Antioxidant Capacity In Vitro and In Vivo

of Various Ecotypes of Mexican Plum (Spondias

purpurea L.)
Juan Manuel Villa-Hernández, Gabriela Mendoza-Cardoso, José Alberto Mendoza-Espinoza, Cristián Vela-Hinojosa ,
Fernando Dı́az de León-Sánchez, Fernando Rivera-Cabrera, Irán Alia-Tejacal, and Laura J. Pérez-Flores

Abstract: Spondias purpurea L. is a fruit native to Mexico, however, it is found as far away as Brazil. It possesses a high
commercial potential owing to its sensorial and nutritional qualities and its low cost of production. There exists a variety
of ecotypes that have not been characterized and their adequate selection process, according to their strongest functional
characteristics, will allow the establishment of improvement programs for this genetic resource. The object of this study was
the chemical characterization and the determination of the in vitro and in vivo antioxidant capacity of 7 Spondias purpurea
L. ecotypes. Differences were observed in the antioxidant capacity and the content of functional compounds among all

Food Chemistry
the ecotypes analyzed. A high total phenolic content and a low flavonoid and carotenoid content were found, both in
the epicarp and in the pulp. In each ecotype, the hydrophilic phase presented up to 40 times greater antioxidant capacity
compared to the lipophilic phase. The hydrophilic phase of the epicarp of “Costeña Tierra Colorada” had the greatest
antioxidant capacity and highest total phenolic content, whereas “Jocote” presented the lowest antioxidant capacity and
total phenolic content. A positive correlation was observed between phenol levels and the antioxidant capacity in the
epicarp. Regarding antioxidant activity in vivo, it was observed that in all analyzed concentrations of hydrophilic extracts of
the epicarp of “Costeña Tierra Colorada” and in the highest “Jocote” concentrations, they provided thermo-protection
against heat stress as well as a general well-being to the worm as evidenced by their high mobility.

Keywords: antioxidant capacity, Caenorhabditis elegans, functional compounds, Spondias purpurea L

Introduction Despite the importance of this genetic resource, research in

Mexican plum (Spondias purpurea L.) is a tropical species of the
Anacardiaceae family, and it is distributed along the west coast
and the southeast part of the country. It is considered native to
Mexico and forms part of the low deciduous and semi-deciduous
forests. Mexican plum fruits possess a high commercial potential
thanks to its sensorial and nutritional qualities and low cost of
production because it grows spontaneously and adapts to poor,
thin soils in which other plants do not grow, and also because it
possesses resistance to drought by defoliation (Avitia and others
2000). Mexican plum fruit is an oblong drupe, round or ovoid,
with a size ranging between 2 and 5 cm and a mass of between
4 and 33 g. They exhibit a thin, smooth to semi-smooth epicarp,
that may turn can be reddish, yellow, red-brown, orange, or purple
when ripe; a rough, fibrous endocarp and a tasty and aromatic
mesocarp. Fruits are consumed ripe and/or in an unripe stage.
Quality and functional compound tests have shown they contain
vitamin C, minerals and antioxidant compounds such as phenolic
compounds and carotenoids (Avitia and others 2000; Ramı́rez and
others 2008; Almeida and others 2011).
JFDS-2017-0691 Submitted 4/27/2017, Accepted 8/2/2017. Authors Villa-
Hernández and Alia-Tejacal are with Facult. de Ciencias Agropecuarias, Univ.
Autónoma del Estado de Morelos. Av. Univ. 1001, Chamilpa 62209, Cuernavaca,
Morelos, México. Authors Villa-Hernández, Mendoza-Cardoso, Mendoza-Espinoza,
Vela-Hinojosa, Dı́az de León-Sánchez, Rivera-Cabrera, Pérez-Flores are with Dept.
de Ciencias de la Salud, Univ. Autónoma Metropolitana, Av. San Rafael Atlixco
186, Col. Vicentina, CP 09340, D. F. México, Mexico. Author Mendoza-
Espinoza is also with Colegio de Ciencias y Humanidades, Univ. Autónoma de
la Ciudad de México, México, Mexico. Direct inquiries to author, Alia-Tejacal,
(E-mail: iran.alia@uaem.mx).
Mexico around its genetic diversity and agronomic use is scarce.
In this regard, studies into its morphological, physical and chemical
characteristics have been developed in fruits collected in the states
of Guerrero, Morelos, and Chiapas. In these studies, the fruit
weight fluctuated between 4.0 and 43.2 g. In relation to color,
ripe fruits can be red, orange, yellow or green. Values of total
soluble solids (°Brix) were from 9.7 to 15.0, while titratable acidity
values varied from 0.4 to 1.0. Altogether, these results are evidence
of a high variability among the studied ecotypes. On the other
hand, studies carried out on 11 ecotypes of Mexican plum from
the states of Morelos, Guerrero, and Chiapas, harvested at ripe
stage, showed that total sugar, phenolic compounds, carotenoids,
and vitamin C contents vary between them. With respect to the
antioxidant capacity of the fruit, it was observed that it was always
higher in the epicarp than in the pulp and the ecotype that had
the highest antioxidant capacity originated in the state of Guerrero
(Alia-Tejacal and others 2012; Solorzano-Morán and others 2015).
Functional and nutraceutical foods are one of the fastest growing
sectors in the food industry. This is due to an ageing population,
the increased cost of healthcare and people understanding more
and more the link between diet and health. With the ageing pop-
ulation comes an increase in the prevalence of diseases associated
with ageing, such as diabetes and neurodegenerative diseases. It is
well documented that free radicals play a crucial role in diseases
associated with ageing. In this context, studies on the effects of
food ingredients with antioxidant properties are of great inter-
est owing to their redox properties. There are different in vitro
methods to determine the antioxidant capacity of those products
(Apak and others 2016). However, these methods do not reflect

