Food Research International 44 (2011) 3087–3093

Contents lists available at SciVerse ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Bio-guided fractionation of the antimutagenic activity of methanolic extract from the

fruit of Randia echinocarpa (Sessé et Mociño) against 1-nitropyrene
M.C. Cano-Campos a, b, S.P. Díaz-Camacho a, c, M.J. Uribe-Beltrán c, G. López-Angulo b, J. Montes-Avila b,
O. Paredes-López c, d, F. Delgado-Vargas a, b,⁎
a
Universidad Autónoma de Sinaloa, Facultad de Ciencias Químico Biológicas, Programa Regional del Noroeste para el Doctorado en Biotecnología, Boulevard de las Américas s/n,
Culiacán, Sinaloa, CP 80010, Mexico
b
Universidad Autónoma de Sinaloa, Facultad de Ciencias Químico Biológicas, Maestría en Ciencia y Tecnología de Alimentos, Boulevard de las Américas s/n, Culiacán, Sinaloa,
CP 80010, Mexico
c
Universidad Autónoma de Sinaloa, Facultad de Ciencias Químico Biológicas, Maestría en Ciencias Biomédicas, Boulevard de las Américas s/n, Culiacán, Sinaloa, CP 80010, Mexico
d
Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, CP 36821, Mexico

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

Article history: Randia echinocarpa is a native plant from Mexico that produces an edible fruit with several ethnopharmaco-
Received 4 April 2011 logical uses (e.g. cancer, kidney ailments, and diabetes). Extracts of this fruit have shown antimutagenic ac-
Accepted 10 August 2011 tivity. In this report, a methanolic extract of R. echinocarpa and a bio-guided chromatographic strategy
were used to obtain an hexanic fraction (HF) with strong antimutagenic activity (microsuspension assay
Keywords:
with Salmonella typhimurium YG1024) using 1-nitropyrene as mutagen (1-NP, 50 and 100 ng/tube). The HF
Antimutagenic activity
Randia echinocarpa fruit
(500 ng/tube) showed the highest inhibition percentage of mutagenic activity (PI) (75%, 1-NP 50 ng/tube;
Linoleic acid 84%, 1-NP 100 ng/tube). HF chromatography with silica produced HF1 which was further separated to pro-
Palmitic acid duce the fractions with the highest antimutagenic activities (HF1–1 and HF1–2, PI ≥ 60%). These fractions
β-sitosterol were chemically characterized by chromatography and gas chromatography–mass spectrometry; among
Functional food the main components of HF, HF1–1 and HF1–2 were registered linoleic acid, palmitic acid and β-sitosterol,
which could be responsible for the antimutagenic activity of R. echinocarpa fruit. The samples evaluated
were neither toxic nor mutagenic. Randia echinocarpa is an underutilized plant and its fruit has been used tra-
ditionally as food/medicine; fruit consumption could provide human health benefits and it has potential to be
exploited under conditions of ecological sustainability.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction Randia is considered as a Neotropical genus with 60 to 70 species;

Nowadays, cancer disease represents one of the most important
public health concerns. This illness of multifactor etiology can affect
every organ and system in the body, and the symptomatology and
treatment responses are highly variable. Consequently, early detection
and prevention are the best strategies for disease control and new ther-
apeutic/preventive agents are needed (Thun, DeLancey, Center, Jemal, &
Ward, 2010).
Plants are the main source of bioactive compounds (Cragg,
Grothaus, & Newman, 2009), especially of anticancer compounds.
Mexico has a large floristic diversity but it is practically unknown
(SEMARNAT, 2008).
⁎ Corresponding author at: Universidad Autónoma de Sinaloa, Facultad de Ciencias
Químico Biológicas, Programa Regional del Noroeste para el Doctorado en Biotecnolo-
gía, Boulevard de las Américas s/n, Culiacán, Sinaloa, CP 80010, México. Tel./fax: + 52
667 713 6615.
E-mail addresses: fdelgado@uas.uasnet.mx, fcodelgadovargas@gmail.com
(F. Delgado-Vargas).
it is distributed from Southern United States to South America and
Mexico represents the area of the highest diversity with 33 species
(Bye et al., 1991; Lorence & Nee, 1987).
Randia echinocarpa Sessé et Mociño (Rubiaceae) is a shrub or
small tree of 2–6 m high, distributed along the Pacific Ocean Shore
of Mexico. The fruit of R. echinocarpa is known as “papache”. The
pulp of the ripe fruit is dark, with a large number of rounded seeds
(see Fig. S1 as supplementary data), the isolated compounds from
the fruit of R. echinocarpa are mannitol, β-sitosterol, and quinovic,
oxoquinovic, ursolic and oleanolic acids (Bye et al., 1991). R. echino-
carpa fruit has a sweet/bitter flavor, it is mainly consumed by people
of rural areas as food/medicine, and only commercialized in Mexican
traditional markets (Cortés, 2000).
R. echinocarpa has been employed in Mexican traditional remedies
for cancer, malaria and diabetes; as well as for kidney, pulmonary, circu-
latory and gastro-intestinal ailments (Bye et al., 1991; Cortés, 2000).
These ethnopharmacological properties have not been scientifically
confirmed. On the other hand, a positive effect has been experimentally
demonstrated for an aqueous extract of the fruit, it improves the

