Journal of Chromatography B, 942–943 (2013) 37–45

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Activity-guided identification of acetogenins as novel lipophilic

antioxidants present in avocado pulp (Persea americana)
Dariana Rodríguez-Sánchez a,b , Christian Silva-Platas b , Rocío P. Rojo b , Noemí García b,c ,
Luis Cisneros-Zevallos d , Gerardo García-Rivas b,c , Carmen Hernández-Brenes a,c,∗
a
Department of Biotechnology and Food Engineering, School of Biotechnology and Food, Tecnológico de Monterrey-Campus Monterrey, E. Garza Sada 2501
Sur, C.P. 64849, Monterrey, NL, Mexico
b
Endowed Chair in Cardiology. School of Medicine. Tecnológico de Monterrey-Campus Monterrey, Monterrey, NL, Mexico
c
Basic and Translational Research Department. Instituto de Cardiología y Medicina Vascular. Hospital Zambrano-Hellion.Tec-Salud del Sistema Tecnológico
de Monterrey, San Pedro Garza García, NL, Mexico
d
Department of Horticultural Sciences, Texas A&M University, College Station, TX, USA

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

Article history: Avocado fruit is a rich source of health-related lipophilic phytochemicals such as monounsaturated fatty
Received 6 May 2013 acids, tocopherols, carotenes, acetogenins and sterols. However, limited information is available on the
Accepted 10 October 2013 contribution of specific phytochemicals to the overall antioxidant capacity (AOC) of the fruit. Centrifugal
Available online 18 October 2013
partition chromatography was used as fractionation tool, guided by an in vitro chemical assay of oxygen
radical absorbance capacity (ORAC). Subsequent experiments focused on isolation and characterization
Keywords:
of the chemical nature of the main contributors to lipophilic AOC of avocado pulp. ORAC values obtained
Acetogenins
for acetogenins were contrasted with results from an isolated kidney mitochondria membrane lipid
Avocado
Antioxidant capacity (AOC)
peroxidation bioassay. The present study established that lipophilic AOC of the pulp was significantly
Centrifugal partition chromatography higher than its hydrophilic AOC. Our results confirmed the presence of acetogenins in the fractions with
Activity-guided isolation highest lipophilic AOC, and for the first time linked them as contributors to lipophilic-ORAC values.
Further HPLC-PDA/MS-TOF analysis led to structural elucidation of two novel acetogenins, not previously
reported as present in avocado pulp, along with five already known related-compounds. Antioxidant
properties observed for avocado pulp acetogenins by the ORAC assay suggested that, in the presence of
an emulsifying agent, acetogenins could serve as novel lipophilic antioxidants in a food matrix. Results
from isolated mitochondria lipid peroxidation bioassay, indicated that L-ORAC values which may have
relevance for food matrix applications, should not be interpreted to have a direct relevance in health-
related claims, compounds need to be evaluated considering the complexity of biological systems.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Contributions of known and novel lipophilic antioxidants to diet

Accumulating evidence suggests that increased oxidative
stress is associated with the development of many chronic and
degenerative diseases [1,2]. Epidemiological studies have also
demonstrated that reduced risk of cardiovascular diseases and cer-
tain forms of cancer are correlated with higher intakes of fruits and
vegetables; health benefits that have been attributed to their high
antioxidant contents [3,4]. In this sense, hydrophilic antioxidants,
such as phenolics and ascorbic acid, are recognized as the major
contributors to the antioxidant capacity (AOC) found in fruits and
vegetables [5,6]. However, lipophilic antioxidants can penetrate
cell membranes, and reach higher levels of bioavailability than
hydrophilic antioxidants, which are generally excreted in urine [7].
∗ Corresponding author. Tel.: +52 818 358 14 00x4817; fax: +52 818 328 4136.
E-mail address: chbrenes@itesm.mx (C. Hernández-Brenes).
therefore continue to be relevant, and a promising research field
for the understanding of their potential roles in human health.
Avocado fruit is recognized as a particularly rich source of
health-related lipophilic phytochemicals like monounsaturated
fatty acids, tocopherols, carotenes, sterols and acetogenins [8,9].
A few studies have been conducted on the correlation of avo-
cado AOC with the concentrations of specific groups of lipophilic
phytochemicals. Prior authors have reported non-significant cor-
relations between AOC and the corresponding concentrations of
carotenoids [10] or sterols [11]. And there are discrepancies on
the correlations between AOC values, assessed by the DPPH (2,2-
diphenyl-1-picrylhydrazyl) assay, and concentrations of fatty acids.
Plaza et al. [11] found a low, but slightly higher correlation with
stearic acid (R2 < 0.37), from a group of saturated, monounsaturated
and polyunsaturated fatty acids. Whereas a more recent report
suggests stronger correlations (R2 > 0.78) between AOC values and
oleic, linoleic and ␣-linolenic acids concentrations [12]. Therefore,

