder cancer), and in the non-
cancerous cell line LLC-PKI (kidney epithelial cells) obtain-
ing values of ­IC50 of 0.26 and 0.55 mg/mL, demonstrating a
potential selectivity of the extract compounds on the cancer
cell line. In the same study, cytotoxic activity of the shell
extracts and walnut paste in rat neurons was evaluated, it was
found that nutshell extract was more cytotoxic compared to
the extract of walnut paste (­ IC50 values of 1.8 and 4.6 mg/
mL, respectively).
On comparing the information obtained in the present
study with those of previously mentioned studies, the ­IC50
values obtained for the cell line HeLa were lower than those
­IC50 values obtained in the other cancer cell lines of the
studies mentioned, which indicates a greater potential of the
shell extracts in the antiproliferative activity. Data generated
in the present study indicated that this activity might be due
to the proanthocyanidins found in the shell extract. B-type
proanthocyanidins cause typical morphological apoptotic
features. However, more studies to elucidate the responsi-
ble compounds and molecular mechanisms are necessary.
Antibacterial Activity
Nutshell extract of both varieties showed antibacterial activ-
ity on S. aureus with diameter of inhibition zones of 13 ± 1.0
and 13.33 ± 1.53 mm for Wichita and Western varieties,
respectively. Neither of extracts presented antimicrobial
activity for E. coli. Caxambú et al. [31] reported inhibition
for the growth of S. aureus in a study of aqueous extract
of Carya illinoinensis on lettuce leaves. Husk extracts of

13
Waste and Biomass Valorization

Table 4  Phenolic substances in the methanolic extract of shell from Western nut

Substances Precursor ion Ion products ESI-MSn (m/z)a
[M–H] (m/z)

1 Malic acid [45] 133 115 (100)

2 Protocatechuic aldehyde [43] 137 109 (100)
3 Cinnamic acid 147 119 (100)
4 Coumaric acid [44] 163 119 (100)
5 Vanillic acid [43] 167 123 (100)
6 Gallic acid [44] 169 125 (100)
7 Caffeic acid [44] 179 135 (81), 179 (24)
8 Quinic acid [57] 191 111 (43), 127 (44), 173 (47)
9 Ferulic acid [44] 193 111 (67), 121 (89), 137 (100), 175 (46)
10 Synaptic acid [58] 223 149 (12), 179 (100)
11 Resveratrol [49] 227 121 (21), 143 (21), 185 (20)
12 [Epi]catechin [43] 289 203 (31), 227 (28), 245 (100)
13 Ellagic acid [43] 301 185 (100), 229 (16), 257 (26)
14 Dihydroquercetin [48] 303 285 (100)
15 [Epi]gallocatechin [43] 305 179 (71), 221 (100)
16 Methyl-ellagic acid [11] 315 185 (100), 300 (1)
17 Laricitrin [50] 331 169 (90), 257 (19), 315 (38)
18 Elagic-pentose acid [47] 433 300 (100), 301 (51)
19 [Epi]afzelechin-3-O-glucoside [51] 435 273 (100), 301 (98)
20 Isoramnetin 7-O-pentose [59] 447 300 (13), 315 (100)
21 Myricetin-3-O-pentoside [52] 449 316 (56), 317 (100)
22 Myricetin 3-O-rhamnoside [43] 463 300 (68), 301 (43), 316 (100)
23 Methyl-ellagic-hexose acid 477 301 (30), 315 (82)
24 HHDP-hexose [45] 481 275 (15), 301 (100)
25 Proanthocyanidin Type B [Epi]catechin dimer [53] 577 289 (46), 407 (100), 425 (43), 451 (67)
26 [Epi]gallocatechin-[epi]catechin [53] 593 289 (64), 303 (75), 407 (64), 423 (31)
27 [Epi]gallocatechin dimer [53] 609 305 (91), 423 (100), 441 (21)
28 HHDP-galloyl-glucose [45] 633 301 (100)
29 [Epi]catechin trimer [53] 865 543 (100), 575 (59), 713 (81), 739 (46)
30 [Epi]catechin-[epi]gallocatechin-[Epi]gallocatechin [54] 897 303 (24), 465 (26), 593 (38), 711 (100)
31 [Epi]galocatechin-O-gallate-[epi]gallocatechin-O-gallate [53] 913 305 (8), 423 (13), 607 (8), 727 (100), 787 (39)
a
 Relative abundance of ion fragments is in parentheses

both varieties showed no antibacterial activity against E. coli
and S. aureus. Oliveira et al. [32] and Orue et al. [33] did
not find antimicrobial activity of Juglans regia L and Carya
illinoinensis extracts for E. coli. It has been observed that S.
aureus is more susceptible to treatments using bark and shell
extracts of different types of walnut than E. coli [31, 34–36].
MICs of the extracts that were positive in the disk dif-
fusion test for S. aureus on Mueller–Hinton agar were
0.89 ± 0.13 and 0.92 ± 0.17 mg/mL for Western and Wichita,
respectively. These concentrations were the lowest in which
no bacterial growth after 24 h of incubation at 37 °C was
detected. Dah et al. [21] reported a MIC of 1.25 mg/mL for
S. aureus with Cola nitida shell. Chabi et al. [34] reported a
MIC of 0.313 mg/mL with bark and leaves of Anacardium
occidentale L.
MBCs of the shell extracts of the two walnut varie-
ties were 5.77 ± 1.43 and 3.66 ± 0.71 mg/mL for Western
and Wichita, respectively. Chabi et al. [34] found a MBC
of 1.25 mg/mL with the bark and leaves of the Indian nut
(Anacardium occidentale L.) for S. aureus. Xiong et al.
[37] proposed that the antimicrobial activity of phenolic
compounds could be due to iron deprivation, the binding to
proteins such as enzymes, and other interactions with car-
bohydrates. Ferulic and gallic acids exhibit activity over a
range of bacteria causing irreversible changes in membrane