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.08.006
3088 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093
cicatrizing speed in skin of wounded rats (Pérez, Pérez, Pérez-González,
& Vargas, 1993); negative effects are also registered for the oral admin-
istration of this extract in rats, increased diuretic activity and urolith for-
mation (Vargas Solis & Pérez Gutiérrez, 2002). In a previous report, an
aqueous extract of R. echinocarpa fruit (500 μg/tube) showed a moder-
ate antimutagenic activity in the microsuspension assay with YG1024
strain, PI of 32 and 53% at 50 and 100 ng/tube of 1-NP, respectively; re-
markably, the extract acts by desmutagenic and bioantimutagenic
mechanisms (Santos-Cervantes, Ibarra-Zazueta, Loarca-Pina, Paredes-
Lopez, & Delgado-Vargas, 2007).
R. echinocarpa plants were widely distributed along the State of Si-
naloa and nowadays their populations have been diminished. Based
on the traditional uses of R. echinocarpa, it could be considered an
underutilized plant and an improved knowledge about its chemical
composition and biological activities will contribute to use this natu-
ral resource under conditions of ecological sustainability (Jaenicke &
Höschle-Zeledon, 2006).
The objective of this study was to characterize the responsible
compounds of the antimutagenicity in the fraction with the highest
activity obtained from the crude methanolic extract of R. echinocarpa
fruit (ME).
2. Materials and methods
2.1. Plant material
The R. echinocarpa fruits were collected from the municipality of
Badiraguato, Sinaloa, México. The plant material was identified and
a voucher specimen (var 9035) was deposited in the herbarium of
the Faculty of Agronomy, Autonomous University of Sinaloa.
Seeds were eliminated and fruit pulp was freeze dried (VirTis 25
EL, VirTis Co., Gardiner, NY). Dried pulp, highly hygroscopic, was
crushed by hand and immediately frozen at −80 °C until required.
2.2. Chemical reagents
Silica gel 60 (particle size 0.035–0.070 mm, 220–440 mesh, Fluka
Chemika), 1-nitropyrene (1-NP), dimethylsulfoxide (DMSO), and
chemical standards (sterols mix, β-sitosterol, palmitic acid and lino-
leic acid) were obtained from Sigma Chemical Co. (St. Louis, MO).
Stock solutions of 1-NP were prepared in DMSO at concentrations of
2.5, 5, 7.5, 10, 15 and 20 ng/μL and stored at 4 °C. All reactive grade or-
ganic solvents (methanol, MeOH; hexane; chloroform, CHCl3; and
ethyl acetate, EtOAc) were obtained from Merck (Edo. de Mexico,
Mexico) and were distilled before their use.
2.3. Mutagenicity and antimutagenicity testing
The microsuspension assay of the Ames test was used (Kado,
Langley, & Eisenstadt, 1983). Salmonella typhimurium YG1024 was
the tester strain and 1-NP the mutagen. Toxicity and mutagenicity
was evaluated for all the fractions obtained (i.e. ME, HF, CF, EAF,
AqF, HF1, HF2, HF3, HF4, HF5, HF1–1 and HF1–2; see Section 2.4 for de-
scription) and those resulting negative were analyzed for their
antimutagenicity.
Tester strain YG1024 was grown overnight in Oxoid Nutrient Broth
No. 2 (Oxoid Ltd, Hants, UK) to approximately 1–2 × 10 9 cells mL−1
and harvested by centrifugation (3000 ×g, 4 °C, 10 min). Bacteria cells
(1× 1010 cells/mL) were resuspended in ice-cold PBS (0.15 M, pH 7.4)
after which ingredients were added in the following order to
10× 100 mm sterile glass culture tubes kept on ice: 0.1 mL cocktail,
0.1 mL of bacteria (1× 1010 cells/mL PBS), 0.01 mL 1-NP (25, 50, 75,
100, 150 and 200 ng/tube) or 0.005 mL of the evaluated samples (100,
200, 300 and 500 μg/tube) and 0.005 mL 1-NP (50 and 100 ng/tube).
The mixture was incubated in the dark at 37 °C with vigorous shaking
(90 min). After 90 min, the tubes were placed in an ice bath. Tubes
were removed one at time, and 2 mL of molten top agar containing
90 nmol histidine and biotin were added (Kado et al., 1983). The com-
bined solutions were vortex mixed and poured onto minimal glucose
plates. Plates were incubated at 37 °C in the dark for 48 h and the colo-
nies were counted (SOL-BAT Model Q-20, SOL-BAT Co., Puebla, Mexico).
Samples were tested in triplicate for two independent experiments.
Strain markers and bacterial survival were routinely monitored for
each experiment. The mutagen and all samples were dissolved in
DMSO but the AqF was dissolved in deionized water. Samples were
sterilized by filtration (0.22 μm). Mutagenic index (MI) was calculated
as the ratio of induced revertants by the evaluated substance to sponta-
neous revertants. The evaluated sample is considered mutagenic if
MI ≥ 2 and cytotoxic if MI ≤ 0.6 (Fernandes & Vargas, 2003; Maron &
Ames, 1983; Moreira-Rosa et al., 2006).
Antimutagenic activity was calculated as inhibition percentage of