C 2017 Institute of Food Technologists

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doi: 10.1111/1750-3841.13862 Vol. 00, Nr. 00, 2017 r Journal of Food Science 1
Further reproduction without permission is prohibited
 Antioxidant capacity of S. purpurea L . . .
Table 1–Ecotypes and location of fruit collection.
Ecotype Sampling location in México
Atzompa Oaxaca
Conserva de Iguala Tepecoacuilco, Guerrero
Costeña Tierra Colorada Acapulco, Guerrero
Jocote Chiapa de Corzo, Chiapas
Morada de Cocula Cocula, Guerrero
Conservera de Tlaxmalac Huitzuco, Guerrero
Amarilla Huitzuco, Guerrero
physiological cellular conditions since absorption, bioavailability
and metabolism of antioxidant compounds are not considered.
The necessity to demonstrate the effects of bioactive compounds
requires the availability of fast, reliable, and inexpensive animal
models as well as an integrated strategy of multiple disciplines to
Food Chemistry
elucidate the health-promoting properties of foods (Martorell and
others 2011; Granato and others 2017). The nematode Caenorhab-
ditis elegans is a small popular model, easy to manipulate in the
laboratory, with a simple anatomy, a well-described embryology,
and a short lifespan (21 d) and it can be frozen and preserved
for several years. Its genome is small and completely sequenced.
It presents a high similarity percentage to the mammal genome
and various mutants are available for study. Due to these char-
acteristics, nematode C. elegans seems a very interesting model
for analyzing in vivo antioxidant capacity of bioactive compounds
from fruits such as plum in a fast and low-cost way (Navarro-
González 2003; Martorell and others 2011; Di Paola Naranjo and
others 2016).
Since Mexican plum fruit is a very abundant natural resource
available in various states of the country, its proper exploitation
and marketing will permit the generation of greater profits for
producers and will provide the consumer with a product of higher
quality, abundant in functional antioxidant compounds. Therefore,
the right selection of ecotypes with better functional character-
istics will allow the establishment of improvement programs for
this resource. Based on the above, the aim of this study was to
characterize genotypes of Mexican plum originating in the states
of Chiapas, Guerrero and Oaxaca to select those that have greater
in vitro antioxidant capacity and functional compounds, as well as
to evaluate their antioxidant capacities in vivo, for improvement
and conservation programs.
Materials and Methods
Biological material
Between 50 and 60 ripe fruits of 7 ecotypes of Mexican plum
(Spondias purpurea L.) free of parasites or physical or mechanical
damage were collected. They were stored in previously tagged
plastic “zipper” bags and transported to the laboratory in sealed
containers at a temperature of 4 to 6 °C until use. Subsequently,
they were disinfected with a 1% (v/v) sodium hypochlorite solu-
tion and rinsed 3 times with distilled water. The pulp and epicarp
of each ecotype were separated, frozen in liquid nitrogen and the
samples were stored at –70 °C until use. Table 1 shows the an-
alyzed ecotypes and location of the origin site. In the pulp of
the studied ecotypes values of total soluble solids (°Brix) varied
from 7.17 to 18.20, those of titratable acidity varied from 0.26
to 0.59, while pH varied from 3.12 to 3.98. These values are
in the range of previous reports for S. purpurea and S. mombin
(Maldonado-Astudillo and others 2014; Solórzano-Morán and
others 2015).
2 Journal of Food Science r Vol. 00, Nr. 00, 2017
Determination of the antioxidant capacity in vitro
The hydrophilic and lipophilic phases were obtained from 1 g
of tissue (pulp or epicarp) from each ecotype studied. Pulp and
epicarp tissues were frozen and pulverized with liquid nitrogen,
4 mL of methanol were added and the mixture was homogenized.
Subsequently, 3.3 mL of dichloromethane were added and the
mixture was homogenized for 3 min in vortex. 4 mL of Tris-HCI
(50 mM pH 7.5) buffer were added, the mixture was stirred for
1 min in vortex and centrifuged at 1050 x g for 5 min at 25 °C.
The upper methanolic phase (hydrophilic phase) was recovered,
measured and stored at –70 °C. 3.3 mL of dichloromethane were
added to the lower phase and it was homogenized for 3 min, and
4 mL of Tris-HCI (50 mM pH 7.5) buffer were added and it was
stirred for 1 min. The mixture was centrifuged at 1050 x g for 5 min
at 25 °C and the lower phase was recovered. The extraction process
of the lipophilic phase was repeated twice. The recovered volume
of the 3 extractions was measured. In vitro antioxidant capacity was
determined in hydrophilic and lipophilic phases, using the ABTS
technique (Re and others 1999). The ABTS radical cation (2,2’-
azinobis 3-ethylbenzothiazoline-6-sulphonic acid) was prepared at
a 7 mM concentration and was mixed with 2.45 mM potassium
persulfate (K2 S2 O8 ) at a ratio of 1:1 (v/v) (stock solution). This
solution was stored for 16 h at room temperature. Subsequently, a
dilution was made mixing 700 μL of stock solution with 15 mL
of PBS 1X pH 7.4 buffer for the hydrophilic phase or 15 mL of
methanol for the lipophilic phase and the absorption was adjusted
to 0.7 ± 0.02 at 734 nm (daily solution). For the determination
of antioxidant capacity at the hydrophilic and lipophilic phases,
100 μL of the sample (adjusted dilutions) was mixed with 1 mL of
the daily solution. The reaction was incubated for 15 min and the
absorption was read at 734 nm. The standard Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid) curve was linear
between 0 and 20.45 μM. The results were expressed as mM
equivalents of Trolox/g of fresh weight (gfw).
Total phenolic and total flavonoid content
The total phenolic and total flavonoid content were determined
in the hydrophilic phase of the pulp and epicarp. Total phenolic
content was determined using the Folin-Ciocalteau method (Mar-
graf and others 2015). 1 mL of Folin-Ciocalteau reagent (1:10 v/v,
diluted with distilled water) was mixed with 100 μL of each of the
samples (adjusted dilutions) and 0.8 mL of sodium carbonate 7.5%
(w/v). The mixture was stirred and incubated for 1 h at room
temperature. Absorbance was read at 765 nm. A standard curve
was prepared using gallic acid and the results were expressed as μg
gallic acid equivalents/gfw.
Flavonoids were determined according to Whisky and Salatino
method (1998). 0.5 mL of each sample was mixed with 1.5 mL
of 95% (v/v) ethanol, 0.1 mL of 10% (w/v) aluminum chloride
and 0.1 mL of 1 mol/L potassium acetate in 5 mL final volume.
The reaction was incubated for 30 min at room temperature and
absorbance was read at 415 nm. A standard curve was prepared
using quercetin and the results were expressed as μg equivalents
of quercetin/gfw.
Carotenoid content
Carotenoid content was determined in the lipophilic phase.
20 μL of each sample previously filtered (0.45 μm nylon filter)
were separated by HPLC into an Agilent Technology instrument,
1200 Series, equipped with a multiple wavelength detector
(MWL). The separation was carried out on a C18 (250 ×
4.6 mm) column, with 5 μm particle size, with acetonitrile
Antioxidant capacity of S. purpurea L . . .

