Waste Management 88 (2019) 319–327

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Biological activity and chemical characterization of Pouteria lucuma

seeds: A possible use of an agricultural waste
Pedro Guerrero-Castillo a, Sarita Reyes b, Juana Robles a, Mario J. Simirgiotis c, Beatriz Sepulveda d,
Ronald Fernandez-Burgos e, Carlos Areche e,⇑
a
Sección Química, Pontificia Universidad Católica del Perú, Lima, Peru
b
Facultad de Química e Ing. Química, Universidad Nacional Mayor de San Marcos, Lima, Peru
c
Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia 5090000, Chile
d
Departamento de Ciencias Químicas, Universidad Andres Bello, Campus Viña del Mar, Quillota 980, Viña del Mar 2520000, Chile
e
Departamento de Química, Facultad de Ciencias, Universidad de Chile, Santiago, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Pouteria lucuma fruit is widely used to prepare cakes, ice creams and juice or also commercialized as pulp
Received 28 November 2018 and flour. As result of this business thousands of tons of seeds are generated as an agricultural waste. This
Revised 2 March 2019 study presents the antioxidant and antiulcer activities, and the identification of secondary metabolites by
Accepted 25 March 2019
UHPLC/ESI/MS/MS of an agroindustrial waste of Pouteria lucuma seeds. Fifty-nine compounds were ten-
Available online 30 March 2019
tatively identified including eight aminoacids, five organic acids, one nucleoside, five phenolic acids, five
phenolic alcohols, nineteen flavonoids, six lipids, and seven unknowns in the methanol extract of P.
Keywords:
lucuma seeds. The total phenolic content of the seeds was 52.82 ± 0.09 lmol GAE/g dry weight, while
Agroindustrial waste
Flavonoids
total flavonoid content was 5.99 ± 0.01 lmol Q/g dry weight. The antioxidant activity was 58.14 ± 0.05,
Lucuma 66.97 ± 0.00, 272.50 ± 0.00, and 67.02 ± 2.23 for the DPPH, ABTS, FRAP, and superoxide anion assays,
Phenolics respectively. The highest gastroprotective activity was obtained at 100 mg/kg (78%), which as higher than
LC-MS/MS the positive control lansoprazole (75%). Our findings showed that P. lucuma seed extracts have moderate
Pouteria to high antioxidant activity and gastroprotective properties. Therefore, it was demostrated that lucuma
UHPLC-Q-exactive focus seeds commonly eliminated as an agricultural industry waste, could be useful for the preparation of
nutritional supplements.
Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Pouteria lucuma is a plant whose fruit is widely used to prepare

Several plants are rich in useful antioxidant metabolites which
can be present in different parts of the plant such as leaves, crust,
peels, flowers, fruits and seeds. These metabolites have been asso-
ciated with multiple beneficial properties for human health; par-
ticularly, phenolic compounds are exhaustively studied due to
antioxidant capacities attributed to these molecules (Hsu, 2005;
Tanwar and Modgil, 2012). Indeed, the human body is affected
by reactive oxidant species (ROS), which are produced during the
metabolism, when the balance of production and neutralization
of ROS is disturbed, an unpaired electron of ROS reacts with near
molecules and harm the human cell, this process is called oxidative
stress (OS) (Coronado et al., 2015), phenolic compounds counteract
the OS process because these metabolites donate a hydrogen to
stabilize those radicals (Villa-Rodriguez et al., 2015).
⇑ Corresponding author.
E-mail address: areche@uchile.cl (C. Areche).
desserts (cakes, ice creams and others) or commercialized as pulp
and flour both in Peru and in other countries such as USA and
members of European Union. Indeed, thousands of tons of seeds
are generated as an agricultural waste due to the high demands.
Besides, there are few researches focused on the content of this
fruit (Pinto et al., 2009; Dini, 2011; Fuentealba et al., 2016;
García Ríos, 2016), and only few studies were reported about its
seeds. Moreover, there is only one research study where a great
number of fatty acids (FAs) were reported in Pouteria lucuma seeds
oil; the highest content were linoleic acid, oleic acid and palmitic
acid; additionally, skin regeneration properties of this oil was evi-
denced by in vivo analysis (Rojo et al., 2010).
However, we were not able to find any study focusing on the
discovery and characterization of the metabolites in an hydrophilic
extract from Pouteria lucuma seeds and the biological properties of
these extract related to its chemical content.
Among the analytical methods, ultra-high-performance liquid
chromatography (UHPLC) coupled with mass spectrometry (MS)