mutagenic activity (PI): PI = 1−y1 =y2  100; where, y1 = number
of revertants per plate in the presence of the evaluated substance
(antimutagen), and y2 = number of revertants per plate in the ab-
sence of the evaluated substance (antimutagen).
Antimutagenicity was classified according to the PI values: 0–20%
negative, 20–40% weak, 40–60% positive, 60–90% strong, N90% sus-
pected toxicity (Fernandes & Vargas, 2003; Moreira-Rosa et al.,
2006; Wall, Wani, Hughes, & Taylor, 1988).
2.4. Preparation of the methanolic extract and its fractions
2.4.1. Preparation of the crude methanolic extract of R. echinocarpa fruit
The crude methanolic extract of R. echinocarpa fruit (ME) was
obtained as described by Wall et al. (1996) (Fig. 1). Dried fruits
(200 g) were extracted with MeOH (1:5 w/v) by stirring at room tem-
perature (30 °C); the extraction was carried out during 3 days, the
suspension was filtered daily, the liquid phase recovered and the res-
idue was re-extracted with fresh solvent; the liquid phases were
pooled and concentrated by rotary evaporation (40 °C) to obtain ME.
2.4.2. Liquid–liquid partition of ME
The ME was fractionated by liquid–liquid partition (Wall et al.,
1996) (Fig. 1). Fractions in hexane, CHCl3 and EtOAc were concentrat-
ed by rotary evaporation (40 °C) and aqueous fraction by freeze dry-
ing; the fractions obtained (hexanic, HF; chloroformic, CF; ethyl
acetate, EAF; and aqueous, AqF) were stored at − 20 °C and protected
from light until used.
2.4.3. Silica gel chromatography of the HF
HF (5 g) was dissolved in hexane and loaded onto a silica gel 60
(122 g) column (12 × 6 cm i.d.). HF was eluted using the following sol-
vents: hexane (650 mL), hexane/CHCl3 (1:1 v/v, 200 mL), hexane/
EtOAc (6:4 v/v, 450 mL), EtOAc (250 mL) and EtOAc/MeOH (9:1 v/v,
300 mL). Thirty seven fractions (50 mL each) were collected and ana-
lyzed by thin layer chromatography (TLC); those with similar mobility
patterns were pooled and concentrated to get five fractions (HF1–HF5,
yielding 0.091, 0.255, 0.091, 0.0179 and 0.0376% d.w., respectively).
HF1 (0.5 g) was further fractionated by column chromatography
(silica gel 60, 14.83 g; 13 × 2 cm i.d.), eluting with hexane (50 mL)
and hexane/MeOH (9:1 v/v, 50 mL). After TLC analyses, two fractions
were obtained HF1–1 (0.072% d.w.) and HF1–2 (0.018% d.w.).
2.5. Phytochemical screening and thin layer chromatography (TLC)
ME, HF, CF, EAF and AqF were analyzed for the presence of main
chemical groups (i.e. alkaloids, coumarins, tannins, flavonoids, sapo-
nins, sterols and/or triterpenes) by using standard phytochemical
techniques (Domínguez, 1998).
Column fractions were analyzed by TLC on precoated silica gel 60
F254 plates (Sigma Chemical Co.) and the bands were observed under
UV-light or developed with Ce(SO4)2. Samples with fatty acids (i.e. HF
 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093 3089

Dried fruit of papache

200 g

Methanol (1 L)

Methanolic extract (ME) 1

Residue 161.34 g
(80.64%)*
MeOH:H2O:Hexane (0.9:0.1:1 v/v)

Methanolic phase
Hexanic fraction
(HF)1
CHCl3 :H2O(1:1 v/v)
1.24 g
(0.62%)

Chloroformic phase Aqueous phase

EtOAc (1:1 v/v)

Aq NaCl 10% (1:1 v/v)

Chloroformic fraction Aqueous fraction Ethyl acetate fraction

(AqF)2
(CF)1 4.24 g 152.80 g (EAF)1 3.06 g
(2.12%) (76.40%) (1.53%)

Fig. 1. Diagram shows the preparation of the crude methanolic extract of R. echinocarpa fruit (ME), as well as the liquid–liquid partition strategy to obtain the organic (HF, CF, EAF)
and aqueous (AqF) fractions. Sample concentration was carried out by rotary evaporation1 (40 °C) or freeze drying2. ME was dissolved in methanol:water (9:1 v/v, 100 mL) to start
the partition; methanolic phase was concentrated to eliminate methanol and residue was dissolved in water (100 mL) to continue the liquid–liquid fractionation. *Percent yield on
dry weight basis is indicated in parentheses.

and its chromatographic subfractions) were methylated using tri-
methylsilildiazomethane (Norio, Toyohiro, & Takayuki, 1981). The
presence of sterols in the HF and its chromatographic subfractions
was confirmed by the Liebermann–Burchard test (Domínguez,
1998) and of β-sitosterol by that described by Fierro, Vásquez,
Reyes, and Sepúlveda (2004); β-sitosterol shows a purple light color
band (Rf = 0.5) by TLC using a two-solvents system, first with
CHCl3:CH3OH:H2O (10:1:0.05 v/v) and then with CHCl3.
2.6. Gas chromatography–mass spectrometry (GC/MS) analysis
HF, HF1–HF4, HF1–1 and HF1–2 were analyzed by GC/MS with a HP
6890 GC instrument equipped with a 5973 mass spectrometer (Agilent
Technologies, Wilmington, NC). The sample was separated in a fused
silica capillary column (Quadrex-series-007, 30 m × 0.25 mm
i.d. × 0.25 μm, QUADREX CORP., Woodbridge, CT), with He as carrier
gas (flow 0.6 mL/min). Temperature conditions were as follows: injec-
tor port 250 °C; and oven temperature 60 °C for 1 min, 3 °C/min to
160 °C, 160 °C for 1 min, 15 °C/min to 240 °C, 240 °C for 15 min,
10 °C/min to 290 °C, and 290 °C for 15 min. The sample (5 μL) was
injected in splitless mode. The MS was operated in the Electron Impact
(EI) ionization mode with an electron energy of 70 eV. The ion source
and quadrupole temperatures were maintained at 280 and 150 °C, re-
spectively. Compounds were identified using the libraries AMDIS 32
In agreement with the dose–response curve for the 1-NP mutage-
nicity against YG1024, doses of 50 and 100 ng/tube of 1-NP were cho-
sen for the antimutagenicity assays (Fig. 2). 1-NP was not toxic in the
range of concentrations tested; a uniform lawn of bacteria (micro-
colonies) was observed in every plate of the mutagenic assay and
the number of revertants never was lower than that of the spontane-
ous revertants.
The ME was neither toxic nor mutagenic at 1000 μg/tube against
the YG1024 strain, the number of revertants in the range of tested
concentrations was similar to that of the negative control (evalua-
tions without extract/fraction); the same behavior was registered
for most of the R. echinocarpa fractions (i.e. HF, CF, EAF, AqF, HF1,
HF2, HF3, HF4, HF5 and HF1–1) up to 500 μg/tube but HF1–2 up to
300 μg/tube (see Fig. S2 as supplementary data).
The ME did not show antimutagenic effect in the range of 0 to
500 μg/tube and the effect was weak in the range 600–1000 μg/tube
(Table 1). On the other hand, the antimutagenicity of R. echinocarpa
fractions (i.e. HF, CF and EAF) was dose-dependent up to 500 μg/tube.
The highest PI was registered for the HF with 75% (50 ng/tube 1-NP)
and 84% (100 ng/tube 1-NP) (Table 2). At the highest concentrations,
800
700
Number of revertants/plate