13
 Waste and Biomass Valorization

Table 5  Phenolic substances in Substances Precursor ion Ion products ESI-MSn (m/z)a

the methanolic extract of husk [M–H] (m/z)
from Western nut
1 Malic acid [45] 133 115 (100)
2 Protocatechuic aldehyde [43] 137 109 (46), 135 (92)
3 Cinnamic acid 147 118 (100), 119 (69)
4 Protocatechuic acid [44] 153 109 (100)
5 Coumaric acid [44] 163 119 (39)
6 Vanillic acid [43] 167 123 (47)
7 Gallic acid [43] 169 125 (100)
8 Syringaldehyde [45] 181 166 (100)
9 Quinic acid [57] 191 111 (7), 173 (63)
10 Daidzein [60] 253 135 (46), 183 (65), 198 (42), 211(33), 225 (100)
11 Formononetin [48] 267 113 (55), 173 (24), 211(100)
12 Trihydroxyflavone [45] 269 225 (21), 141 (66)
13 Phloretin [45] 273 151 (56), 167 (79)
14 [Epi] catechin [43] 289 179 (58), 203 (20), 205 (27), 229 (100), 245 (58)
15 Quercetin [43] 301 151 (33), 179 (30), 257 (38), 283 (91), 285 (71)
16 Dihydroquercetin [48] 303 285 (100)
17 [Epi] gallocatechin [43] 305 133(100), 179 (14), 219 (15, 261 (89)
18 Methyl-ellagic acid [11] 315 185 (30), 300 (90)
19 Dimethyl-elagic acid [61] 329 301 (14), 314 (44)
20 Laricitrin [50] 331 169 (9), 287 (56), 315 (100)
21 Quercetin 3-O-pentose [62] 433 301 (90), 407 (100)
22 Elagic-pentose acid [47] 433 259 (23), 301 (28)
23 Quercetin 3-O-rhamnose [55] 447 301 (13), 387 (100)
24 Diosmetin-8-C-glucoside [56] 461 281 (64), 341 (100), 371 (23), 446 (40)
25 6-Hydroxy-7-methoxyquerce- 463 301 (32), 331 (8), 433 (100), 445 (25)
tin-3-O-pentose [63]
a
 Relative abundance of ion fragments is in parentheses

properties [38]. [Epi]gallocatechin-O-gallate has shown a
strong antimicrobial activity against S. aureus [39].
Antimicrobial activities of the extracts are attributed to
the presence of proanthocyanidins mainly to B-type proan-
thocyanidins. Mayer et al. [40] reported that proanthocyani-
dins showed higher antibacterial activity as compared to its
monomer. In addition the liking method of proanthocyani-
dins is involved in the variation of biological activities being
more effective the B-type proanthocyanidins [41]. Thus,
in this study, the nutshell extracts that are rich in B-type
proanthocyanidins exhibit a major antibacterial activity.
It has been proven that B-type proanthocyanidins alter the
morphology of bacterial due to the destruction of the cell
walls and membranes. The treatment with B-type proan-
thocyanidins produces an increase of alkaline phosphatase,
bacterial fluid conductivity and ATPase activity. Moreover,
it generates a depletion in the energy metabolic systems in
the bacteria [42].
Conclusions
The results indicated that the content of phenolic com-
pounds and antioxidant activity in nutshell was greater than
in husk. Therefore, an antiproliferative effect of the nut-
shell extracts of both cultivars in the HeLa gynecological
cell line was found. Furthermore, nutshell extracts of both
varieties showed antibacterial activity on S. aureus; while
husk extracts of both varieties did not show any antibacte-
rial activity against E. coli and S. aureus. These differential
effects may be due to the specific components of the extracts.
Nutshell extracts were rich in resveratrol, phenolic acids, and
derivatives of gallic acid and especially in proanthocyani-
dins of high molecular weight, whereas the extracts of husk

13
Waste and Biomass Valorization

Fig. 1  Mass spectra of phenolic acids in nutshell: a ferulic acid; b gallic acid; c ellagic acid; d methyl-ellagic acid

13
 Waste and Biomass Valorization

13
 Waste and Biomass Valorization

◂Fig. 2  Mass spectra of phenolic substances present in nutshell: a Res-
veratrol; b HHDP-galloyl-glucose; c [Epi]gallocatechin-O-gallate-
[epi]gallocatechin-O-gallate; d [Epi]catechin trimer
mainly contained phenolic acids and flavonoids. In scientific
literature have been reported that proanthocyanidins higher
antibacterial activity as compared to its monomer, and
these compounds may trigger the cell death by activation

Fig. 3  Antiproliferative activity of nutshell and husk extracts from Western pecan nut (mean ± SD, n = 3)

Fig. 4  Antiproliferative activity of nutshell and husk extracts from Wichita pecan nut (mean ± SD, n = 3)

13

of apoptosis, and the bacterial cell by destruction of cell
wall and membrane, the mechanism has not been elucidated.
Therefore, the agroindustrial waste of pecan nut (nutshell)
to obtain bioactive compounds could be used.
Acknowledgements  The authors to thank Grupo Alta S.A. de C.V.
(www.grupoa​ lta.com) for providing the pecan nut, Consejo Nacional de
Ciencia y Tecnologia of Mexico for the fellowship for the first author.
Authors also to thank Ph.D. Kristin Whitney, who provided help with
language.
Compliance with Ethical Standards  genh.) C. Koch] Shell Infusion. Grasas Aceites. 60, 330–335
Conflict of interest  The authors declare no conflict of interest.
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