/methanol/dichloromethane (43:43:14) (v/v) as mobile phase, at Table 2–Antioxidant capacity in vitro.

a flow rate of 1 mL/min with a 20 min run time. Absorbance was Hydrophilic phase Lipophilic phase
measured at 485 nm. Lutein, β-carotene and lycopene were used
as standards (Sigma Aldrich). Ecotype Epicarp Pulp Epicarp Pulp
Atzompa 90 ± 12c 6.5e 1.6b 0.1d
Antioxidant capacity in vivo Conserva de Iguala 163 ± 38b 9 ± 1c 2 ± 1b 0.2c
The in vivo antioxidant capacity of the Mexican plum extracts Costeña Tierra Colorada 269 ± 18ª 15b 2b 0.8a
was evaluated by means of the survival (%) in the nematode C. Jocote 43 ± 6d 4.9 ± 1f 1.1b 0.1d
Morada de Cocula 81 ± 31c 9d 4 ± 1a 0.3b
elegans (wild-type strain Bristol N2, kindly provided by Rosa Estela Tlaxmalac 116 ± 12c 28 ± 2a 0.2c ND
Navarro and Laura Silvia Salinas, obtained from CGC) at growth ∗ Amarilla 35 ± 11 1.1
stage L4 subjected to heat shock at a temperature of 35 °C.
Antioxidant capacity of hydrophilic and lipophilic phases [mM equivalents of
Synchronization of the nematode growth stage. Animals Trolox/gfw] of the epicarp and the pulp of the 7 ecotypes analyzed. Mean values
were maintained at 20 °C on petri dishes containing Nematode followed by the same letter in each column are not significantly different between the
ecotypes analyzed according to the Tukey’s multiple comparison test. Values are the
Growth Medium (NGM) seeded with a dead E.coli strain OP50 average of 3 independent experiments ± standard deviation. ND = Not detected.
(kindly provided by Rosa Estela Navarro and Laura Silvia Salinas,
obtained from CGC) as the food source according to the method
Statistical analysis

outlined by Stiernagle (2006), for approximately 7 d. Nematodes
of all stages were collected (eggs, larva, L1, L2, L3, L4, and adult)
with 1 mL of M9 medium and placed in a sterile 2 mL Eppen-
dorf tube (approximately 800 μL of the medium was recovered).
400 μL of sodium hypochlorite and 200 μL of 5 N NaOH, were
added, the mixture was stirred in vortex for 5 min. Subsequently,
it was centrifuged for 1 min at 3765 x g at ambient temperature.
The supernatant was discarded and 1 mL of M9 medium was
added, it was stirred in vortex for 30 s and centrifuged for 1 min at
3765 x g (this step was repeated twice at room temperature). The
supernatant was discarded and the eggs were resuspended in 1 mL
of M9 medium, and they were placed in a 10 mL beaker, 3 mL
of M9 medium were added (final volume of 4 mL), the container
was sealed with parafilm and incubated overnight at 20 °C. Sub-
sequently, 1 mL of the medium with L1 larvae was centrifuged
for 1 min at 3765 x g at room temperature. The supernatant was
discarded and the obtained larvae were quantified. 300 larvae at
the L1 stage were spread on a petri dish with NGM medium.
Obtention of S. purpurea L. hydrophilic extracts from
epicarp. The extracts with higher and lower antioxidant capac-
ity in vitro, were selected. 4 mL of hydrophilic extract from the
epicarp of the “Costeña Tierra Colorada” ecotype and 5 mL of
hydrophilic extract of the epicarp of “Jocote” were lyophilized.
78.2 mg of “Costeña Tierra Colorada” extract and 68.7 mg of
“Jocote” extract were obtained. The residues were re-suspended
in 10% (v/v) ethanol at a concentration of 10 mg/mL. From this
extracts, dilutions were made in serial form to obtain a final con-
centration of 0.05, 0.12, 0.25, 0.3, 0.37, 0.5, 0.75, 2.5, 5, 7.5,
and 10 μg/mL. Previous reports demonstrated that quercetin has
a protective effect in nematodes against heat shock and oxidative