https://doi.org/10.1016/j.wasman.2019.03.055
0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
320 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327
is one of the most powerful tools to detect and recognize metabo-
lites. In fact, there were a lot of studies, which used this technique
to identify compounds from various natural sources (Ramirez et al.,
2013; Simirgiotis et al., 2016).
In the present work we report the antioxidant and antiulcer
properties plus the identification of secondary metabolites by
UHPLC coupled to high resolution mass spectrometry of an agroin-
dustrial waste of Pouteria lucuma seeds for the first time.
2. Materials and methods
2.1. Chemicals
UHPLC-MS grade solvents as hexane, methanol, formic acid, and
acetonitrile were purchased from Merck (Santiago, Chile). Ultra-
pure water was obtained from a Millipore water purification sys-
tem (Milli-Q Merck Millipore, Chile). HPLC standards (purity
higher than 95% by HPLC) were purchased either from Sigma
Aldrich (Saint Louis, Mo, USA), ChromaDex (Santa Ana, CA, USA),
or Extrasynthèse (Genay, France). Folin-Ciocalteu phenol reagent
(2 N), reagent grade Na2CO3, AlCl3, HCl, FeCl3, NaNO2, NaOH, quer-
cetin, trichloroacetic acid, sodium acetate, Gallic acid, 2,4,6-tri(2-
pyridyl)-s-triazine (TPTZ), Trolox (6-hydroxy-2,5,7,8-tetramethyl
chroman-2-carboxylic acid), nitroblue tetrazolium, xanthine oxi-
dase, and DPPH (1,1-diphenyl-2-picrylhydrazyl radical) were pur-
chased from Sigma-Aldrich Chemical Co.
2.2. Plant material
Pouteria lucuma fresh seeds were obtained from OVNI factory in
Chilca, Cañete-Perú, during harvest stage of the fruits in November
2017. The brown pericarp was removed and then immersed in liq-
uid nitrogen. Finally, it was powdered on a hammer mill (INQUI-
MET) and stored at
18 °C until used it.
2.3. Extraction
Dried and milled seeds of P. lucuma (50 g) were extracted with
methanol (200 mL, per 3 times in the dark, 72 Hs each time) at
room temperature and concentrated in vacuum below 40 °C to
yield 5.82 g of a dark gummy extract.
2.4. UHPLC-DAD-MS instrument
Thermo Scientific Dionex Ultimate 3000 UHPLC system hyphen-
ated with a Thermo Q exactive focus was used in our study. 1.0 mg
of the extract was dissolved in 1 mL of methanol, filtered (PTFE fil-
ter) and 10 lL were injected in the instrument, with all specifica-
tions set as previously reported (Simirgiotis et al., 2016).
2.5. LC parameters and MS parameters
Liquid chromatography was performed using an UHPLC C18
column (Acclaim, 150 mm  4.6 mm ID, 2.5 lm, Thermo Fisher
Scientific, Bremen, Germany) operated at 22 °C. The detection
wavelengths were 254, 280, 330, and 354 nm, and DAD was
recorded from 200 to 800 nm for peak characterization. Mobile
phases were 1% formic aqueous solution (A) and acetonitrile (B).
The gradient program (time (min), % B) was: (0.00, 5); (5.00, 5);
(10.00, 30); (15.00, 30); (20.00, 70); (25.00, 70); (35.00, 5) and
12 min for column equilibration before each injection. The flow
rate was 1.00 mL min
1, and the injection volume was 10 lL. Stan-
dards and extract dissolved in methanol were kept at 10 °C during
storage in the autosampler. The HESI II and Orbitrap spectrometer
parameters were optimized as previously reported (Garneau et. al.,
2013; Simirgiotis et al., 2016). We used a hybrid machine equipped
with quadrupole and orbitrap (quadrupole with orbital trap, with a
resolution of 70.000 at m/z 200) and HCD (high resolution) cell,
using independent variable data acquisition (vDIA) for a qualitative
coverage that allows detecting unknown compounds (untargeted
analysis). The daughter ions are formed in the HCD cell, with a col-
lition energy of 5 ev, getting high sensitivity, ions with five deci-
mals and getting 0.1–10 ppm of accuracy. ESI-MS in negative
mode with 0 ev of energy is the best for the detection of ionizable
phenolic compounds (selecting all ions from 50 to 2000 Da) which
are the mayor compounds in the extract. All ions are formed after
the heated electrospray probe (HESI II), which are detected in the
orbitrap and fragmented in the HCD cell. After detection, we can
analyse the neutral losses, the base peak spectra and full TIC spec-
tra with daughter MS2 ions, using Thermo Xcalibur 3.1 software.
2.6. Animals
Animals were acquired from the Chilean Institute of Health,
Chile, Santiago. Swiss albino mice weighing 30 ± 3 g were fasted
for 24 h before the experiments. The animals were fed on certified
Champion diet with free access to water under standard conditions
of 12 h dark-light period, 50% relative humidity and room tempera-
ture (22 C). The protocols were approved by the Animal Use and
Care Committee of the Universidad de Chile following the recom-
mendations of the Canadian Council on Animal Care as stated pre-
viously (Areche et al., 2013).
2.7. Gastroprotective effects
The gastroprotective activity of the extract was performed in
the HCl/ EtOH-induced lesion model as described previously
(Areche et al., 2013). Briefly, mice were distributed into groups of
seven animals each and fasted for 24 h with free access to water
prior to the experiments. Fifty min after oral administration of
the extracts (10, 25, 50, and 100 mg/kg), lansoprazole (30 mg/kg)
or 12% Tween 80 (10 mL/kg), all groups were orally treated with
0.2 mL of a solution containing 0.3 M HCl/60% ethanol for gastric
lesion induction. Animals were sacrificed 1 h after the administra-
tion of HCl/EtOH, and the stomachs were excised and inflated by
injection of saline (1 mL). The ulcerated stomachs were fixed in
5% formalin for 30 min and opened along the greater curvature.
Gastric damage visible to the naked eye was observed in the gastric
mucosa as elongated black-red lines, parallel to the long axis of the
stomach. The length (mm) of each lesion was measured, and the
lesion index was expressed as the sum of the length of all lesions.
2.8. Polyphenol and flavonoid contents
The analyses of total phenolic compounds (TPC) was based on
(Ramirez et al., 2013) with some modifications in a microplate
reader. To some 12 lL of extract to be measured, 168 lL of the
1% Folin-Ciocalteu reagent (Merck, Santiago) were added to well
of a microplate reader. The mixture was allowed to react for
5 min, then 120 lL of 10% sodium carbonate was added. The mix-
ture was incubated at room temperature for 30 min in darkness.
Absorbance was then taken at 765 nm using an UV–Visible multi-
plate reader (Synergy HTX, Biotek, USA). The obtained absorbance
values were replaced in the equation of the standard curve of gallic
acid (lmol/L). The content regarding phenolic compounds was
then expressed as gallic acid micromoles per gram of dry weight
(lmol GAE/g extract). The aluminum chloride method was used
for the determination of the total content of flavonoids (Ramirez
et al., 2013). For this test, 30 lL of the filtered sample (2 mg/mL)
was added to 159 mL of 5% NaNO2. After 5 min of rest, 18 lL of
10% AlCl3 was additioned to the mixture. At the sixth minute
 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327
18 lL of 1 M NaOH was completed and the absorbance measured
at 510 nm using an UV–Visible multiplate reader (Synergy HTX,
Biotek, USA). Flavonoid content (TFC) was calculated using a quer-
cetin standard calibration curve (25–150 ppm). Results were
expressed as quercetin micromoles per gram of dry sample (lmol
Q/g dry weight).
2.9. Antioxidant assays
2.9.1. DPPH cation radical discoloration test
The capturing capacity of the DPPH radical was evaluated by
the decolorization method (Ramirez et al., 2013). Briefly, 9 lL of
extract, (2 mg/mL), plus 341 lL of methanol DPPH solution
(400 lM) were adjusted with the solvent methanol to an absor-
bance of 1.10 ± 0.02 at 517 nm. The mixture was homogenized
using a vortex, allowed to react in the dark at room temperature
for 20 min, after which time absorbance was measured at
517 nm in a Synergy HTX monochromator (Biotek, USA). The per-
centage of discoloration of the DPPH moiety was obtained by mea-
suring the change in absorbance at 517 nm, the values obtained
converted to percent inhibition of the DPPH moiety using the
following:
 