1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2013.10.013
38 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45
and to the best of our knowledge, no prior studies have been
conducted on the chemical isolation and characterization of avo-
cado compounds for the assessment of their contribution to overall
lipophilic AOC of avocado pulp.
In our preliminary studies, fractions obtained by centrifu-
gal partition chromatography (CPC) separation of an acetone
crude extract (E001) from avocado pulp, exhibited significant
higher lipophilic-ORAC (L-ORAC) values than their corresponding
hydrophilic-ORAC (H-ORAC) values. Preliminary chemical identifi-
cation of the compounds present in fractions with highest L-ORAC
values presumably indicated the presence of lipid derivatives from
a family of compounds known as acetogenins. In the present study
we performed the activity-guided fractionation of an acetogenin-
enriched crude extract (E003) from avocado pulp, in order to
confirm acetogenin contribution to avocado lipophilic AOC and
characterize their chemical structures. Finally, acetogenin ORAC
values were contrasted with results from an isolated mitochondria
lipid peroxidation bioassay, in order to study their performance
and lipophilic antioxidant properties in a more complex biological
system.
2. Materials and methods
2.1. Plant material
Avocado fruits (Persea americana Mill, cv. var. Hass) were
obtained by Avomex International, S.A de C.V. (Sabinas, COA,
Mexico) from Uruapan, Michoacán, México. Pulp was manually
separated from seeds, homogenized, vacuum packed and stored at
−80 ◦ C. Frozen avocado paste was freeze-dried in a VirTis Freeze-
mobile Model 25EL (SP Industry, Inc. Gardier, NY, EUA) stored at
−20 ◦ C prior to its use.
2.2. Preparation of an acetogenin-enriched extract
Freeze-dried paste (120 g) was homogenized with acetone (1:6,
w/v) using an Ultra-Turrax T25 homogenizer (IKA Works, Willm-
ington, NC, US) at 11,000 rpm for 3 min, sonicated during 5 min
and centrifuged at 3000 rpm at 4 ◦ C for 10 min. Supernatant was
collected and precipitate was extracted 5 more times, as abovemen-
tioned. Supernatants were mixed and evaporated under reduced
pressure at 30 ◦ C, yielding an acetone crude extract E001, which
was subsequently partitioned in a heptane:methanol (1:1 v/v)
biphasic system at an extract-to-each-phase of the solvent system
ratio of 1:6 w/v. Phases were separated and concentrated under
reduced pressure, yielding heptane-soluble and methanol-soluble
semi-crude subfractions, designated as E002 and E003 extracts,
respectively. Extracts E001 to E003 were subjected to HPLC-PDA
analysis as described in Section 2.6.
2.3. CPC fractionation of acetogenins-enriched extract
After HPLC-PDA evaluation, E003 extract was further fraction-
ated in a 1L CPC system (Kromaton Technologies, Angers, France).
A hexane:methylene chloride:methanol:water (10:5:7:3) biphasic
solvent system was used for the separation, and its upper phase
(UP) served as stationary phase. CPC column (set at 800 rpm) was
entirely filled with stationary phase, and lower phase (LP) was
pumped at a 10 ml/min flow rate to establish hydrodynamic equi-
librium (80% retention of the SP). E003 extract was dissolved in
100 ml of LP and injected. LP was pumped during 120 min and then
UP for other 100 min to finally collect 220 fractions (10 ml each).
Fractions were individually evaporated and their corresponding
partition coefficients (KD ) were calculated as described by Berthod
et al. [13]. HPLC-PDA analysis of the fractions was carried out as
described in Section 2.6. Based on the similarities of their chro-
matographic profiles, consecutive fractions were mixed together
to finally obtain seven different groups of fractions (designated as
purified fractions GF01 to GF07) that were stored at −80 ◦ C, until
use. Subsequently, AOCs of the 7 groups of fractions obtained were
further characterized using the assays described in Sections 2.4 and
2.5.
2.4. Chemical antioxidant capacity screening
Chemical AOC screening was initially carried out by using
H-ORAC and L-ORAC assays, as previously reported [14]. Dried
samples were adjusted to 200 ␮g/ml using phosphate buffer for
H-ORAC or 7% of randomly methylated ␤-cyclodextrin (Fisher Sci-
entific Int., Winnipeg, MB., Canada) in acetone:water (1:1) for
L-ORAC. Fluorescence was recorded every 2 min after the addi-
tion of AAPH and during one hour, using a microplate reader
Synergy HT (Bio-Tek® Instruments, Winooski, Vermont, EUA.) set
at a constant temperature of 37 ◦ C. Results were expressed as
␮mol of Trolox Equivalents (TE)/g of solids on dry weight (dw)
basis.
2.5. Isolated-mitochondria lipid peroxidation bioassay
2.5.1. Animal use
Experiments were performed in accordance to the animal care
guidelines of the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication
No. 85–23, revised 1996). All procedures were approved by the
animal use and care committee at Medical School Tecnológico de
Monterrey.
2.5.2. Isolated mitochondria experiments
Male Wistar rats (200–230 g/8 week old) were euthanized by
cervical dislocation and kidneys were immediately removed and
placed in ice-cold medium containing 250 mM sucrose, 10 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and
1 mM EGTA (ethylene glycol-bis (␤-aminoethyl ether)-N,N,N ,N -
tetraacetic acid), pH 7.4. Tissue was minced with sharp scissors,
rinsed and gently homogenized using a glass homogenizer. The
mitochondrial fraction was obtained by differential centrifugation,
as previously described [15], with all steps carried out at 4 ◦ C.
The mitochondrial pellet was re-suspended in isolation medium
EGTA-free, and the protein concentration was determined using
the method described by Lowry et al. [16]. Mitochondrial oxygen
consumption was measured using a Clark-type oxygen electrode
as previously reported [17]. The experiments were carried out in
0.5 ml of assay medium, containing 125 mM KCl, 10 mM HEPES and
3 mM KH2 PO4 –TRIS, pH 7.3. A state 4 respiration was evaluated
in the presence of 10 mM succinate plus 1 ␮g/ml rotenone. State
3 respiration was measured after addition of 200 ␮M ADP. Respi-
ratory control ratio (RCR) as parameter of mitochondria integrity
was determined as respiration in state 3 rate divided by that in
state 4.
2.5.3. Antioxidant capacity bioassay
The extent of lipid peroxidation was determined by measuring
the thiobarbituric acid reactive species (TBARS), such a malondi-
aldehyde (MDA), generated by FeCl2 in mitochondria. Succinate
was used as substrate, since it has been reported that acetogenins
inhibit mitochondrial nicotinamide adenine dinucleotide (NADH)-
ubiquinone oxidoreductase activity [17]. Mitochondrial protein
(0.3 mg) was added to 0.5 ml of respiration assay medium. Mixture
was supplemented with 200 ␮M FeCl2 and 50 ␮M CaCl2 and incu-
bated at 37 ◦ C for 20 min, in presence or absence of each evaluated
sample (200 ␮g of sample/mg mitochondrial protein). The reaction
 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45
was stopped with 0.4% BHT (3,5-di-tert-butyl-4-hydroxytoluene).
Mitochondrial suspension was incubated in 0.5 ml 0.15 M phos-
phate buffer, pH 7.0 at 37 ◦ C for 20 min. Then, it was further