(v. 2006, Agilent) and NIST98 (Wiley, V.A.O7.01); the identity of the 600
main components was corroborated by using the retention times and
500
mass spectra of chemical standards processed as the samples studied.
Analyses were carried out by duplicate. 400

2.7. Statistical analysis 300

200
Statistical differences (P b 0.05) were established by the Dunnett's
test using Minitab (1998 v. 13.1). Graphs were designed with Sigma 100
plot (2001 v. 7.0).
0
0 20 40 60 80 100 120 140 160 180 200
3. Results
1-NP concentration (ng/tube)
The yield for the ME was high (80.67%) and liquid–liquid partition Fig. 2. Dose–response curve of 1-nitropyrene (1-NP) mutagenicity in YG1024. Each
produced a higher yield of polar (AqF, 76.40%) than non polar frac- data point represents the average of three independent experiments. The number of
tions (Fig. 1). spontaneous revertants/plate was 18 ± 7.
3090 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093

100
Table 1 A

Percentage of inhibition(PI)
Effect of the crude methanolic extract (ME), obtained from the edible portion of R. echi- 90
nocarpa fruit, against the mutagenicity of 1-NP to Salmonella typhimurium YG1024. a
80
b Strong
ME dose (μg/tube) Revertants/plate Inhibition (%) 70
0 368 ± 55c 0 60
100 347 ± 50c 6 50 Positive
200 321 ± 78c 13
300 308 ± 75c 16 40
500 300 ± 73c 18 30 Weak
600 271 ± 48d 26
20
800 252 ± 42d 31 HF1
10 HF4 Negative
1000 250 ± 42d 32 HF2
HF3 HF5
a
The mutagen (1-NP) was used at 100 ng/tube. Spontaneous reversion: 20 ± 0
0 100 200 300 400 500
6 revertants/plate. 1-NP stands for 1-nitropyrene.
b
Results represent the average of two independent experiments ± SD, each experi- Concentration of fraction (µg/tube)
ment was done per triplicate. Different letters in the second column show significant
differences (ρ b 0.05). 100 B

Percentage of inhibition (PI)

90
80
Strong
70
CF and EAF showed PI values in the range 40–60 (Table 2). By contrast,
the AqF showed a co-mutagenic effect (data not shown). Thus, HF was 60
the most potent fraction and was chosen to continue with the bio- 50 Positive
guided fractionation. 40
The antimutagenic effect of the silica chromatographic fractions of 30 Weak
the HF (HF1, HF2, HF3, HF4, HF5, HF1–1 and HF1–2) was dose- 20
dependent; and PI, evaluated at 50 and 100 ng/tube 1-NP, was the HF1
HF4
10 HF2 Negative
highest for HF1 (83% and 87%), HF4 (76% and 81%) and HF1–2 (80% HF3
HF5
0
and 95%) (Fig. 3). HF1–2 resulted in the strongest antimutagenic effect
0 100 200 300 400 500
since the registered values were for 300 μg/tube (Fig. 3C).
Concentration of fraction (µg/tube)
The phytochemical analysis for ME showed a high presence of sa-
ponins; the non-polar fractions of ME (HF and CF) of saponins and 100 C
terpenes/sterols, whereas the AqF was depleted in these components
Percentage of inhibition (PI)