S:A
Percentage Inhibition ¼ 1
 100
B:A
where S.A. is sample absorbance and B.A. is blank absorbance.
A curve was prepared with different dilutions of the extract, and
the results were expressed as IC50 in microg per mL. Standard
antioxidant compounds gallic acid (from 1.0 to 125.0 mg/mL,
R2 = 0.991) and quercetin (from 1.0 to 125.0 mg/mL, R2 = 0.993)
were used as standard antioxidant compounds, and were
determined to have IC50 values of 1.12 ± 0.01 mg/mL
(6.58 ± 0.05 mmol/L) and 7.36 ± 0.01 mg/mL (24.37 ± 0.03 mmol/L),
respectively.
2.9.2. Bleaching test with the cationic radical ABTS+
The capturing capacity of the ABTS+ radical was evaluated by
the decolorization method developed by Ramirez et al. (2013).
The radical ABTS+ is generated chemically by the oxidation of ABTS
with potassium persulfate after 16 h of incubation at room temper-
ature in the dark. Briefly, each well of the microplate reader (Syn-
ergy HTX, Biotek USA) was filled with 273 lL of the prepared
ABTS+ solution (previously adjusted with 80% methanol to obtain
an absorbance of 0.70 ± 0.02 at 734 nm), and 27 lL of the extract
(at a concentration of 2 mg/mL), and the mixture allowed to react
in darkness at room temperature for 45 min. The absorbance was
then measured at 734 nm and the values obtained converted to %
inhibition of the ABTS+ radical and a curve was prepared with dif-
ferent dilutions of the extract, and the results were expressed as
IC50 in lg/mL.
2.9.3. Ferric reduction ability-antioxidant power test (FRAP)
For the FRAP test, the methodology was performed with
slight modifications for use in a microplate reader (Ramirez
et al., 2013). Briefly, to 10 lL of the dissolved extract (2 mg/
mL), 290 lL of the FRAP solution was added and mixed in the
well of the microplate, allowed to react in the dark at room
temperature for 5 min. The absorbance measurement of the col-
ored Fe-TPTZ complex was performed at 595 nm. Absorbance
values were replaced in the Trolox standard curve equation
(lmol/L). The results were determined as equivalents of Trolox
(TE), in Trolox micromoles per gram of dry weight (lmol
Trolox/g dry weight).
321
2.9.4. Superoxide anion bleaching activity
The method is based in the activity of the enzyme xanthine oxi-
dase which generates superoxide anion radical (O
2 ) by oxidation
of reduced products from intracellular ATP metabolism in vivo.
The superoxide generated in this way reduces the nitro blue tetra-
zolium dye (NBT), leading to a chromophore with a maximum of
absorption at 560 nm. Superoxide anion scavengers reduce the
speed of generation of the chromophore. The activity of Lucuma
was measured in a microplate reader as reported previously
(Ramirez et al., 2013). All compounds and crude alcoholic extract
were evaluated at 100 g/mL. Values are presented as mean ± stan-
dard deviation of three determinations.
2.10. Statistical analysis
The statistical analysis was carried out using the originPro 9.1
software packages (Origin lab Corporation, Northampton, MA,
USA). The determination was repeated at least three times for each
sample solution. Analysis of variance was performed using ANOVA.
Significant differences between means were determined by Dun-
nett comparison test (p values < 0.05 were regarded as significant).
3. Results and discussion
3.1. Full identification by UHPLC-PDA-MS of the methanolic extract of
P. lucuma seeds
Fifty-nine compounds were tentatively identified including
eight aminoacids (peaks 1, 5, 7–11, 16, and 20), five organic acids
(peaks 2, 3, 6, 12, and 18), a disaccharide (peak 4), one nucleoside
(peak 13), five phenolic acids (peaks 24, 31, 32, 39, and 42), five
phenolic alcohols (peak 15, 26, 30, 36, and 49), one gallic acid (peak
14), nineteen flavonoids (peaks 17, 19, 21–23, 25, 27–29, 33–35,
37–39, 41, 43–45), a cinnamic acid derivative (peak 40), six lipids
(peaks 51–53, 56, 58–59) and seven unknown compounds, (peaks
46–48, 50, 54–55, and 57) in the chromatogram of the methanol
extract of P. lucuma (Table 1). These compounds appeared in the
following order of increasing retention time (RT) according to its
decreasing hydrophilicity (amino acids, organic acids, nucleosides,
phenolic acids, tannins, flavonoids and triterpenoids). A tendency
in the elution order of the metabolites based on their chemical
structural class was observed in agreement with literature
(Gómez-Romero et al., 2010; Iswaldi et al., 2013; Abu-Reidah
et al., 2013). Up to date, there is no study about the metabolomic
profile of Pouteria lucuma seeds using UHPLC-ESI-MS/MS. There-
fore, the compounds found in this extract will give value to this
agroindustrial waste as well as could lead to the formulation of a
supplement food.
3.2. Amino acids and derivatives
Eight free amino acids such as asparagine (m/z 131.04579; peak
1), pyroglutamic acid (m/z 128.03467; peak 7), glutamine (m/z
146.04524; peak 8), leucine (m/z 130.08659; peak 9), tyrosine
(m/z 180.06613; peak 10), isoleucine (m/z 130.08685; peak 11),
phenylalanine (m/z 164.07126; peak 16) and tryptophan (m/z
203.08229; peak 20) were tentative characterized from methanolic
extract of lucuma seed (Table 1). These compounds were detected
from RT 1.27 to 6.94 min (Fig. 1). Generally, these aminoacids lose
NH3 (17.0265 u), CO2 (43.9898 u) and H2O (18.0106 u) (Abu-
Reidah et al., 2013). At the peak 20, the indole group of tryptophan
(m/z 116.04983) was the most abundant fragment detected. Addi-
tionally, peak 5 was assigned to pyroglutamic acid glycoside (hex-
ose), which lost bound hexose (162.05376 u) and as a result,
pyroglutamic acid (m/z 128.03453) was detected.
322 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327

Table 1
Identification of secondary metabolites of methanolic extract from Pouteria lucuma seed by UHPLC-DAD-ESI-MS/MS.