stress (Pietsch and others 2009; Surco-Laos and others 2011). In
this work 10 μg/mL of quercetin was applied to compare with
the potential protective effects of fruit extract.
Stress resistance assays. The toxicity of the extracts, as well as
the necessary concentrations for which the survival of the C. elegans
nematodes exposed to the extracts were similar to those obtained
with control without heat shock treatment were determined.
The nematodes were placed with the plum extracts at concen-
trations of 0.3, 2.5, 5, 7.5, and 10 μg/mL and were subjected
to heat shock (35 °C). Subsequently, the total number of worms
present on the plate was counted and also the number of live
worms. The survival (%) was obtained using the following equa-
tion:
Survival (%) = (Number of live worms) (100)
/Total number of worms on the plate
Food Chemistry
Determinations of antioxidant capacity, total phenolic and total
flavonoid content of each of the ecotypes studied were carried
out in triplicate. In each case, a one-way variance analysis and
mean comparison were carried out using Tukey–Krammer (P ࣘ
0.05 using the statistical package NCSS v.1.0.). A Pearson correla-
tion analysis was applied for epicarp antioxidant capacity and total
phenolic content (P ࣘ 0.05).
Results and Discussion
In general, it was observed that in every ecotype studied the
antioxidant capacity was greater in the epicarp compared with the
pulp, in the hydrophilic phase as much as in the lipophilic phase.
Likewise, the antioxidant capacity in the hydrophilic phase was
superior to that obtained in the lipophilic phase (Table 2). These
results are similar to those previously reported by Usenik and
others (2013), Ahmed and others (2014), and Solorzano-Morán
and others (2015) where they demonstrated that the total antiox-
idant capacity of the epicarp was greater in comparison with the
pulp for P. domestica L., L. siceraria, and S. purpurea fruits, respec-
tively. In these studies, the antioxidant capacity of the hydrophilic
and lipophilic phases was not determined separately.
The result of the hydrophilic phase of the epicarp showed that
“Costeña Tierra Colorada” and “Jocote” presented the highest
and lowest antioxidant capacities (269 and 43 mM equivalents
of Trolox/gfw, respectively), while in the hydrophilic phase of
the pulp of the ecotypes “Tlaxmalac” and “Jocote” presented
the highest and lowest antioxidant capacities (28 and 4.9 mM
equivalents of Trolox/gfw, respectively). It is worth mentioning
that “Jocote” was the ecotype that presented the lowest antioxi-
dant capacity both in the epicarp and the pulp. In the case of the
“Amarilla” ecotype, in which separation of epicarp and pulp was
not achieved, the antioxidant capacity of the whole fruit showed
values inferior to those obtained from the other ecotypes (Table 2).
Solorzano-Morán and others (2015) determined the antioxidant
capacity by DPPH method in 11 ecotypes of Mexican plum
and demonstrated that “Conservera de Tlaxmalac” and “Costeña
Tierra Colorada” were among the ecotypes that presented the
highest antioxidant capacity both in the epicarp and the pulp,
while “Jocote” and “Amarilla” were among those with the lowest
antioxidant capacity, which coincides with the trend found in
the current study. Highly variable range values for antioxidant
capacity in pulp or whole fruit have been reported for plums
grown in the same regions, but at different harvest times. Almeida
and others (2011) reported a difference in antioxidant capacity
for whole plums up to 6 times greater using the ABTS method