incubated with 0.8% (w/v) thiobarbituric acid and 20% (v/v) acetic
acid; reaction was allowed to develop at 100 ◦ C for 45 min. The reac-
tion was stopped by adding 2% (w/v) KCl at 4 ◦ C. Finally, TBARS were
extracted with one volume of n-butanol and measured at 532 nm.
A standard curve was made with tetraetoxipropane. Results were
recorded as nmol TBARs/mg of protein. Data are expressed as inhi-
bition percentage of lipid peroxidation in presence of each group
of fractions, considering the amount of TBARS formed in a sample
exposed to FeCl2 in the absence of any group of fractions, as 0%
inhibition.
2.6. Acetogenin characterization and quantification by HPLC-PDA
Evaluated samples, at different degrees of purification, were
adjusted to 2 mg/ml using isopropanol HPLC grade. Analyses were
performed in an Agilent 1100 HPLC system (Agilent Technolo-
gies, Santa Clara, CA, US) coupled to a photodiode array (PDA)
detector G1315D, set at a wavelength of 220 nm. Chromatographic
separation was carried out in a Zorbax Extend-C18 (100 × 3 mm,
3.5 ␮m) column, with mobile phases that consisted of water 100%
(phase A) and methanol 100% (phase B). Gradient elution was
set as follows: 0–2 min, 50–70% B linear at 0.3 ml/min; 2–5 min,
70–80% B linear at 0.3–0.38 ml/min; 5–25 min, 80% B isocratic at
0.38–0.5 ml/min; 25–28 min, 80–100% B linear at 0.5–0.55 ml/min;
28–38, 100% B isocratic at 0.55–0.6 ml/min, followed by 10 min of
re-equilibration between injections. Acetogenin quantification was
performed by external calibration in triplicate using Persenone-
A (isolated and purified in our laboratory) as standard. Results
were expressed as mmol of Persenone-A equivalents/g of solids
(dw). Total acetogenins recovered from freeze-dried avocado pulp,
for each extract, were calculated from the multiplication of ace-
togenin concentrations by the recovered solids, and expressed as
mmol of Persenone-A equivalents/g of freeze-dried avocado pulp
(dw).
2.7. HPLC–MS analysis of active compounds
Mass spectra analysis of common compounds present in the
most potent antioxidant fractions was carried out in an Agilent
1100 HPLC system coupled to a time-of-flight mass spectrome-
ter (MS-TOF) G1969A, equipped with an electrospray ionization
(ESI) interface. The chromatographic separation was performed as
described in Section 2.6, but adding 0.1% formic acid to mobile
phases. ESI conditions included analysis in positive-ion mode; N2
at 45 psi as nebulizing gas; drying gas flow rate and temperature of
8 l/min and 300 ◦ C, respectively; voltage at the capillary entrance
set at 2500 V; fragmentation, skimmer and Oct RFV voltage at 100,
60 and 250 V, respectively. Mass spectra were recorded from an
m/z of 100–1000. Chemical identity was assigned by comparison of
spectroscopic data with values reported in the literature and with
data from standards isolated in our laboratory from avocado seed
[18].
2.8. Statistical analysis
All samples were analyzed in triplicate and data was expressed
as mean ± standard deviation. Mean separation was conducted
using the one-way ANOVA procedure and significant differences
assessed by the LSMean Student’s t-test (P < 0.05). Non-linear
correlations between ORAC values and isolated mitochondria
experiments values were calculated. Data were analyzed using JMP
software software version 5.0 (SAS Institute Inc., Cary, NC, USA).
39
3. Results and discussion
3.1. Preparation of acetogenin-enriched extracts
Preliminary studies on the fractionation of an acetone crude
extract (E001) from avocado pulp revealed that L-ORAC values were
significantly higher than H-ORAC values (Fig. S1 of Supplementary
data). The sum of L-ORAC values for evaluated fractions was 4.7-fold
higher than the corresponding H-ORAC values. Results that were
in agreement with prior AOC evaluations of avocado pulp [12,19]
and with our preliminary study (Fig. S1 of Supplementary data).
On the contrary, Wu et al. [20] reported that H-ORAC values of avo-
cado pulp were 2.5-fold higher than L-ORAC values; differences
that may be attributed to variations in growing and postharvest
handling practices.
A spectrophotometric characterization of fractions that pre-
sented the highest L-ORAC and H-ORAC values (KD = 1.01 and
0.46, respectively), in the preliminary work, was also conducted.
Absorbance values were recorded, at the characteristic wave-
lengths of maximum absorption (max ), for different families
of phytochemicals with known antioxidant properties. Results
revealed that flavones, ascorbic acid and phenolics appeared to
be present in the fraction that presented the highest H-ORAC
value (KD = 0.46) (Fig. S2 of Supplementary data). Fraction with
a KD = 1.01 presented the highest L-ORAC value, and moderate
absorbances at characteristic wavelengths for ascorbic acid, chloro-
phylls, carotenes and phenolics. However, values recorded at 205
and 220 nm, which are characteristic for Persin and Persenones
(two members of the acetogenin family), were significantly higher
(∼7 to 25-fold) than those recorded at any other wavelength (Fig.
S2 of Supplementary data). Preliminary confirmation of acetogenin
presence, in the grouped fraction (KD = 0.95–1.26) that presented
the highest L-ORAC values, was also conducted by HPLC-PDA
analysis. PDA spectrums of the chromatographic peaks obtained,
presented max values at 205, and 224–227 nm, characteristic
for acetogenins [21,22] (Fig. S3 and Table S1 of Supplementary
data).
Because preliminary data strongly suggested that acetogenins
were predominantly found in fractions that presented the highest
lipophilic AOC values; in the present work an optimized analytical
HPLC-PAD method was developed to selectively track and quan-
tify their concentrations during an activity-guided identification
procedure. For this task, a new acetone crude extract E001 was
prepared and partitioned in the same biphasic system, yielding
heptane-soluble and methanol-soluble semi-crude sub-fractions,
designated as E002 and E003 extracts, respectively. As it can be
observed in Table 1, about 90% of the E001 extract initial weight
was further recovered into E002. From a recovery perspective hep-
tane sub-fraction (E002) isolated higher concentrations of total
acetogenins (1.3-fold) than E003. Results that were expected, due
to the lipophilic nature of acetogenins and affinity for less polar
solvents [23]. However from a purity perspective, although E003
extract contained the least recovered solids (5.1%), they were highly
enriched in acetogenins (8.1-fold higher than E002). Lipid contents
of avocado pulp account for more than the 50% of its dry matter
[24], therefore, fatty acids and non-polar compounds, other than
acetogenins, also partitioned into the E002 extract, possibly dis-
placing acetogenins into E003. Consequently, because of its purity,
E003 extract was selected for further CPC-fractionation.
3.2. CPC fractionation of an acetogenin-enriched extract
CPC was used to fractionate the acetogenin-enriched extract of
avocado pulp (E003). A series of solvent systems were evaluated
for the separation (data not shown), and hexane:methylene
chloride:methanol:water (10:5:7:3) system was found to perform
40 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45