Peak Tentative UV Elemental tR Theoretical Measured Accuracy MSn ions Class Reference
# identification max composition (min.) mass mass (dppm)
[M–H]
(m/z) (m/z)
1 Asn – C4H7O3N
2 1.27 131.04622 131.04579 3.28 113.03494 Amino acid Abu-Reidah et al. (2013)
2 Quinic acid – C7H11O

6 1.34 191.05611 191.05573 1.99 108.90097 Organic acid Spínola et al. (2014)
173.09271
3 Citric acid – C6H7O

7 1.53 191.01972 191.01942 1.57 111.0087 Organic acid Gómez-Romero, Segura-Carretero, &
Fernández-Gutiérrez (2010)
4 Sucrose – C12H21O

11 1.74 341.10894 341.10916 0.65 162.52010 Carbohidrate Gómez-Romero, Segura-Carretero, &
Fernández-Gutiérrez (2010)


5 Glp-hexose 245 C11H16O8N 1.78 290.08814 290.08829 0.52 128.03453 Amino acid Abu-Reidah et al. (2013)
200.05573
6 Isocitric acid – C6H7O

7 1.80 191.01973 191.01935 1.99 111.00790 Organic acid Gómez-Romero, Segura-Carretero, and
Fernández-Gutiérrez (2010)
7 Pyroglutamic 245 C5H6O3N 1.83 128.03532 128.03467 5.07 n.d. Amino acid Gómez-Romero, Segura-Carretero, and
acid Fernández-Gutiérrez (2010)
8 Glutamine 245 C5H8O4N
1.88 146.04588 146.04524 4.38 128.03453 Amino acid Gómez-Romero, Segura-Carretero, and
Fernández-Gutiérrez (2010) and Abu-
Reidah et al. (2013)
9 Leu/Ile – C6H12O2N
1.91 130.08735 130.08659 5.84 n.d. Amino acid Abu-Reidah et al. (2013)
10 Tyr 280 C9H10O3N
1.98 180.06662 180.06613 2.72 163.03958 Amino acid Abu-Reidah et al. (2013)
11 Leu/Ile 2 – C6H12O2N
2.05 130.08735 130.08685 3.84 n.d. Amino acid Abu-Reidah et al. (2013)
12 Succinic acid 245 C4H5O
4 2.16 117.01933 117.01862 6.06 n.d. Organic acid Abu-Reidah et al. (2013)
13 Methyl 255 C12H15O9N

2 2.50 331.07830 331.07843 0.39 111.00804 Nucleoside –
uridine-5- 127.00278
oxyacetate
14 Gallic acid 272 C7H5O
5 2.79 169.01425 169.01385 2.37 125.02361 Tannin Sandhu and Gu (2010)
15 Pyrogallol C6H5O3
3.27 125.02442 125.02377 5.20 n.d. Phenolic –
alcohol
16 Phe 260 C9H10O2N
3.34 164.07170 164.07126 2.68 147.04454 Amino acid Abu-Reidah et al. (2013)
17 Gallocatechin- 279 C21H23O

12 3.65 467.11950 467.11969 0.41 125.02372 Flavonoid –
hexose 1 137.02350 glycoside
167.03450
18 Pantothenic 235 C9H16O5N
4.49 218.10340 218.10316 1.10 146.00438 Vitamine Gómez-Romero, Segura-Carretero, and
acid Fernández-Gutiérrez (2010) and
Abu-Reidah et al. (2013)

Peak Tentative UV Elemental tR Theoretical Measured Accuracy MSn ions Class Reference
# identification max composition (min.) mass mass (dppm)
[M-H]
(m/z) (m/z)
19 Gallocatechin 1 283 C15H13O

7 5.67 305.06668 305.06683 0.49 125.02369 Flavonoid Sandhu and Gu (2010)
137.02368
219.06622
20 Trp 280 C11H11O2N
2 6.94 203.08260 203.08229 1.53 116.04983 Amino acid Abu-Reidah et al. (2013)
21 Gallocatechin- 279 C21H23O

12 8.31 467.11950 467.11981 0.66 125.02357 Flavonoid –
hexose 2 137.02409 glicosidade
167.03468
245.52000
22 Gallocatechin- 280 C21H23O

12 8.70 467.11950 467.11990 0.86 125.02363 Flavonoid –
hexose 3 137.02367 glicosidade
167.03435
23 Gallocatechin 2 280 C15H13O

7 9.23 305.06668 305.06680 0.39 125.02364 Flavonoid Sandhu and Gu (2010)
137.02368
219.06563
24 Protocatechuic 258– C7H5O

4 9.27 153.01933 153.01877 3.66 109.02861 Hydroxybenzoic Gómez-Romero, Segura-
acid or Gentisic 293 acid Carretero, and Fernández-
acid Gutiérrez (2010)
25 Catechin 279 C15H13O

6 9.58 289.07176 289.07184 0.28 109.02863 Flavonoid Gómez-Romero et al. (2011)
123.04440
125.02366
137.02371
151.03970
26 Dihydroxyphenyl 265 C8H7O

3 9,78 151,04006 151,03958 3.18 109,02870 Phenolic alcohol –
methyl ketone 137,02380 derivative
27 Vitexin 269– C21H19O

10 9.94 431.09837 431.09879 0.60 312.18161 Flavonoid Lin et al. (2015)
327 glycoside
28 Epicatechin 280 C15H13O

6 10.27 289.07176 289.07208 1.11 109.02865 Flavonoid Gómez-Romero et al. (2011)
123.04442
125.02362
137.02370
151.03946
29 Epigallocatechin- 280 C22H17O