90
but enriched in free anthracenic derivatives (Table 3). EAF showed 80
slight presence of coumarins, flavonoids, tannins and anthracenic de- Strong
70
rivatives. The fractions of HF (HF1, HF2, HF3, HF4, HF5 and HF1–2) were
60
rich in sterols and it was corroborated by the Liebermann–Burchard
50 Positive
test (bluish green color).
Thin layer chromatography of the HF showed the presence of 40
three main bands with similar intensity: sterols (Rf = 0.47), unknown 30 Weak
(Rf = 0.87) and fatty acids (Rf = 0.97); in the chosen mobile phase 20
palmitic and linoleic methyl esters showed similar mobility. The HF1-1 HF1-1
10 Negative
HF1-2 HF1-2
0
0 100 200 300 400 500
Table 2 Concentration of fraction (µg/tube)
Effect of fractions of the crude methanolic extract (ME) of Randia echinocarpa fruit
against the mutagenicity of 1-NP to Salmonella typhimurium YG1024.a
Fig. 3. Antimutagenic activity of the fractions obtained by column chromatography in
Fraction 1-NP, 50 ng/tube 1-NP, 100 ng/tube silica gel: HF1–HF5 from HF using (A) 1-NP 50 ng/tube; (B) 1-NP 100 ng/tube; and
(C) HF1–1 and HF1–2 from HF1 using 1-NP at 50 ng/tube (circles) and 100 ng/tube
Name Dose Revertants/platec Inhibition Revertants/platec Inhibition (squares). Samples can be classified as negative, weak, positive or strong based on
(μg/tube) (%) (%) the antimutagenic effect. Data are the average of two experiments by triplicate, bars
HF 0b 203 ± 60d 372 ± 66d show the standard deviation. Spontaneous reversion was 23 ± 6 revertants/plate. HF
100 77 ± 15e 62 90 ± 26e 76 stands for hexanic fraction; and 1-NP for 1-nitropyrene.
200 63 ± 12e 69 100 ± 32e 73
300 53 ± 11e 74 100 ± 22e 73
500 51 ± 8e 75 61 ± 17f 84
CF 0b 199 ± 29d 384 ± 69d HF1–1 presented mainly sterols (Rf = 0.47) and fatty acids in minor
100 136 ± 32d,e 32 294 ± 54d,e 24 proportion (Rf = 0.97) whereas in the HF1–2 only fatty acids were ob-
200 101 ± 17e 49 170 ± 19e 56 served (Rf = 0.97); consequently, the fatty acids showed a higher
300 99 ± 24e 50 176 ± 20e 54 contribution to the antimutagenic activity of the HF (Fig. 3C). The oc-
500 100 ± 42e 50 207 ± 21e 46
currence of β-sitosterol as the main component of samples was con-
EAF 0b 173 ± 38d 323 ± 41d
100 146 ± 21d,e 16 238 ± 22e 26 firmed as described by Fierro et al. (2004).
200 116 ± 16e 33 209 ± 41e,f 35 The GC-MS analyses demonstrated that fatty acids and sterols
300 108 ± 15e,f 38 169 ± 25f 48 were the main components of the HF (Fig. 4), aldehydes were also
500 104 ± 15f 40 153 ± 28f 53
identified but must be minor components based on the TLC analyses;
a
The evaluated fractions of the crude methanolic extract were the hexanic (HF), the samples separated by TLC and stained with cerium sulfate, a general
chloroformic (CF) and the ethyl acetate fraction (EAF). Spontaneous reversion: 20 ± 6 stain, did not show any other major compound. The main compo-
revertants / plate.
b
Results represent the average of two independent experiments ± SD. The muta-
nents of the HF were palmitic acid, linoleic acid, β-sitosterol, campes-
genicity control, induced revertants by 1-NP. terol, and stigmasterol (Fig. 4A). Data are not presented for HF1–HF5
c
Different letters in the same column/fraction show significant differences(ρ b 0.05). fractions, but the same fatty acids and sterols were found in the
 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093
Table 3
Phytochemical screening of fractions obtained from the edible portion of Randia echinocarpa fruit.a
Fractions Alkaloids Coumarins Flavonoids Tannins
ME − + + +
HF − + − −
CF − − + +
EAF − + + +
AqF − − + +
a
ME, crude methanolic extract; HF, hexanic fraction; CF, chloroformic fraction; EAF, fraction in ethyl acetate; AqF, aqueous fraction. The relative quantity of metabolite is estab-
lished as abundant (+++), moderate (++), poor presence (+) and complete absence (−).
HF1; sterols (mainly β-sitosterol) in HF2 and HF3; and palmitic and
linoleic acids in HF4; GC-MS analysis of HF5 was not carried out due
to its polarity. The HF1–1 was enriched in sterols (i.e. campesterol
and β-sitosterol) with minor contribution of fatty acids (Fig. 4B);
fatty acids (i.e. palmitic and linoleic) were the main components of
HF1–2 with low concentration of sterols (Fig. 4C).
4. Discussion
In a previous report, we established the antioxidant and antimuta-
genic activity of an aqueous extract of R. echinocarpa fruit, the anti-
mutagenic PI at 500 μg/tube was 32% (50 ng/tube 1-NP) and 53%
(100 ng/tube 1-NP) (Santos-Cervantes et al., 2007). These values
were lower than the registered in this study for the HF, 75% and
84%, respectively. In addition, CF and EAF were positive antimutagens
(Table 2) with values similar to those reported for the aqueous ex-
tract of papache fruit (Santos-Cervantes et al., 2007).
The antimutagenic activity of plant products (extracts or com-
pounds) has been evaluated using YG1024 as tester strain and 1-NP
as mutagen and these data can be contrasted with our results. A hot
aqueous extract (HAE 10 mg/mL, as procatechuic acid equivalents) is
a better antimutagen than other extracts of Hibiscus sabdariffa (1-NP
0.1 μg/tube, PI 74%) (Olvera Garcia et al., 2008). Ellagic acid (EA,
300 μg/tube) shows antimutagenic characteristics (1-NP 100 ng/plate,
PI of 83%) (González-de-Mejía, Castaño-Tostado, & Loarca-Piña, 1999).
Phenolics and carbohydrates of Lupinus campestris seeds have shown
a dose–response antimutagenic effect, and at the highest concentration
tested the PI values were 86% (phenolics 200 μg/tube) and 76% (carbo-
hydrates 512 μg/tube) (Jimenez-Martínez, Loarca-Pina, & Davila-Ortiz,
2003). Our results showed a higher antimutagenicity for HF and
its sub-fractions (Table 2 and Fig. 3) than that reported for HAE of
H. sabdariffa and they were similar to those of pure EA and phenolics
and carbohydrates of L. campestris.
The most active fractions (HF, HF1, HF1–1 and HF1–2) obtained from
the edible portion of R. echinocarpa fruit contained palmitic acid, linoleic
acid, campesterol and β-sitosterol as the main components (Fig. 4); and
they could be responsible of the associated antimutagenicity (Fig. 3C).
The antimutagenic activity of these compounds has been previously
reported. Palmitic acid is the straight fatty acid of yogurt with a high ac-
tivity against the mutagen N-methyl-N′-nitro-N-nitroguanidine
(MNNG) (2 μM), with Salmonella typhimurium TA100 as tester strain;
palmitic acid (12.5–37.5 μg/plate) was the active antimutagen against
3,2′-dimethyl-4-aminobiphenyl (DMAB) (30 μM) (PI 19.1%–29.6%)
(Nadathur, Carney, Gould, & Bakalinsky, 1996). β-sitosterol-glucoside
and linoleic acid are active compounds in Doenjang (Korean fermented
soypaste) showing antimutagenic effects (Park, Jung, Rhee, & Choi,
2003). Linoleic acid (1.25 μg/plate) was a stronger antimutagen than
β-sitosterol-glucoside (1.25 μg/plate) against the mutagen aflatoxin
B1 (AFB1, 0.75 μg/plate) and MNNG (0.36 μg/plate) on S. typhimurium
TA100; the PI values were 80% (AFB1) and 60% (MNNG). Linoleic acid
(0.005–2.5 mg per plate) was neither toxic nor mutagenic on S. typhi-
murium TA100, with or without S9 activation (Park et al., 2003). The
antimutagenic activity of linoleic acid was also demonstrated by other
3091
Saponins Terpenes and/or sterols Free anthracenic derivatives
+++ + −
++ ++ −
+ ++ −
− − +
− − ++
assays (i.e. SOS chromotest and Drosophila wing spot) and with other
mutagens (Lim, Rhee & Park, 1997; Park et al., 2003).
A bio-guided strategy has been used to characterize a methanolic
extract of Gleditsia sinensis thorns as antimutagen, using the muta-
gens 4-nitro-o-fenilenediamina (NPD, 1 μg/plate) and sodium azide
(NaN3 1 μg/plate) and S. typhimurium TA98 (NPD) and TA100
(NaN3), without S9. The methanolic extract and fractions were evalu-
ated at 0.1 mg/plate. The inhibition rate for the methanolic extract
was 17.1% and fractionation produced a methylene chloride fraction
(MeChlF) that showed the highest inhibition rate (42.6% on TA98;
40.7% on TA100); MeChlF chromatography showed the presence of
sterols, including stigmasterol, the most abundant, and β-sitosterol;
stigmasterol and β-sitosterol, both at 10 μg/tube, were the most po-
tent antimutagens in the SOS chromotest using MNNG (1 μg/tube)
and 4-nitroquinoine-1-oxide (1 μg/tube) (Lim et al., 2005).
An aqueous extract of R. echinocarpa fruit showed desmutagenic
and bioantimutagenic activities on YG1024 using 1-NP as mutagen
(Santos-Cervantes et al., 2007). In this study, we have established
that the main components of the HF were fatty acids and sterols. It
has been proposed that the antimutagenic activity of fatty acids is
mediated by desmutagenic mechanisms. The anti-MNNG activity of
fatty acids could be associated with the entrapment of MNNG in
fatty acid micelles and with the modification of membrane perme-
ability which interfere with the cell uptake of mutagens (Lim et al.,
1997; Nadathur et al., 1996). On the other hand, it has been registered
that β-sitosterol is incorporated into cell membranes resulting in a
significant decrease of phospholipids and membrane fluidity; conse-
quently, membrane permeability is modified and it has been sug-
gested that cell metabolism is affected by changes in protein–
protein interactions and enzyme activities (A.B. Awad & Fink, 2000).
Palmitic acid, linoleic acid and β-sitosterol have shown antitu-
moral activities on different cancer cell lines (Awad, Bartal, Fink &
Bradford, 2008; Kong & Rabkin, 2002; Kwon, Kim, Park, Ryu, & Choi,
2008) and anticarcinogenic activities on mouse skin model (Guevara
et al., 1999; Yasukawa, Takido, Matsumoto, Takeuchi, & Nakagawa,
1991).
The consumption of exotic fruits is being promoted around the
world. These materials contain a large number of compounds with bi-
ological activity such as sterols, fatty acids, phenolics and pigments,
among others. Some exotic fruits (e.g. Vaccinium spp., Hylocereus
polyrhizus, Durio zibethinus, Annona muricata) have shown potential
to be used for cancer prevention/treatment (Dembitsky et al., 2011).
Antitumoral activity has been attributed to the fruit of Annona muri-
cata and different breeding strategies are being used in order to im-
prove this and other of its nutraceutical characteristics; in addition,
it has been proposed that such strategies could be used to improve
other underutilized exotic fruit crops (Rodríguez-Burruezo, Prohens,
& Fita, 2011).
Cancer begins with a mutation in a single cell, according with the
somatic mutation theory, which is passed to its progeny generating a
clone of malignant cells (Vaux, 2011). Moreover, in agreement with
the Ames test, it exists a positive association among carcinogenicity
and mutagenicity; thus antimutagenic compounds have been associ-
ated with prevention/treatment of cancer (Maron & Ames, 1983;
3092 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093