Table 1
Percentage of recovered solids, acetogenin concentrations and total acetogenins recovered that resulted from the extraction of freeze-dried avocado pulp to obtain an acetone
crude extract (E001) and two semi-crude sub-fractions.

Extraction system Fraction labela Recovered solids (%)b,c Acetogenin concentrations Total acetogenins recovered
(Extraction solvent) (mmol eq. Persenone-A/g of solids) (mmol eq. Persenone-A/g of
freeze-dried avocado pulp)

Crude extract E001 (acetone) 61.19 ± 0.29 ad 0.046 ± 0.003 b 0.028 ± 0.002 a
Biphasic system 1 E002 (heptane) 56.02 ± 0.85 b 0.028 ± 0.002 c 0.016 ± 0.001 b
E003 (MeOH) 5.11 ± 0.87 c 0.227 ± 0.014 a 0.012 ± 0.003 c
a
E001 is an acetone crude extract from freeze-dried avocado pulp that was further partitioned in biphasic system 1 (heptane:methanol, 1:1) to obtain semi-crude
sub-fractions E002 and E003.
b
Data expressed as g of solids/100 g of freeze-dried avocado pulp.
c
Values represent mean ± standard deviation (n = 3).
d
Same letters within columns indicate that values are not significantly different from each other (LSMean Student’s t- test, P < 0.05).

better for E003. CPC separation yielded 220 fractions that were
analyzed by HPLC-PDA at 220 nm. Chromatographs collected
at 220 nm showed that fractions contained six common peaks,
which were labeled as Peaks A-F (Fig. 1A). Based on similarities
in their chromatographic profiles, consecutive fractions were
mixed together to finally obtain seven different groups (Fig. 1B),
designated as purified fractions GF01 to GF07. Relative concentra-
tions of peaks present in the purified fractions (Table 2), indicated
that GF03 contained peaks with elution times (tE ) lower than
18 min, and was preferably enriched in Peak A. While acetogenin
contents in GF04 decreased, and then increased again in GF05,
showing the presence of Peaks B-D (tE between 19 and 30 min),

Peak D
A
Absorbance (mAU)