11 10.37 457.07764 457.07809 0.99 125.02364 Flavonoid Spínola et al. (2014)
3-O-gallate 169.01370
305.06729
 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327 323

Table 1 (continued)

Peak Tentative UV Elemental tR Theoretical Measured Accuracy MSn ions Class Reference
# identification max composition (min.) mass mass (dppm)
[M-H]
(m/z) (m/z)
30 Methoxy 280 C12H17O

4 10.73 225.11323 225.11307 0.71 109.02869 Phenolic alcohol –
penthoxy 125.02364 derivative
catechol 139.03918
31 Hidroxy benzoic 265 C7H5O

3 11.05 137.02442 137.02383 4.31 n.d. Hydroxybenzoic Abu-Reidah et al. (2013)
acid 1 acid
32 Ethyl gallate 273 C9H9O

5 11.11 197.04555 197.04524 1.57 125.02356 Hydroxybenzoic –
derivative
33 Myricitrin 254– C21H19O

12 11.15 463.08820 463.08844 0.52 137.02364 Flavonoid Sandhu and Gu (2010)
354 151.03947 glycoside
271.02490
316.02094
34 Phloretin 265 C21H23O

10 11.61 435.12967 435.13007 0.92 119.04995 Flavonoid Ibdah and Gang (2014)
hexoside 123.04432 glycoside
35 Taxifolin I 290– C15H11O

7 11.71 303.05103 303.05130 0.89 125.02371 Flavonoid Gómez-Romero et al. (2011)
327
36 Methoxy 280 C7H7O

4 12.03 155.03498 155.03447 3.29 125.02390 Phenolic alcohol –
catechol derivative
37 Taxifolin II 290– C15H11O

7 12.30 303.05103 303.05127 0.79 125.02351 Flavonoid Gómez-Romero et al. (2011)
327
38 Myricetin 254– C15H9O

8 12.43 317.03029 317.03052 0.73 107.01299 Flavonoid Gómez-Romero, Segura-
355 137.02377 Carretero, and Fernández-
151.00305 Gutiérrez (2010)
39 Quercetin 254– C15H9O

7 12.54 301.03538 301.03555 0.57 121.02872 Flavonoid Gómez-Romero et al. (2011)
354 151.03957
40 Syringin 225– C17H23O

9 12.57 371.13476 371.13516 1.08 209.08185 Hidroxicinnamic –
265 acid derivative
41 Eriodictyol 245 C15H11O

6 12.63 287.05611 287.05637 0.91 125.02360 Flavonoid Gómez-Romero, Segura-
chalcone Carretero, and Fernández-
Gutiérrez (2010)
42 Hydroxybenzoic 255 C7H5O

3 13.05 137.02442 137.02377 4.74 n.d. Hydroxybenzoic Abu-Reidah et al. (2013)
acid 2
43 Eridictyol 279 C15H11O

6 14.05 287.05611 287.05618 0.24 107.01305 Flavonoid Gómez-Romero, Segura-
135.04427 Carretero, and Fernández-
151.00316 Gutiérrez (2010)
44 Apigenin 267– C15H9O

5 16.76 269.04555 269.04550 0.19 151.03949 Flavonoid Gómez-Romero et al. (2011)
335

Peak Tentative identification UV Elemental tR Theoretical Measured Accuracy MSn ions Class Reference
# max composition (min.) mass (m/z) mass (m/z) (dppm)
[M-H]

45 Naringenin 279 C15H11O

5 16.99 271.06120 271.06134 0.52 107.01289 Flavonoid Gómez-Romero
119.04941 et al. (2011)
151.00276
46 Unknown – C30H45O

7 17.42 517.31708 517.31714 0.12 – Taraxane –
triterpenoid
47 Unknown – C30H45O

6 18.35 501.32216 501.32236 0.40 – Taraxane –
triterpenoid
48 Unknown – C29H41O

6 19.02 485.29086 485.29108 0.45 – Taraxane –
triterpenoid
49 methoxyelemicin 257 C13H17O

4 19.43 237.11323 237.11319 0.17 – Phenolic –
derivative
50 Unknown – C16H32O3N
20.03 286.23877 286.23892 0.52 – – –
51 Tetrahydroxytricosanoic acid 245 C23H45O

6 20.31 417.32216 417.32248 0.77 – Tentative –
fatty acid
52 Trihydroxyheptadecatetraenoic 255 C17H25O

5 20.41 309.17075 309.17102 0.87 – Tentative –
acid fatty acid
53 dihydroxyheptadecatetraenoic 248 C17H25O

4 20.61 293.17583 293.17603 0.68 – Tentative –
acid fatty acid
54 Unknown 280 C26H31O

9 20.72 487.19736 487.19754 0.37 – Tentative –
steroid
55 Unknown – C26H31O

9 20.93 487.19736 487.19754 0.37 – Tentative –
steroid
56 dihydroxyoxoheptadecatrienoic 245 C17H25O

5 21.14 309.17075 309.17096 0.68 – Tentative –
acid fatty acid
57 Unknown 250 C30H55O14N

6 21.79 723.37817 723.37939 1.68 – – –
58 Hydroxyundecanoic acid 245 C11H21O

3 23.01 201.14962 201.14929 1.64 183.01161 Tentative –
fatty acid
59 Nonadecanedioic acid 245 C19H35O