100 A 1. Palmitic acid a,b 9 traditionally used for cancer treatment (Bye et al., 1991), and the reg-
2. Linoleic acid a,b istered antimutagenicity could give support to this traditional use. In
3. Pentadecanal a
this report, we observed that HF1–2 was toxic for the YG1024 strain, at
4. Hexadecanal a
the highest evaluated concentration (500 ng/tube) (Fig. 3C). The use
5. Octadecanal a
75 of R. echinocarpa as a functional food by considering its antimutagenic
6. Squalene a
7. Campesterol a,b 7 activity must result of the combination of several compounds; the ac-
tivity was registered for several fractions, HF was the most active but
Abundance

8. Stigmasterol a,b 8
9. β -sitosterol a,b other fractions (i.e. CF and EAF) with higher yields were also antimu-
50 tagenic (Table 2, Fig. 1); thus, other compounds with this activity re-
1
main to be characterized from R. echinocarpa fruit.

5. Conclusions
25
2
3 The fruit of papache contains compounds with antimutagenic ac-
tivity. The antimutagenicity was highest in the non-polar fractions
4 5
(HF, HF1–1 and HF1–2) which were enriched with sterols (campesterol
0 and β-sitosterol, HF1–1) and fatty acids (palmitic and linoleic, HF1–2).
9.97 17.74 25.52 33.29 41.06 48.84 56.61 64.38 72.15
Time (min) Previous reports have shown antimutagenicity, antitumoral and
anticarcinogenic properties for these compounds; thus, papache anti-
100 B 3 mutagenicity could be associated with these sterols and fatty acids.
R. echinocarpa fruit has been traditionally used as food/medicine, it
contains compounds with in vitro antigenotoxic properties and it
may be considered as a nutraceutical fruit. The consumption of
75 R. echinocarpa fruit could be beneficial for human health and this
underutilized plant could be exploited under conditions of ecological
sustainability.
Abundance

Supplementary materials related to this article can be found on-

50 4 5 line at doi:10.1016/j.foodres.2011.08.006.