Vol. 00, Nr. 00, 2017 r Journal of Food Science 3

 Antioxidant capacity of S. purpurea L . . .
Table 3–Totals phenolic and flavonoid content.
Total phenolic content Flavonoids
Ecotype Epicarp Pulp Epicarp Pulp
Atzompa 0.9 ± 0.4c 0.1d 1c ND
Conservera de 53.2 ± 5.6b 5.4 ± 0.4b 2.4 ± 0.5b ND
Iguala
Costeña Tierra 276.5 ± 38.8ª 40.3 ± 3.3ª 4.5ª 0.4 ± 0.1a
Colorada
Jocote 0.33d 0.2d ND ND
Morada de Cocula 45 ± 2.5b 4.5 ± 0.5b 4.5 ± 0.3ª ND
Tlaxmalac 17.9 ± 1.3c 3.8c 4.7a ND
∗ Amarilla 0.20 ± 0.20 ND
Total phenolic [μg equivalents of gallic acid/gfw] and total flavonoid [μg equivalents of
/gfw] content in the epicarp and the pulp of the 7 ecotypes analyzed. Mean values
followed by the same letter in each column are not significantly different between the
ecotypes analyzed according to the Tukey’s multiple comparison test. Values are the
average of 3 independent experiments ± standard deviation. ND = Not detected.
Food Chemistry
compared to DPPH. This could support the idea that factors
like time of harvest and environmental conditions influence the
antioxidant capacity of these fruits.
In terms of the antioxidant capacity of the lipophilic phase, the
results were lower in comparison with the antioxidant capacity of
the hydrophilic phase (40 times less for the epicarp and 35 times
less for the pulp). This could be due to a low content of lipophilic
antioxidants such as carotenoids in these tissues. The ecotypes
“Morada de Cocula,” Costeña Tierra Colorada,” and “Conserva
de Iguala” were those that presented the highest antioxidant ca-
pacity in the epicarp, while in the pulp “Costeña Tierra Colorada”
and “Morada de Cocula” had the highest values (Table 2).
With respect to total phenolic content, it was observed that
in every ecotype analyzed the epicarp presented a range from
between 4 and 10 times higher in comparison with the pulp.
“Costeña Tierra Colorada,” “Conserva de Iguala,” and “Morada
de Cocula” were the ecotypes that presented the highest con-
tent of total phenolic content, both in the epicarp and in the
pulp, while “Jocote” was the ecoptype that presented the low-
est content in both tissues (Table 3). These results coincide with
those reported by Solorzano-Morán and others (2015), who found
higher phenolic content in the epicarp of the 11 ecotypes analyzed
in comparison with the pulp. However, the values obtained from
the ecotypes analyzed in this study were lower than those reported
by Solorzano-Morán and others (2015). This could indicate that
even though the fruit was harvested at ripeness, there may be other
factors that influence the content of total phenolic content, such
as environmental conditions during cultivation, harvest season,
cultural practices, transportation, and storage conditions. In this
context, Moo-Huchin and others (2014) reported high phenolic
content and low content of flavonoids, carotenoids and vitamin
C in whole Mexican plums cultivated in Yucatán México, which
could indicate that the location and growing conditions influ-
ence the content of functional compounds of these fruits. Variable
ranges in both the antioxidant capacity and the content of total
phenolic content were reported in hydrophilic extracts of pulp and
whole fruits of plums grown in Brazil (Rufino and others 2010;
Contreras-Calderón and others 2011) and in pulp of plums grown
in Colombia (Ferreira-Zielinski and others 2014). The above re-
sults could explain the broad range of values obtained in this study.
The total phenolic content in the epicarp is high and correlates
with the higher antioxidant capacity observed in the hydrophilic
phase of this tissue in the “Costeña Tierra Colorada” ecotype. The
correlation coefficient analysis reveals that a positive significant
correlation exists (r = 0.99) between the antioxidant capacity of
4 Journal of Food Science r Vol. 00, Nr. 00, 2017
the hydrophilic phase and total phenolic content in the epicarp