4 23.41 327.25408 327.25433 0.76 283.26483 Tentative –
fatty acid
324 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327
Fig. 1. UHPLC total ion current chromatogram (TIC) of Lucuma seed methanolic extract.
3.3. Organic acids and derivatives
A total amount of five known organic acids such as quinic acid
(m/z 191.05611; peak 2), isomers citric (m/z 191.01942; peak 3)
and isocitric acid (m/z 191.01935; peak 6), succinic acid (m/z
117.01862; peak 12) and the amino derivative, pantothenic acid
or vitamin B5 (m/z 218.10316; peak 18) were tentatively identified
from hydrophilic extract of lucuma seed (Table 1), and those peaks
were detected from RT 1.34 to 4.49 (Fig. 1). Fragmentation pattern
of citric and isocitric acids were similar to those described by pre-
vious studies (Gómez-Romero et al., 2010; Iswaldi et al., 2013;
Abu-Reidah et al., 2013), generating neutral losses of CO2 from car-
boxylic group. This fragmentation pattern was also found for qui-
nic acid, where a fragment ion [M-H-CO2]
at m/z 173.09314 was
detected from this compound according to (Spínola et al., 2014).
Pantothenic acid produced an ion with m/z value of 146.00438,
which it assigned to the loss of CO2 and two CH2 (Abu-Reidah
et al., 2013).
3.4. Nucleosides
The nucleoside methyluridine-5-oxyacetic acid (peak 13, m/z
331.07843) was identified in the hydrophilic extract (Table 1).
Two daughter ions at m/z 111.00804 and m/z 127.00278 were
the most abundant. Both were generated by breakage of the N-
glycosidic bond and losing ribose (131.0428 u).
3.5. Phenolic acids and derivatives
Among the phenolic acids, two isomers of hydroxybenzoic acid
at m/z 137.0442 (peak 31) and m/z 137.02377 (peak 42) were ten-
tatively identified (Table 1). Although these isomers were not frag-
mented due to our MS/MS conditions, their presence in the
hydrophilic extract is confirmed by their UV data in the range
250–350 nm. Gentisic acid (m/z 153.01933; peak 24) is a dihydrox-
ybenzoic acid, whose fragmentation pattern is the loss of CO2 (m/z
109.02861) (Gómez-Romero et al., 2010). Both hydroxybenzoic
acid and dihydroxybenzoic acid have been shown to have signifi-
cant anti-inflammatory in vitro activity (Choudhary et al., 2009).
Ethyl gallate (m/z 197.04555; peak 32) was characterized based
on the major fragment ion at m/z 125.02356 (loss of C2H4 plus
CO2). It has been proposed that ethyl gallate could be associated
with chemopreventive role in carcinogenesis (Morais et al.,
2010). Peak 39, a glycosylated compound derived from hydrox-
ycinnamic acid, known as syringin (m/z 371.13516) was detected.
Its characterization was made according to the MS data and frag-
mentation pattern by the loss of a hexose (162 u). A previous study
has shown that syringin and its aglycone has anti-inflammatory
and antinociceptive properties (Choi et al., 2004).
3.6. Phenolic alcohol and derivatives
Three compounds (peaks 15, 26, 30, 36, and 49) were tenta-
tively identified among phenolic alcohols and its derivatives
(Table 1). Peak 15 with a [M-H]
ion at m/z 125.02442 was identi-
fied as pyrogallol. Peak 26 with a [M-H]
ion at m/z 151.03958 was
identified as dyhydroxyphenylmethylketone. Peak 36 assigned to
methoxy catechol (m/z 155.03447) was tentatively identified by
the loss of CH2O (30.0111 u). Peak 49 was identified as the natural
occurring phenolic methoxyelemicin. Finally, methoxy penthoxy
catechol (m/z 225.11308; peak 30) was characterized based on
their molecular formula and MS/MS spectra, because there is no
information in the literature. This metabolite produced three frag-
ment ions at m/z 139.03918 (loss of C5H10O), 109.02869 (loss of
CH2O) and 125.02364 (loss of CH2).
3.7. Hydrolysable tannins
Gallic acid (m/z 169.01425; peak 14) was the only hydrolysable
tannin present in hydrophilic extract. This compound lost CO2
(43.9898 u) which yielded the daughter ion at m/z 125.02361. Gal-
lic acid is one of the main constituents of Pouteria genus (Ma et al.,
2004) and possess anti-microbial, antioxidant and cytotoxic prop-
erties, even it could be relationated to chemopreventive role in car-
cinogenesis (Morais et al., 2010).
3.8. Flavonoids and its glycosides
Nineteen flavonoids were tentatively identified from methano-
lic extract (Table 1). The occurrence of phenylalanine in this extract
could explain the presence of flavonoids by shikimate pathway.
Furthermore, a previous study with three different species of
Pouteria fruits reported that compounds such as flavonoids are
commonly associated with this genus (Ma et al., 2004). The pres-
ence of flavonoids and its glycosides in this extract is valuable
due to their properties such as antioxidant, hepatoprotective,
antibacterial, anti-inflammatory and anticancer activities (Kumar
and Pandey, 2013). Our results suggest that P. lucuma seeds could
be a potential source for the isolation of these metabolites. Flavo-
noid aglycones and their glycosides from this extract eluted
between 3 and 17 min (Fig. 1).
Some 13 non-glycosylated flavonoids were tentatively identi-
fied. These belong to the class of flavan-3-ols: 2 isomers of gallo-
catechin at m/z 305.06683 (peak 19) and m/z 305.06680 (peak
23), catechin at m/z 289.07184 (peak 25), epicatechin at m/z
289.07208 (peak 28), and epigallocatechin-3-O-gallate at m/z
457.07809 (peak 29). Flavanonols: 2 isomers of taxifolin at m/z
303.05130 (peak 35) and m/z 303.05127 (peak 37). Flavonols: myr-
icetin at m/z 317.03052 (peak 38) and quercetin at m/z 301.03555
 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327
(peak 39). The eriodictyol chalcone at m/z 287.05637 (peak 41),
two flavanone: eriodictyol at m/z 287.05618 (peak 43) and narin-
genin at m/z 271.06134 (peak 45) and a flavone apigenin at m/z
269.04550 (peak 44). According to the MS data, the most abundant