100

50
Peak C
Peak A

Peak F
Peak E
Peak B

0
0 10 20 30
Time (min)
Peak A

Peak D

Peak E
Peak B
Peak C

Peak F

B
Fraction
GF07

Fraction
GF06

Fraction
GF05

Fraction
GF04

Fraction
GF03

Fraction
GF02

Fraction
GF01
50 mAU

5 min

Fig. 1. (A) Characteristic HPLC-PDA chromatogram (at 220 nm) of sub-fractions obtained by centrifugal partition chromatography of an acetogenin-enriched extract (E003).
Peak letters from A to F were assigned according to the order of elution from a Zorbax Extend-C18 column. (B) HPLC-PDA chromatogram (at 220 nm) of the purified fractions
GF01 to GF07. Sub-fractions were grouped based on similarities in their chromatographic profiles. Partition coefficients (KD ) ranged from 0.14 to 0.40, 0.83 to 1.13, 1.59 to
1.72, 2.03 to 2.41, 4.12 to 5.82, 7.37 to 11.67 and 14 to ∞, for GF01 to GF07, respectively.
 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45
Table 2
Relative concentrations of chromatographic peaks A to F present in the groups of fractions GF01 to GF07.
Fraction (Partition Coefficient) Peak Concentrationa (mmol eq. Persenone-A/g solids, on dry weight basis)
Peak Ab (17.1 min) Peak B (19.9 min)
GF01 (0.14–0.40) ND ND
GF02 (0.83–1.13) ND ND
GF03 (1.59–1.72) 0.73 ± 0.02 ac ND
GF04 (2.03–2.41) ND ND
GF05 (4.12–5.82) 0.053 ± 0.002 b 0.036 ± 0.001 a
GF06 (7.37–11.67) 0.006 ± 0.001 c 0.004 ± 0.001 b
GF07 (14–100) ND ND
a
Values represent mean ± standard deviation (n = 3).
b
Peak letters from A to F were assigned according to their elution time (specified in parenthesis) from a Zorbax Extend-C18 column as shown in Fig. 1B.
c
Same letter within columns indicate that values are not significantly different from each other (LSMean Student’s t- test, P < 0.05).
which were absent in GF03. Finally, total acetogenin concentra-
tions in GF06 decreased again, and fraction was enriched in less
polar compounds, with tE > 23 min (Peaks D-F).
As it can be observed in Fig. 2A, purified fractions GF03,
GF05 and GF06 contained the highest total acetogenin concen-
trations (1.27 ± 0.07, 1.26 ± 0.06, 0.62 ± 0.03 mmol of Persenone-A
equivalents/g of solids (dw), respectively). Total acetogenin con-
centrations, in those three groups, were ∼6-, 6- and 3-fold higher
than E003 levels, respectively. Fractions GF01, GF02 and GF07, in
contrast, contained very low concentrations or non-detectable ace-
togenin levels. CPC separation principles are based on differential
solubility of the analytes of interest between the solvent system
phases [13]. Under the conditions used in the present study, par-
tition resulted in enhancement of acetogenin concentrations and
purity, as well as their separation of into two distinct subgroups.
Acetogenins with higher polarity eluted from CPC column at the
beginning of the extrusion stage (127–138 min) and were purified
in GF03 (Peak A). Less polar compounds abandoned CPC column
later (186–210 min) and were contained in fractions GF05 and
GF06 (Peaks B-F). Similar methods have also been used for the
separation of avocado seed phytochemicals, although not pulp. In
prior works, CPC isolation was used for bioassay-guided isolation
and identification of antimicrobial acetogenins from avocado seed
extracts [18]. High speed CCC, followed by multiple-steps of column
chromatography (normal-, reverse-phase and size-exclusion), was
also successfully applied for isolation of bioactive molecules from
different avocado seed extracts [25,26]. Their studies focused on
evaluating the effects of isolated molecules on physiological mod-
ulation of human skin cells [26]; and others on the identification
of molecules with low toxicity and antioxidant potential, as deter-
mined by the “brine-shrimp” (Artemia salina) lethality assay and the
TEAC (Trolox equivalent antioxidant capacity), DPPH assays [25].
3.3. Determination of antioxidant capacity by the ORAC assay
Chemical AOC assessment (Fig. 2B) revealed that the sum of L-
ORAC values for all purified fractions (GF01-GF07) were 2.9-fold
higher than their corresponding H-ORAC values, confirming obser-
vations from preliminary evaluations with crude extracts. As it can
also be observed in Fig. 2B, fraction GF05 (KD = 4.12–5.82) presented
a significantly higher L-ORAC value (3933.34 ␮mol TE/g of solids),
which was at minimum 2-fold higher than H-ORAC values from
the other purified fractions. Overall, AOC values of purified fractions
were higher than values exhibited by semi-crude subfractions E002
and E003 (Table 1). In a direct comparison, L-ORAC and H-ORAC
values for fractions GF05 and GF03 (Fig. 2B) presented 3.7 and 8.9-
fold better AOC, respectively, than the highest L-ORAC and H-ORAC
values of semi-crude fractions evaluated in our preliminary study
(Fig. S1 of supplementary data). An enhancement in AOC that can be
attributed to the increased purity of the antioxidants compounds.
Initial partition in the heptane:methanol biphasic system resulted
41
Peak C (21.0 min) Peak D (23.6 min) Peak E (28.2 min) Peak F (29.3 min)
ND ND ND ND
ND ND ND ND
ND ND ND ND
0.006 ± 0.001 c 0.017 ± 0.001 c ND 0.080 ± 0.002 a
0.133 ± 0.003 a 0.549 ± 0.021 a 0.084 ± 0.001 a 0.028 ± 0.001 c
0.013 ± 0.001 b 0.278 ± 0.008 b 0.058 ± 0.001 b 0.063 ± 0.001 b
ND ND 0.006 ± 0.001 c ND
in the withdrawal of 91.3% of E001’s total solid weight in order
to obtain the acetogenin-enriched extract E003 (Table 1). Further
fractionation by CPC continued to reduce the complexity of phy-
tochemicals present in each purified fraction, and also resulted in
the enrichment of acetogenins in some of the fractions (Fig. 2A).
Interestingly, GF03-GF04 (KD = 1.59–2.41) showed higher H-ORAC
values, whereas GF05-GF06 (KD = 4.12–11.67) exhibited the highest
L-ORAC values (Fig. 2B). Going back to the principles of the ORAC
assay used to measure both values, it is reminded to the reader that
the same samples are used in the determinations of the L-ORAC and
H-ORAC values. The main analytical difference is the incorporation
␤-cyclodextrin in the determination of the L-ORAC value, which
facilitates the interaction of lipophilic molecules in the assay [27].
Therefore hydrophilic molecules may present dual contributions
to the H- and L-ORAC values, whereas contribution of lipophilic
molecules to the H-ORAC value depends on their individual polar-
ity and ability to participate in the assay reactions under each set
of conditions. The latter being said because, although the frac-
tions contained lipophilic compounds (as indicated by their high
KD values), results from the ORAC assay shown in Fig. 2B indicate
that acetogenins contained in the fractions appear to have differ-
ent polarities among them. Fractions GF03 and GF05 had the same
content of total acetogenins (Fig. 2A), however GF03 presented an
L-ORAC value 1.5-fold lower than GF05 (Fig. 2B). In conformity with
this observation, as previously mentioned, based on their retention
times, GF05 and G0F6 contained less polar acetogenins than GF03
(Table 2). Since acetogenins are lipophilic molecules, nearly insolu-
ble in water [18,28], they require ␤-cyclodextrin for solubility in the
assay, and are very likely to exhibit very low or no participation in
the H-ORAC assay. Therefore, GF05-GF06, with elevated content of
less polar acetogenins (Fig. 1), exhibited the highest L-ORAC value,
but showed no H-ORAC activity (Fig. 2B).
Whereas, GF07, containing lipophilic compounds (KD = 14–100),
shows no acetogenins signal and concomitantly no L-ORAC activ-
ity. This later observation remarked the contribution of less polar
acetogenins to the lipophilic AOC of avocado pulp. The practical
application of this observation established that, in the presence of a
proper emulsifying agent, acetogenins can serve as novel lipophilic
antioxidants in a food matrix.
Finally, the fact that GF03-GF04 showed the highest H-ORAC
activity and considerable L-ORAC activity, suggested the presence
of additional and more polar compounds in the fractions; different
from acetogenins, especially because GF04 showed non-detectable
levels of acetogenins.
3.4. Further characterization of acetogenins
The confirmation of the contribution of acetogenins to the L-
ORAC values of avocado pulp, led to further studies on chemical
structure elucidation. MS/TOF-ESI(+) analyses were performed on
the purified fractions that presented the highest L-ORAC values
42 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45