of this ecotype. This supports the idea that the higher antioxidant
capacity of the hydrophilic phase is owing to these functional
compounds in this tissue in comparison with that of the pulp.
In this sense, Rufino and others (2010) reported in tropical
fruits from Brazil, including yellow plums a positive correlation
between polyphenol content and antioxidant capacity determined
by ABTS method (r = 0.92). Engels and others (2012) also men-
tioned that in pulp, polyphenol amounts are low in comparison
with the peel of S. purpurea fruit cultivated in Costa Rica. They
identified 21 phenol and flavonoid compounds present in the
epicarp of fruits. Among the major compounds present they
found quercetin, kaempferol, gallic acid, 3-caffeoylquinic acid,
3-hydroxibenzoic acid, isorhamnetin, and rhamnetin.
As already mentioned, the Mexican plum fruit varies in color
depending on the ecotype. Solorzano-Morán and others (2015)
reported a high content of total phenolic compounds and vitamin
C in fruits with an epicarp purple or red in color that may be
associated with the increased antioxidant activity of this tissue. In
this regard, the ‘Costeña Tierra Colorada,” “Conserva de Iguala,”
and “Morada de Cocula” ecotypes that displayed a red or red-
purple epicarp respectively, were those that presented the highest
antioxidant activity and the highest total phenolic content in the
hydrophilic phase.
In general, the content of total flavonoids was low in all the
ecotypes studied and it could only be determined in the epicarp
(Table 3). “Conservera de Tlaxmalac,” “Morada de Cocula,” and
“Costeña Tierra Colorada” were the ecotypes that presented the
highest flavonoid content in the epicarp (4.7, 4.5, and 4.5 μg
equivalents of quercetin/gfw, respectively). These results suggest
that flavonoids do not contribute strongly to antioxidant capacity.
With respect to the carotenoid content, the results showed that
the epicarp was the tissue that presented higher concentrations of
these antioxidant compounds in all ecotypes analyzed. Figure 1
shows a representative chromatogram of the epicarp (1A) and of
the pulp (1B) of the ecotype “Costeña Tierra Colorada.” Four
defined peaks were detected in the epicarp, while 5 were de-
tected in the pulp. In general, we see that the epicarp contains a
higher carotenoid content in comparison with the pulp. Among
the most abundant carotenoids in all ecotypes was lutein, followed
by β-carotene, both in the epicarp and the pulp. Lycopene was
not detected. The epicarp of “Conserva de Iguala,” Morada de
Cocula,” and “Costeña Tierra Colorada” were those that pre-
sented higher lutein (6.4, 3.3, and 2.8 ppm) and β-carotene con-
tent (1.4, 2, and 1 ppm, respectively) (Table 4). In this regard, the
results of this work concur with the results of recent studies that
show that the highest concentrations of carotenoids are found in
the epicarp compared with the pulp in S. purpurea fruits (Ferreira-
Zielinski and others 2014; Maldonado-Astudillo and others 2014;
Solorzano-Morán and others 2015). It is important to identify the
compound detected at 2.8 min retention time since it is the most
prominent peak in both tissues (this unidentified peak, as well
as other identified peaks are marked in Figure 1). Tiburski and
others (2011) identified the presence of 5 carotenoids in the pulp
of the S. mombin L. fruit (β-cryptoxanthin, lutein, zeinoxanthin,
α and β-carotene), with β-cryptoxanthin being the carotenoid
found in highest proportion. In addition, Maldonado-Astudillo
and others (2014) mention that aside from phenolic compounds,
S. mombin and S. purpurea fruit contain β-cryptoxanthin as its main
carotenoid, followed by lutein and zeinoxanthin, so one might
think that the 2.8 min peak yet unidentified could correspond to
the β-cryptoxanthin, reported by these authors.
Antioxidant capacity of S. purpurea L . . .