fragment ion among flavan-3-ols was detected at m/z 125.0244
(C6H5O–3), which was generated by the cleavage in the ring C. Addi-
tionally, a daughter ion at m/z 137.0244, represented by [M-H-
C8H6O4]– to gallocatechin was detected. The same ion represented
by [M-H-C8H6O3]– to catechin and epicatechin was detected and
was agreed with the literature (Sandhu and Gu, 2010; Gómez-
Romero et al., 2011). In the case of epigallocatechin-3-O-gallate,
aside from C6H5O–3 (m/z 125.02364), two additional fragments
were observed at m/z 169.01370 and m/z 305.06729 belonging to
gallic acid and epigallocatechin respectively. Flavan-3-ols have
been found in green tea (Camelia sinensis) and their cutaneous pho-
toprotection from UV injury has been proposed, being
epigallocatechin-3-O-gallate (EGCG) the one that showed a poten-
tial activity, following epigallocatechin and epicatechin (Elmets
et al., 2001). Besides, EGCG from Camelia sinensis is the most bio-
logically active preventing the aging of the skin by the release of
free radicals (Inja Bogdan & Baumann, 2009). In similar way to
flavan-3-ols, peak 35 and peak 37, were tentatively identified
based on the fragment ion [M-H-C9H6O4]– at m/z 125.0236, which
was previously reported (Gómez-Romero et al., 2011). Taxifolin
has also been found in Pouteria campechiana as glycosilated forms
(Baky et al., 2016). Myrcetin (peak 38), quercetin (peak 39), eriod-
ictyol (peak 43), apigenin (peak 44), and naringenin (peak 45) were
characterized based on its [M-H]– and MS/MS fragments (Gómez-
Romero et al., 2011). Quercetin, myrcetin and its derivatives such
as myricitrin isolated from Pouteria campechiana leaves and seeds
showed antiulcerogenic and analgesic properties (Aly et al.,
2016). However, a previous work revealed that concentrations over
than 25 lM would act as a carcinogen (Nimse and Pal, 2015); even
certain doses of hydroethanolic extract of Pouteria torta (Sapota-
ceae), which contained derivatives of myrcetin, exhibited a muta-
genic activity (Costa et al., 2014). So, further studies about
methanolic extract of P. lucuma seeds are required to know the
concentration of these metabolites and, in this way, to support this
extract by its beneficial properties. Finally, eriodictyol chalcone
(peak 41) was recognized by the fragment ion at m/z 125.02360.
In the case of glycosylated flavonoids, six were found and identi-
fied according to the MS data and fragmentation patterns due to the
loss of the glycoside moiety. In this sense, 3 fragment ions such as
[M-H-C6H10O5-C9H8O4]– (m/z 125.0235), [M-H–C6H10O5–C8H8O4]–
(m/z 137.0240) and [M-H-C6H10O5-C7H6O3]– (m/z 167.0346) were
observed as the most abundant corresponding to 3 isomers of
gallocatechin-hexose (peaks 17, 21 and 22) at m/z 467.11950. Peak
27 was identified as the natural occurring glycoside vitexin. Myric-
itrin at m/z 463.08844 was tentatively identified by a fragment ion
at m/z 316.02094, which represents their non-glycosylated form
(myricetin). Besides, two fragment ions at m/z 137.02364 and m/z
151.03947 were detected confirming this peak 33. Finally, phloretin
hexose (peak 34), a glycosylated chalcone, was characterized
Table 2
Scavenging of the 1,1-diphenyl-2-picrylhydrazyl Radical (DPPH), radical, ABTS as Trolox equivalent antioxidant capacity, (ABTS), Ferric Reducing Antioxidant Power (FRAP), Total
phenolic content (TPC), Total flavonoid content (TFC), Superoxide anion scavenging Activity (SA) of P. lucuma (n = 5).
Sample DPPHa ABTSa
Lucuma seeds 58.14 ± 0.05 66.97 ± 0.00
Gallic acid 1.12 ± 0.01 8.73 ± 0.00
Quercetin 7.36 ± 0.01 15.32 ± 0.00
a
Antiradical DPPH and ABTS activities are expressed as IC50 in lg/mL.
b
Ferric reducing power expressed as lmol Trolox/g dry weight.
d
Total phenolic content (TPC) expressed as lmol GAE/g dry weight.
e
Total flavonoid content (TFC) expressed as lmol Q/g dry weight.
f
SA is expressed as percent inhibition at 100 lg/ml. Values in the same row marked with the same letter are not significantly different (at p < 0.05).
325
based on their principal fragment ions, according to the literature
(Ibdah and Gang, 2014). Phloretin has been reported in Rosaceae,
Ericaceae, and Symplocaceae as phloretin 20 -glucoside and its anti-
inflammatory activity has been proposed (Perez, 2001).
3.9. Lipidic compounds
Peak 51, 52 and peak 53 showed a [M-H]– at m/z 417.32248,
309.17102 and 293.17603, which were tentatively characterized
as tetrahydroxytricosanoic acid, trihydroxyheptadecatetraenoic
acid, and dihydroxyheptadecatetraenoic acid respectively. In the
same way were identified dihydroxyoxoheptadecatrienoic acid
(peaks 56), hydroxyundecanoic acid (peak 58), and nonadecane-
dioic acid (peak 59).
3.10. Unknown compounds
Seven compounds (peaks 46–48, 50, 54–55, and 57) were
detected and not identified (Table 1). Peaks 50 and 57 were not
identified and could be nitrogen-containing compounds. Peaks
46–48, 54 and 55 could be steroidal or triterpene skeletals based
on their molecular formulas. For example, peak 46 with a molecu-
lar ion [C30H45O7]– (m/z 517.31708), matches to fragment ion
[C28H45O3]– (m/z 429.55469), could probably be attributed to tar-
axe triterpenoid. In addition, triterpenoids such as ursolic acid,
betulinic acid, taraxerol, betulin, a–and b–amyrin among others
were isolated and identified from Pouteria genus (Montenegro
et al., 2006; Silva et al., 2009); thus, peaks 46–48, 54 and 55 could
be derivatives of these compounds. However, these metabolites
remain unidentified due to the huge amount of possible com-
pounds by isomerism. Further studies are required to isolate and
characterize the structure of these metabolites because of the
numerous biological properties assigned to compounds before
mentioned (Silva et al., 2009).
3.11. Antioxidant activity and phenolic and flavonoid content