1.5
A
GF03 GF05

(mmol Persenone A eq./ g)

a a

Total Acetogenins
1.0
GF06
b

0.5
c
d d
GF04 c,d
0.0
GF01 GF02 GF07

0.1 1 10 100

5000
L-ORAC GF05
B
H-ORAC GF04
a
GF06
Antioxidant Activity

b
(µmol of TE/g)

c
GF03
d
2500
GF02
e
e e e
GF01 GF07
f f

f,g f,g,h g,h

0 h
g,h

0.1 1 10 100

GF04
100 a C
Lipid Peroxidation
inhibition (%)

GF07
50 b

GF02 GF03
b,c
c
GF01 GF05
c, d c, d
GF06
d
0

0.1 1 10 100
Partition coefficient (KD)
Fig. 2. Characterization of the purified fractions GF01 to GF07, obtained after centrifugal partition chromatography of an acetogenins-enriched extract (E003). (A). Total
acetogenin concentration expressed in mmol of Persenone-A equivalents/g of solids, on dry weight (dw) basis, (B) Hydrophilic and Lipophilic Oxygen Radical Absorbance
Capacity values (H-ORAC and L-ORAC, respectively) expressed in ␮mol of Trolox Equivalents (TE)/gram of solids (dw). (C) Inhibition of isolated-mitochondria lipid peroxidation
(induced with FeCl2 200 ␮M). Values are the mean ± standard error (n = 3), where error bars values did not shown, they did not exceed the size of the respective symbols.
Means with the same letter are not significantly different from each other (LSMean Student’s t- test, P < 0.05). Horizontal bars indicate the partition coefficients (KD ) ranges of
the compounds comprised in each group of fractions, which are: 0.14–0.40, 0.83–1.13, 1.59–1.72, 2.03–2.41, 4.12–5.82, 7.37–11.67 and 14–∞, for GF01 to GF07, respectively.
 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45 43

Identification methods applied for identify assignment: (I) Identification by comparison with the retention time and wavelengths of maximum absorption in the UV/Vis spectra of standards isolated in our laboratory from
References

[1]
[2]
[2]
[1]
[3]
[4]
[5]

avocado seed [2]; (II) Identification by comparison with the mass spectra of standards isolated in our laboratory (III) Identification comparison with the order of elution and mass spectra reported in the literature.
Identification
methodd

I, II
I, II
I, II
I, II
I, II
I, II
III
351, 311, 269, 251

401, 361, 319, 301

403, 363, 321, 303

Ion pattern (m/z)c

375, 335, 293

375, 335, 293

377, 337, 295

365, 323, 305

[M + H]+ (m/z)
Calculated

329.2692
353.2692
353.2692
379.2848
355.2848

383.3161
381.3005
[M + H]+ (m/z)c
Measured

379.2864
355.2865

383.3178
329.2708
353.2706
353.2708

381.3022

Peak letters from A to F were assigned according to their elution time from a Zorbax Extend-C18 column, as shown in Fig. 1B.
(nm)b
max

225
224
225
227
225
225
227
Identification of acetogenins present in purified avocado seed fractions with the highest L-ORAC values (GF05 and GF06).
Fig. 3. Chemical structure of compounds 1 to 7 (Table 3), present in purified avocado
pulp fractions with the highest L-ORAC values (GF05 and GF06). Molecular

C19 H36 O4
C21 H36 O4
C21 H36 O4
C23 H38 O4
C21 H38 O4
C23 H40 O4
C23 H42 O4
formula

(GF05 and GF06). Compounds contained in those fractions pre-

sented the characteristic ion pattern reported for acetogenins;
consisting of [M + Na]+ and [M + H]+ molecular ions, as well as frag-
ment ions showing the consecutive losses of H2 O and/or acetic
(elution time, min)

acid (C2 H4 O2 ) from the [M + H]+ ion [21,29,30]. As shown in

Wavelengths of maximum absorption in the UV/Vis spectra of each chromatographic peak.

MS/TOF detection using electrospray ionization interface in positive-ion mode of analysis.
Table 3, seven compounds were identified by comparing their
Peak D (23.6)
Peak A (17.1)
Peak B (19.9)
Peak C (21.0)

Peak E (28.2)
Peak E (28.2)
Peak F (29.3)
Peak labela ,

mass spectra with values reported in the literature, and with data
from standards isolated in our laboratory from avocado seed [18].
Active compounds were identified as: 1-acetoxy-2,4-dihydroxy-
n-heptadeca-16-ene (1) [21]; Persediene (2) [18]; Persenone-C
(3) [18]; Persenone-A (4) [21]; Persenone-B (5) [22]; Persin (6)
Persenone-A, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene (4)

[31] and 1-acetoxy-2,4-dihydroxy-heneicosa-12,15-diene (7) [30].