Figure 1–Chromatogram of a sample of epicarp

(A) and pulp (B) of the fruit of “Costeña Tierra
Colorada” ecotype.Identification of lutein,
lycopene and β-carotene based on retention
times of commercial standards (3.5 min, 8.5 min,
12.5 min respectively).

Food Chemistry
From the results of the in vitro antioxidant capacity, the “Costeña thermotolerance of C. elegans. Figure 2B shows the percentage
Tierra Colorada” and “Jocote” ecotypes were selected (with of worm survival in the presence of the extracts. The results ob-
higher and lower antioxidant capacities of the hydrophilic phase tained show that in all concentrations analyzed from both ecotypes,
of the epicarp, respectively) to determine the antioxidant capacity the hydrophilic extracts provided thermotolerance to heat shock
in vivo. The toxicity of the hydrophilic extracts of the fruit epi- at temperatures that cause stress and even death of the worms.
carp, as well as that of the ethanol in the growth of the nematode “Costeña Tierra Colorada” presented a greater protective effect
was first evaluated. Figure 2A shows the curve of the extract’s than “Jocote,” since a higher concentration of the latter extract
effect. It is seen that there are no significant differences in the was required to have a similar effect to extract of “Costeña Tierra
percentage of viable worms grown in the presence of extracts af- Colorada” ecotype and presented a similar protective effect than
ter 12 h of incubation relative to control without treatment in quercetin (Figure 2B). On the other hand, it is important to men-
all the concentrations analyzed (range of 0.05 to 10 μg/mL). On tion that in the higher concentrations of the hydrophilic extract of
the other hand, ethanol did not affect the viability of the worms “Costeña Tierra Colorada,” the worms had high mobility, whereas
(data not shown). From this trial, the concentrations of epicarp in the extracts of “Jocote” they were almost motionless, although
hydrophilic extracts were defined to determine their effect in the they still responded to mechanical stimuli. Thus in the presence of
the hydrophilic extract of the “Costeña Tierra Colorada” epicarp,
the exposure to heat shock did not affect the health of the worm.
Table 4–Total carotenoid content.
These results concur with those reported by Martorell and others
Lutein Lycopene β-carotene (2011) and Vayndorf and others (2013) where they showed that
Ecotype Epicarp Pulp Epicarp Pulp Epicarp Pulp
cocoa phenolic compounds and extracts of whole apples, respec-
tively, gave resistance to different types of stress (heat shock, UV
Atzompa 1.1d 0.4d ND ND 0 0 radiation, oxidative stress, and pathogen infection) to the nema-
Conserva de Iguala 6.4a 1a ND ND 1.4b 1.4a tode C. elegans; in addition, they delayed aging, prolonged the
Costeña Tierra Colorada 2.8c 0.4d ND ND 1c 0.2d
Jocote 1.2 d 0.4 d ND ND 0.3 d 0.05 e lifespan of the worm and improved its health. However, it is im-
Morada de Cocula 3.3b 0.7b ND ND 2a 0.7b portant to note that the thermoprotective effect of plum extracts
Tlaxmalac 3c 0.5c ND ND 1.2c 0.3c was observed at a concentration a thousand times lower than those
∗ Amarilla 1.1 ND 0.3 reported for cocoa and apple extracts.
Carotenoid content in the epicarp and the pulp of the 7 ecotypes analyzed (the results Recent studies have shown that hydrophilic tomato extracts
are expressed as parts per million). Mean values followed by the same letter in each from landraces accessions, cultivated in different regions of Ar-
column are not significantly different between the ecotypes analyzed according to the
Tukey’s multiple comparison test. Values are the average of 3 independent experiments gentina, protect nematode C. elegans from thermal stress of
± standard deviation. ND = Not detected.