The ferric reducing ability or antioxidant power (FRAP), the
trapping of DPPH and ABTS (TEAC) free radicals, and superoxide
anion trapping (SA) assays were used to determine the antioxidant
capacities of P. lucuma seeds for the first time (see Table 2). These
in vitro assays are simple and widely employed for the evaluation
of antioxidant power. Moreover, we determined the phenolic con-
tent in total (TPC) and total flavonoid in total (TFC) calculated in
dried weight of sample (see Table 2), and compared to other fruits
previously analyzed by us and other South American fruits
(Girones-Vilaplana et al., 2014; Ramirez et al., 2013; Simirgiotis
et al., 2016). Phenolic compounds are structurally diverse and com-
prise phenolic acids, flavonoids, tannins, coumarins, etc, most of
them bearing phenolic moieties that give them the capacity to
bleach harmful radicals. Three main classes of compounds are dis-
tinguished in this work, organic acids, phenolic acid and flavo-
noids. The phenolics present in P. Lúcuma are shown in Table 1,
FRAPb TPCd TFCe SAf
272.50 ± 0.00 52.82 ± 0.09 5.99 ± 0.01 67.02 ± 2.23
885.70 ± 0.03 – – 85.15 ± 2.84a
564.20 ± 0.07 – – 78.12 ± 3.45a
326 P. Guerrero-Castillo et al. / Waste Management 88 (2019) 319–327
Table 3
Gastroprotective effect of lucuma seed extracts on HCl/EtOH-induced gastric lesions in mice.
Compound n Lesion index (mm)
LM-1 7 47.3 ± 1.1
LM-2 7 39.4 ± 1.2
LM-3 7 25.4 ± 1.4
LM-4 7 12.4 ± 1.0
Lansoprazole 7 17.6 ± 1.1
Control 7 56.3 ± 1.6
The results are expressed as mean ± sem *P < 0.01; significantly different compared with the control (ANOVA followed by Dunnett’s test). n = number of mice.
and are together with other phenolics such as the flavonols, citric
acid, etc, responsible for the antioxidant power and phenolic con-
tent. Thus, the TPC values of seeds of these fruits, 52.82 ± 0.09 mg
GAE/g dry weight, (Table 2) were closer to those reported for the
Chilean species Ugni molinae, (50 mg GAE/g dry weight) which is
located in the average of antioxidant edible fruits (Balasundram
et al., 2006; Rodriguez-Roque et al., 2014) and Pouteria ovobata
flour (Dini, 2011), besides was higher than peaches, apples and
blueberries (Pinto et al., 2009). Also, the TPC were close to those
measured for Chilean blueberries (45.86 ± 3.46 mg GAE/g)
(Ramirez et al., 2013) and almost twice than the berries of Luma
apiculata (29 mg Q/g) (Simirgiotis et al., 2016), Indeed, the seeds
of lucuma exceed the reported values for food flavonoids (Fink
et al., 2007) and phenolics content as reported for P. ovovata
(Dini, 2011). Regarding flavonoids, several flavones and flavanones
and flavanes were also detected in P. Lúcuma (Table 1), and the
total flavonoid content (5.99 ± 0.01 mg GAE/g), measured spectro-
scopically was lower than the aerial parts of Ugni molinae (28 mg
Q/g) (Rodriguez-Roque et al., 2014) and was lower than the high
antioxidant berries Berberis microphilla (45.72 ± 2.68 mg Q/g). In
the DPPH assay, P. Lúcuma (58.14 ± 0.05 mg/ml) was closer to that
reported for lemon fruits (Brito et al., 2014). The ABTS values
(66.97 lg/ml) were closer to that of various super-antioxidant
fruits, such as maqui, Aristotelia chilensis (254.8 lM TE/g dry
weight: 63. 79 lg/mL) and Acai, Euterpe oleraceae (208.7 lM TE/g
dry weight: 52.23 lg/ml) (Girones-Vilaplana et al., 2014), while
the FRAP values (272 ± 0 lM TE/g dry weight) where higher than
those of Acai (157.9 lM TE/g dry weight) and maqui fruits
(254.2 lM TE/g dry weight). These values can classify these P.
Lúcuma seed extracts as moderate to high antioxidant fruits
derived products, such as plum, cherries, strawberries etc
(Girones-Vilaplana et al., 2014; Ramirez et al., 2013; Rodriguez-
Roque et al., 2014).
3.12. Gastroprotective activity
The results of the gastroprotective activity performed with
Lucuma seed extracts are presented in Table 3. All methanolic
extracts at doses (p.o.) of 10 mg/Kg (LM-1), 25 mg/Kg (LM-2),
50 mg/Kg (LM-3), and 100 (LM-4) mg/Kg showed gastroprotective
effects in the HCl/EtOH-induced gastric lesions model in mice. The
effect showed by LM-4 (78%) was superior to that observed with
Lansoprazole (68%), while LM-1 (16%), LM-2 (30%), and LM-3
(55%) were less active in comparison. In previous study, Aly et al.
(2016) reported that the seed ethanolic extracts of Pouteria cam-
pechiana at 100 mg/Kg (80%) and 200 mg/Kg (90%) showed gastro-
protective activity in the ethanol-induced ulcer model in rats. In
the same study the authors reported the isolation of protocate-
chuic acid, gallic acid, quercetin, myricetin, myricetin-3-O-L-
rhamnoside and myricetin-3-O-D-galactoside, which were linked
to their antiulcerogenic effects. Our result was similar to that
reported with Pouteria campechiana although the ulcerogenic mod-
els were differents (Aly et al., 2016).
% Lesion reduction Dose (mg/Kg)
*
16 10
30* 25
55* 50
78* 100
68* 30
– –
4. Conclusions
In the present study, a total of 59 compounds were detected for
first time using UHPLC-ESI-MS/MS from hydrophilic methanol
extract of the Pouteria lucuma seeds from Perú, which showed high
antioxidant power and gastroprotective compared to other fruit
products. Among the compounds several amino acids, organic
acids, phenolic compounds and flavonoids were identified. This
work demostrated that lucuma seeds, which are commonly elimi-
nated as an agricultural industry waste from lucuma pulp, could be
useful for the preparation of nutritional supplements. However,
more research is necessary to support the seed as a source of
nutraceutical or healthy flavonoids.
Acknowledgements
The authors thank CONCYTEC (Convention 231-2015 FONDE-
CYT) and FONDECYT REGULAR (Grant 1170871 and 1150745) for
financial support.
Conflicts of interest
The authors declare no conflict of interest.
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