Persenone-C, 1-acetoxy-2-hydroxy-4-oxo-nonadeca-5,16-diene (3)
Persediene, 1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene (2)

Structures of the identified compounds are shown Fig. 3. Com-

Persin, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (6)
Persenone-B, 1-acetoxy-2-hydroxy-4-oxo-nonadeca-5-ene (5)

pounds 2 and 3 are being reported here for the first time as present
Trivial and/or chemical nomenclature (compound number)

in avocado pulp.
The analysis of previous publications on acetogenin bioactiv-
1-acetoxy-2,4-dihydroxy-heneicosa-12,15-diene (7)

ities, now viewed in the light of our new observations on their

1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene (1)

lipophilic AOC, allowed us to infer that their prior data confirms our
findings. Particularly studies by Ardi et al. [32], Madi et al. [33] and
Wang et al. [34] indicated that exposure of unripe avocado fruit to
ethylene, cold stress or inoculation with C. gloesporiodie increased
Persin (compound 6) concentrations in avocado peel and increased
their resistance to fungal development. Other authors had previ-
ously established that those stress factors induce H2 O2 and ROS
production [35–39]. All this evidence suggests that ROS generated
during pathogen attack and abiotic stresses, are recognized by avo-
cado fruit as a signal for triggering defense responses. Therefore,
increase in acetogenin concentrations in the unripe fruit, indirectly
suggests their potential antioxidant role in the counteraction of the
fungal and abiotic induced oxidative stresses.
Table 3