Vol. 00, Nr. 00, 2017 r Journal of Food Science 5

 Antioxidant capacity of S. purpurea L . . .

Figure 2–Toxicity effect of the epicarp extracts

and antioxidant capacity in vivo.The toxicity of
plum extracts was determined during the growth
of the nematode after 12 h of treatment (A).
Effect of pretreatment with epicarp plum extract
on the resistance to heat shock in C. elegans (B).
Mean values followed by the same letter in bars at
each concentration are not significantly different
between “Costeña Tierra Colorada” and “Jocote”
ecotypes according to the Tukey’s multiple
comparison test. Values are the average of 3
independent experiments ± standard deviation.
Food Chemistry

37 °C, which generates the accumulation of reactive oxygen the in vitro analysis the extracts from “Jocote” presented the lowest
species leading to the death of the nematodes in a relatively antioxidant capacity. At the highest concentrations of hydrophilic
short time (approximately 5 h). Tomato extracts protected the plum extract tested, the nematodes showed higher stress tolerance
nematodes from thermal stress and these results were attributed and health span as demonstrated by the lower loss of mobility. The
to phenolic compounds like chlorogenic acid, quercetin-3-O- phenolic compounds contained in the epicarp could be responsi-
rutinoside (most abundant components) as well as to derivatives ble for providing protection against heat shock. Based in this result
of caffeoylquinic acid (to a lesser extent), present in landraces ac- the “Costeña Tierra Colorada” ecotype could be considered as
cession of tomato (Di Paola Naranjo and others 2016). In this having the potential to be selected for conservation and breeding
regard, within the phenolic compounds present in the hydrophilic program for this fruit.
extracts of the S. purpurea epicarp, chlorogenic acid, among others,
was identified (data not showed). Thus, the phenolic compounds Acknowledgments
present in extract of S. purpurea could be responsible for the ther- The authors thank Rosa Estela Navarro and Laura Silvia Salinas
motolerance observed in the nematodes. who kindly provided the C. elegans and E. coli OP50 strains and
assisted in the establishment of methodologies. This work is part of
the JMVH postdoctoral position in Univ. Autónoma del Estado de
Conclusions Morelos. This research was funded by PRODEP Red de Cuerpos
The results show a high variability in antioxidant capacity and Académicos Ciencia y Tecnologı́a Pre y Postcosecha. The authors
the content of functional compounds between the ecotypes an- declare no conflict of interest.
alyzed, both in the epicarp and in the pulp. The epicarp of the
“Costeña Tierra Colorada” and “Jocote” ecotypes had the high- Author Contributions
est and lowest antioxidant capacity, as well as the highest and Juan Manuel Villa-Hernández: designed and performed the de-
lowest total phenolic content, respectively. A positive correlation terminations of quantification of total phenolic and total flavonoid
was observed between the antioxidant capacity and total phenolic content, carotenoid and antioxidant capacity in vitro and in vivo.
content. When testing the thermoprotective effect of these ex- Integrated, interpreted all data and wrote this article.
tracts in animal model C. elegans, it was found that both provided Gabriela Mendoza-Cardoso: contributed to the determination
the nematode with protection from thermal stress, even though in of total phenolic content and in vivo antioxidant capacity.

6 Journal of Food Science r Vol. 00, Nr. 00, 2017

Antioxidant capacity of S. purpurea L . . .
José Alberto Mendoza-Espinoza and Cristián Vela-Hinojosa:
helped with the quantification of carotenoids by HPLC and ana-
lyzed the data.
Fernando Dı́az de León-Sánchez and Fernando Rivera-
Cabrera: contributed to analyze all data and to revise the
manuscript
Irán Alia-Tejacal and Laura Josefina Pérez-Flores: designed the
project, directed the experimental work and contributed to the
analysis of data and to elaborate the manuscript.
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