9
Abbreviations
1-NP 1-nitropyrene
25 7 AFB1 aflatoxin B1
AqF aqueous fractions
CF chloroformic fraction
1 2
DMAB 3,2′-dimethyl-4-aminobiphenyl
0 DMSO dimethylsulfoxide
9.97 17.74 25.52 33.29 41.06 48.84 56.61 64.38 72.15
EA ellagic acid
Time (min)
EAF ethyl acetate fraction
100 C 1 EtOAc ethyl acetate
HAE hot aqueous extract of Hibiscus sabdariffa
HF hexanic fraction
ME crude methanolic extract of papache
75 MeChlF methylene chloride fraction of the methanol extract of Gle-
ditsia sinensis thorns
MeOH methanol
Abundance

MI mutagenic index
6 MNNG N-methyl-N′-nitro-N-nitroguanidine
50
2
NPD 4-nitro-o-fenilenediamina
PI percentage of inhibition of mutagenic activity

25
3 Acknowledgments

7 9 Authors acknowledge the financial support from Consejo Nacional

0 de Ciencia y Tecnologia (CONACyT), Consejo Estatal de Ciencia y Tecno-
9.97 17.74 25.52 33.29 41.06 48.84 56.61 64.38 72.15
logia del Estado de Sinaloa (CECyT) and from Universidad Autónoma de
Time (min)
Sinaloa (PROFAPI); the identification of R. echinocarpa by Dr. Rito Vega-
Fig. 4. Gas chromatography–Mass spectrometry chromatogram of: (A) HF, (B) HF1–1 Aviña, Faculty of Agronomy, Universidad Autónoma de Sinaloa (UAS);
and (C) HF1–2. Numbered peaks were identified by using: athe libraries AMDIS 32 and technical assistance by Dr. José Ángel López-Valenzuela.
(v. 2006, Agilent) and NIST98 (Wiley, V.A.O7.01), and bauthentic standards. HF stands
for hexanic fraction.
References