As previously mentioned, Villa-Rodriguez et al. [12] also

a

reported that avocado pulp presented higher L- than H-ORAC

44 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45
values. And that some unsaturated fatty acids were correlated with
AOC measured by DPPH and TEAC assays, but no correlations were
found with results from ORAC assay. Differences were attributed to
the dissimilar reaction mechanisms of each assay. As ORAC assay
is based on a hydrogen atom transfer reaction, whereas DPPH and
TEAC assays are based on an electron transfer mechanism [40]. In
this regard, our findings showed that total acetogenin concentra-
tions in the different purified fractions had a non-linear correlation
(R2 = 0.79) with the L-ORAC values. Suggesting that opposite to
mechanism described by Villa-Rodriguez et al. [12] for fatty acids,
acetogenins may quench free radicals by hydrogen donation.
This last hypothesis is possibly strengthened by results reported
by our group [17], establishing that acetogenins from avocado seed
act as mitochondrial uncoupling agents, transporting protons from
the mitochondrial inter-membrane to the matrix. We proposed
that aliphatic chain, together with a trans-enone group (unsatu-
ration at C5–C6 conjugated to a keto group), present in some
acetogenins, can stabilize anionic species through delocalization
of the charge within its structure. Furthermore, Mendoza-Wilson,
et al. [41] proposed that the keto group of acetogenins confers acid-
ity to the hydrogens present in ␣ carbons C-3 and C-5 facilitating the
loss of the protons. Consequently an enolate anion is stabilized by
resonance and it can react with free radicals for neutralizing them.
These observations could also explain the higher L-ORAC values
observed for fractions GF05 and GF06 (Fig. 2B); as they contained
the highest concentrations of compounds 3, 4 and 5, all of them
exhibiting a trans-enone group in their structures (Fig. 3).
Moreover, Ramos-Jerz [25] reported that a methanolic extract
from avocado seeds exhibited higher AOC levels than a
petroleum ether extract. Further CCC fraction these extracts, fol-
lowed by preparative-HPLC isolation allowed confirmation of
flavan-3ols (proanthocyanidins B-1, B-2, A2-(+)-catechin and A2-
(+)-epicatechin) as the responsible for the AOC of methanol extract;
whereas acetogenins, including compounds 1, 4 and 5, were the
main constituents of petroleum ether extract. However, in this
study, AOC evaluation was carried out by using TEAC and DPPH
assays. This evidence supports our hypothesis that acetogenins sta-
bilize free radicals through hydrogen donation, and not electron
transfer mechanisms.
Additionally, it is important to remark that fractionation of
petroleum ether extract, carried out by Ramos-Jerz [25], was guided
by the A salina lethality assay, and finally led to the identification
of eight acetogenin as responsible for the cytotoxic effect. How-
ever, toxic acetogenins were constituted of C19 , and among them,
those containing hydroxyl groups, both in C-2 and C-4 positions,
exhibited a highest toxicity. As shown in Fig. 3, from the aceto-
genins identified in the present study, only compound 1 matches
their description.
Observations made by Silva-Platas et al. [17] and Ramos-Jerz
[25] suggest that among acetogenins characterized herein, if the
proposed stabilization mechanism is correct, compounds 3, 4 and
5, may have a higher potential as antioxidants in food systems. Since
they contain the trans-enone group necessary to stabilize anionic
species and support hydrogen donation, and they contain more
than C19 and no hydroxyl groups in C-4 (Fig. 3), avoiding possible
toxic effects.
3.5. Isolated-mitochondria lipid peroxidation bioassay
The use of chemical antioxidant capacity in vitro assays, that
require little test material and enable the handling of multiple
samples, is strongly recommended in the initial screening stages
of an activity-guided fractionation approach [42]. Therefore, in
the present study, the ORAC assay was initially used to identify
lipophilic compounds with antioxidant potential. However com-
monly used AOC in vitro assays (such as the ORAC, DPPH• , ABTS•+ ,
FRAP, TEAC) have the limitation of being based on oxidative chem-
ical reactions taking place in a solution. Other biological variables
such as bioavailability, transport and accumulation in tissues, sta-
bility in vivo and overall ability to react in situ are not considered
in those in vitro AOC assays [40].
Aware of ORAC assay limitations, in this work the lipophilic AOC
of acetogenins was further evaluated in an isolated mitochondria
lipid peroxidation bioassay, in order to document their behavior
in a more complex biological system. Rat kidney mitochondria
were chosen as an oxidative biological model, since they are the
most important endogenous source of ROS in animals, and are
derived from electro transport chain leakage [43]. At first, we
measured mitochondria respiration, using succinate plus rotenone
as oxidative substrate, since it has been previously reported that
acetogenins are potent inhibitors of (NADH)-ubiquinone oxidore-
ductase activity [17]. As shown in supplemental Fig. S4A, we
observed the effects of purified fractions GF01, GF03 and GFO7
on respiratory control ratio (RCR). A dose-dependent response was
not evident, suggesting that at least 300 ␮g/mg of protein of these
purified fractions did not elicit a significant inhibition of elec-
tron transport chain or produced an uncoupling effect. In addition,
fractions GF01 to GF07 (200 ␮g/mg of protein) neither produced
a significant effect on state 3 respiration, indicating that in the
basal state without FeCl2 our mitochondrial sample are undamaged
(Fig. S4B of supplementary data). Therefore, the capability of the
purified fractions GF01 to GF07 obtained in Section 2.3, to inhibit
isolated-mitochondria lipid peroxidation caused by mitochondrial
ROS production (induced by 200 ␮M FeCl2 ) was further assessed
by means of TBARs assay.
As shown in Fig. 2C, inhibition of lipid peroxidation pro-
gressively increased as the KD increased, reaching a maximum
inhibition value of 93 ± 1.43% at KD values of 2.03 to 2.41 (GF04),
a fraction that contained significantly lower acetogenin concen-
trations (Table 2). This percentage of lipid peroxidation inhibition
was significantly higher (P < 0.05) than values displayed by Trolox
(73.35 ± 0.53%, data not shown), a Vitamin E analogue tested at
the same concentration (200 ␮g/mg of protein). Fractions that con-
tained the highest concentrations of acetogenins (GF05 and GF06)
presented very low levels of lipid peroxidation inhibition (4.66 and
−3.91%, respectively), and GF06 actually promoted peroxidation.
Finally, fraction GF07 (KD = 14–∞) that contained a very low ace-
togenin concentration, resulted a better AOC in the mitochondria
bioassay, since it inhibit lipid peroxidation by ∼40%.
Results therefore indicated that an opposite behavior was
observed for acetogenins in the L-ORAC assay and in the isolated-
mitochondria lipid peroxidation bioassay. Using values from all
purified acetogenin fractions (GF01 to GF07), attempts to explain
the relationship between both bioassays using non-linear regres-
sion models showed very low association among them (R2 = 0.37).
Other authors have also documented that in vitro chemical
reaction-based assays often do not correlate with the ability of com-
pounds to inhibit oxidative deterioration in biological systems [44].
Mainly because, as previously mentioned, factors like environmen-
tal conditions, physical location of the antioxidants and interactions
with other macromolecules are not taken into account.
On the other hand, a very recent study evaluated the effects of
dietary supplementation with avocado oil during 90 days (oral dose
of 4 ml/kg of body weight) on rat mitochondria isolated from kidney
[45]. Prior characterization of the avocado oil used in their stud-
ies indicated that, as expected, >70% of the total fatty acids were
monounsaturated fatty acids (MUFAs) mainly comprised of oleic
acid. Researchers observed that lipo peroxidation induced by Fe2 +
in the isolated-mitochondria bioassay was successfully inhibited
(∼50%) in the avocado oil treatments and in reference to con-
trols. Additionally, they observed a significant increase (24.8%) in
total MUFA composition of mitochondria. Researchers attributed
 D. Rodríguez-Sánchez et al. / J. Chromatogr. B 942–943 (2013) 37–45
the observed protective effects, to the high MUFAs concentrations
present in the membranes; since it is well known that stability of
fatty acids to peroxidation increases as their unsaturation degree
decreases. Similar remarks have been stated for yeast mitochondria
having a native MUFA predominant composition [46]; and eukary-
otic mitochondria, in which MUFA predominant composition was
induced through dietary supplementation of olive oil [45]. Differ-
ing from MUFAs, acetogenins are polyunsatured fatty acid (PUFA)
derivatives [47], which makes them more susceptible to lipid per-
oxidation. Therefore, it can be suggested that acetogenins increased
unsaturation levels of mitochondria membrane, increasing their
susceptibility to peroxidation; as purified fractions with high ace-
togenin concentration (GF05 and GF06) did not offer protection
against lipid peroxidation (Fig. 2C).
In conclusion, the present study established an association of
the AOC evaluated by means of L-ORAC and acetogenins present in
avocado pulp. This observation suggested that, in the presence of a
proper emulsifying agent, acetogenins may act as lipophilic antiox-
idant molecules with potential applications for the stabilization of
food matrixes. The use of CPC in combination with the ORAC assay,
proved to be an effective strategy for the isolation and identifica-
tion elucidation of the bioactive compounds. Two new acetogenins
and five already known compounds were found to be present in the
fractions with the highest L-ORAC values. Differences in the results
obtained by the ORAC and some other chemical reactions-based
assays, such as TEAC and DPPH, can be attributed to the differ-
ences in their free radicals stabilizing mechanisms. Acetogenin
AOC, established by the L-ORAC assay, was not correlated with their
ability to inhibit lipid peroxidation in the isolated-mitochondria
bioassay. Differences in results can be attributed to the polyunsat-
urated nature of acetogenins. Results from the L-ORAC assay and
evaluation of prior literature, seems to indicate that compounds
3, 4 and 5 may have a higher potential as antioxidants in food
systems; since they contain the structural features needed to sta-
bilize anionic species and support hydrogen donation (trans-enone
group) and also lack features linked to toxicity (no hydroxyl groups
in C-4). Further characterization of fraction GF04 would be nec-
essary to determine the chemical nature of the molecules therein
contained, since they successfully inhibited lipid peroxidation.
Acknowledgment
This research was possible thanks to the research funds from
the CONACYT-TAMU Research Initiative and from the Tecnológico
de Monterrey-Research Chair Initiatives on Micronutrientes (CAT-
198) and Cardiology (CAT-131). As well as CONACYT-México grants
133591 (G. García-Rivas) and the scholarship provided by CONACYT
for author D.G. Rodríguez-Sánchez (228072). We also would like
to thank Avomex Intl., S.A. de C.V. (Sabinas, Coahuila, México) for
providing avocado fruits.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.jchromb.2013.10.013.
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