Nobili et al., 2009). The antimutagenic activity of papache fruit was
previously demonstrated (Santos-Cervantes et al., 2007) and in this
study we have identified the main compounds in the most antimuta-
genic fraction of this fruit (hexanic fraction). R. echinocarpa has been
Awad, A. B., & Fink, C. S. (2000). Phytosterols as anticancer dietary components: Evi-
dence and mechanism of action. The Journal of Nutrition, 130, 2127–2130.
Awad, A. B., Bartal, S. L., Fink, C. S., & Bradford, P. G. (2008). Beta-sitosterol enhances
tamoxifen effectiveness on breast cancer cells by affecting ceramide metabolism.
Molecular Nutrition & Food Research, 52(4), 419–426.
 M.C. Cano-Campos et al. / Food Research International 44 (2011) 3087–3093
Bye, R., Linares, E., Mata, R., Albor, C., Castañeda, P. C., & Delgado, G. (1991). Ethnobo-
tanical and phytochemical investigation of Randia echinocarpa (Rubiaceae). Anales
del Instituto de Biología de la Universidad Nacional Autónoma de México, Serie Botán-
ica, 62(1), 87–106.
Cortés, F. (2000). Medicine, myths and magic the folk healers of a Mexican market.
Economy Botany, 54(4), 427–438.
Cragg, G. M., Grothaus, P. G., & Newman, D. J. (2009). Impact of natural products on de-
veloping new anti-cancer agents. Chemical Reviews, 109, 3012–3043.
Dembitsky, V. M., Poovarodom, S., Leontowicz, H., Leontowicz, M., Vearasilp, S., Trakhtenberg,
S., et al. (2011). The multiple nutrition properties of some exotic fruits: Biological activity
and active metabolites. Food Research International, 44, 1671–1701.
Domínguez, A. X. (1998). Métodos de investigación fitoquímica (4th. ed.). D.F., Mexico:
Limusa.
Fernandes, J. B. F., & Vargas, V. M. F. (2003). Mutagenic and antimutagenic potential of
the medicinal plants M. laevigata and C. xanthocarpa. Phytotherapy Research, 17(3),
269–273.
Fierro, A., Vásquez, Y., Reyes, P. M., & Sepúlveda, B. S. (2004). Quantitative determina-
tion of ß-sitosterol in dietary vegetables. Possible implications for its preventive
use in susceptible populations. Clínica y Ciencia, 2, 43–48.
González-de-Mejía, E., Castaño-Tostado, E., & Loarca-Piña, G. (1999). Antimutagenic ef-
fects of natural phenolic compounds in beans. Mutation Research, 441(1), 1–9.
Guevara, A. P., Vargas, C., Sakurai, H., Fujiwara, Y., Hashimoto, K., Maoka, T., et al. (1999). An
antitumor promoter from Moringa oleifera Lam. Mutation Research, 440(2), 181–188.
Jaenicke, H., & Höschle-Zeledon, I. (2006). Strategic framework for underutilized plant
species research and development, with special reference to Asia and the Pacific,
and to Sub-Saharan Africa. Rome, Italy: International Centre for Underutilised
Crops, Colombo, Sri Lanka and Global Facilitation Unit for Underutilized Species.
Jimenez-Martínez, C., Loarca-Pina, G., & Davila-Ortiz, G. (2003). Antimutagenic activity
of phenolic compounds, oligosaccharides and quinolizidinic alkaloids from Lupinus
campestris seeds. Food Additives and Contaminants, 20(10), 940–948.
Kado, N. Y., Langley, D., & Eisenstadt, E. (1983). A simple modification of the Salmonella
liquid-incubation assay. Increased sensitivity for detecting mutagens in human
urine. Mutation Research, 121(1), 25–32.
Kong, J. Y., & Rabkin, S. W. (2002). Lovastatin does not accentuate but is rather additive
to palmitate-induced apoptosis in cardiomyocytes. Prostaglandins, Leukotrienes,
and Essential Fatty Acids, 67(5), 293–302.
Kwon, J. I., Kim, G. Y., Park, K. Y., Ryu, C. H., & Choi, Y. H. (2008). Induction of apoptosis
by linoleic acid is associated with the modulation of Bcl-2 family and Fas/FasL sys-
tem and activation of caspases in AGS human gastric adenocarcinoma cells. Journal
of Medicinal Food, 11(1), 1–8.
Lim, S. Y., Rhee, S. H., & Park, K. Y. (1997). Antimutagenic effects of linoleic acid. Journal
of Food Science and Nutrition, 2(1), 29–34.
Lim, J. C., Park, J. H., Budesinsky, M., Kasal, A., Han, Y. H., Koo, B. S., et al. (2005). Anti-
mutagenic constituents from the thorns of Gleditsia sinensis. Chemical & Pharma-
ceutical Bulletin, 53(5), 561–564.
Lorence, D. H., & Nee, M. (1987). Randia retroflexa (Rubiaceae), a new species from
Southern Mexico. Brittonia, 39(3), 371–375.
Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity
test. Mutation Research, 113(3–4), 173–215.
Moreira-Rosa, R., Melecchi, M. I. S., Da-Costa-Halmenschlager, R., Abad, F. C., Rosat-
Simoni, C., Carmao, E. B., et al. (2006). Antioxidant and antimutagenic properties
3093
of Hibiscus tiliaceus L. methanolic extract. Journal of Agricultural and Food Chemistry,
54, 7324–7330.
Nadathur, S. R., Carney, J. R., Gould, S. J., & Bakalinsky, A. T. (1996). Palmitic acid is the
major fatty acid responsible for significant anti-N-methyl-N′-nitro-N-nitrosogua-
nidine (MNNG) activity in yogurt. Mutation Research, 359(3), 179–189.
Nobili, S., Lippi, D., Witort, E., Donnini, M., Bausi, L., Mini, E., et al. (2009). Natural com-
pounds for cancer treatment and prevention. Pharmacological Research, 59,
365–378.
Norio, H., Toyohiro, A., & Takayuki, S. (1981). New methods and reagents in organic
synthesis. 14. A simple efficient preparation of methyl esters with trimethylsilyl-
diazomethane (TMSCHN2) and its application to gas chromatographic analysis of
fatty acids. Chemical & Pharmaceutical Bulletin, 29(5), 1475–1478.
Olvera Garcia, V., Castaño Tostado, E., Rezendiz Lopez, R. I., Reynoso Camacho, R., González
de Mejia, E., Elizondo, G., et al. (2008). Hibiscus sabdariffa L. extracts inhibit the
mutagenicity in microsuspension assay and the proliferation of HeLa cells. Jour-
nal of Food Science, 73(5), T75–T81.
Park, K. Y., Jung, K. O., Rhee, S. H., & Choi, Y. H. (2003). Antimutagenic effects of doen-
jang (Korean fermented soypaste) and its active compounds. Mutation Research,
523, 43–53.
Pérez, S., Pérez, R. M., Pérez-González, C., & Vargas, R. (1993). Cicatrizing activity of
Randia echinocarpa in gastric ulcers. Phyton, 54(2), 157–162.
Rodríguez-Burruezo, A., Prohens, J., & Fita, A. M. (2011). Breeding strategies for im-
proving the performance and fruit quality of the pepino (Solanum muricatum): A
model for the enhancement of underutilized exotic fruits. Food Research Interna-
tional, 44, 1927–1935.
Santos-Cervantes, M. E., Ibarra-Zazueta, M. E., Loarca-Pina, G., Paredes-Lopez, O., & Delgado-
Vargas, F. (2007). Antioxidant and antimutagenic activities of Randia echinocarpa fruit.
Plant Foods for Human Nutrition, 62(2), 71–77.
SEMARNAT (2008). Informe de la Situación del Medio Ambiente en México. In D. G. d. E. e. I.
Ambiental (Ed.), Compendio de Estadísticas Ambientales (Edición 2008). México, D.F.:
Secretaría del Medio Ambiente y Recursos Naturales.
Thun, M. J., DeLancey, J. O., Center, M. M., Jemal, A., & Ward, E. M. (2010). The global
burden of cancer: Priorities for prevention. Carcinogenesis, 31(1), 100–110.
Vargas Solis, R., & Pérez Gutiérrez, R. M. (2002). Diuretic and urolithiatic activities of
the aqueous extract of the fruit of Randia echinocarpa on rats. Journal of Ethnophar-
macology, 83, 145–147.
Vaux, D. L. (2011). In defense of the somatic mutation theory of cancer. BioEssays, 33,
341–343.
Wall, M. E., Wani, M. C., Hughes, T. J., & Taylor, H. (1988). Plant antimutagenic agents, 1.
General bioassay and isolation procedures. Journal of Natural Products, 51(5),
866–873.
Wall, M. E., Wani, M. C., Brown, D. M., Fullas, F., Olwald, J. B., Josephson, F. F., et al.
(1996). Effect of tannins on screening of plant extracts for enzyme inhibitory activ-
ity and techniques for their removal. Phytomedicine, 3(3), 281–285.
Yasukawa, K., Takido, M., Matsumoto, T., Takeuchi, M., & Nakagawa, S. (1991). Sterol
and triterpene derivatives from plants inhibit the effects of a tumor promoter,
and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-
stage carcinogenesis. Oncology, 48, 72–76.