Volume 84, Issue 4, December 2013, Pages 1298–1304
Open Access
Antioxidant compounds in hawthorn fruits (Crataegus spp.) of Mexico
Compuestos antioxidantes en frutos de tejocote (Crataegus spp.) de México
- Open Access funded by Universidad Nacional Autónoma de México, Instituto de Biología
- Under a Creative Commons license
Abstract
The content of phytochemicals associated with the antioxidant activity of the fruits of species of hawthorn (Crataegus
spp.; Rosacea) located in Mexico is unknown. The objective of the
present study was to evaluate the content of phenolic compounds,
flavonoids, vitamin C and the antioxidant activity in a selection of
Mexican hawthorn species. A quantification was made of total phenols,
flavonoids and vitamin C (expressed on mg of phenol, quercetin and
ascorbic acid per 100 g of fresh weight, respectively), in 10 g of
fruits selected from each genotype; a total of 20 genotypes were
sampled, these located in the germplasm bank of the Universidad Autónoma
Chapingo, Mexico. The antioxidant activity was evaluated by the DPPH
method, expressed as mean inhibitory concentration (IC50).
Content of total phenols, flavonoids and vitamin C cannot be associated
with the origin and species of the samples. Some genotypes from the
state of Chiapas could be considered to have a higher potential for
commercial use and consumption due to their nutraceutical quality. Most
of the fruits of the 20 genotypes of hawthorn presented a content of
phenolic compounds higher than that described for other fruits (lychee
fruits, peaches and strawberries); these nutraceutical characteristics
provide an added value to the fruit.
Resumen
El contenido de fitoquímicos asociado con la actividad antioxidante de los frutos de especies de tejocote (Crataegus
spp.; Rosacea) localizadas en México es desconocido. El objetivo del
presente estudio fue evaluar el contenido de compuestos fenólicos,
flavonoides, vitamina C y la actividad antioxidante, en una selección de
especies de tejocote mexicano. La cuantificación de fenoles totales,
flavonoides y vitamina C (expresada en mg de ácido gálico, quercetina y
ácido ascórbico por 100 gr de peso fresco, respectivamente) en 10 gr de
frutos seleccionados de cada genotipo, se muestrearon 20 genotipos
localizados en el banco de germoplasma de la Universidad Autónoma
Chapingo, México. La actividad antioxidante se evaluó de acuerdo al
método del DPPH y se expresó como concentración media inhibitoria (CI50).
No se observó una relación del contenido de fenólicos, flavonoides y
vitamina C con el origen y la especie de las muestras. Algunos genotipos
del estado de Chiapas por su calidad nutracéutica podrían ser
consideradas para uso comercial y consumo. La mayoría de los 20
genotipos de tejocote presentaron un contenido de compuestos fenólicos
más alto que el descrito para otros frutos (lichi, durazno y fresa),
estas características proporcionan un valor agregado a la fruta.
Key words
- Mexican hawthorn species;
- antioxidants;
- phenolic compounds;
- flavonoids;
- vitamin C
Palabras clave
- especies de tejocote mexicanas;
- antioxidantes;
- compuestos fenólicos;
- flavonoides;
- vitamina C
Introduction
Recently
in Mexico there has been a growing interest in knowledge and management
of underutilized fruits, also known as minor, secondary or alternative
fruits, such as the case of hawthorn fruits (Crataegus spp.) ( Nieto-Ángel, 2007). There are descriptions of 150-200 species of this genus (Crataegus) in the world, approximately 13 have been identified in the north and center of Mexico, Phipps (1997) indicates 9 endemic species ( Phipps, 1997 and Phipps, 1998). Nieto-Ángel (2007)
mentions that there is great diversity and variability in its genotypic
and phenotypic characteristics. Hawthorn belongs to the Rosacea family,
is located principally in cold and temperate climates ( Nieto-Ángel, 2007). Tejocote is the most widely used term and comes from the nahuatl language, in which tetl-xocotl means wild or hard sour fruit; the Nahoas (ancestors of the mexicas) referred to them as texococuahutl, which means the tree of Indian apple ( Martínez, 1994) depending on the region where it is located, the tejocote has adopted different common names ( Martínez, 1994).
In
Mexico it is mainly used as animal feed, ornamental plant; in
agro-industry it is used to make regional sweets, in the preparation of
punches and conserves because of its high pectin content; it has a high
demand mainly in the south-southeast-central region of Mexico in the
traditional festivals of “All Saints” because it is put on the table as
an offering and consumed as fruit (offerings and piñatas) (Nieto-Ángel, 2007). In traditional Mexican medicine the flowers, leaves, root and the fruits have numerous uses (Martínez, 1994; Ody, 1994).
There
are a number of medicinally active phytochemicals that have been
isolated from hawthorn plants with most of the data generated in studies
of those species that are native to Europe and Asia. Little is known
about the North American and Canadian (Edwards et al., 2012), and more specifically about the Mexican Crataegus species. One possible barrier to chemical studies of Crataegus, has been the perception that Mexican Crataegus are taxonomically problematic ( Phipps, 1997).
The high contents of phenolic compounds such as flavonoids,
proanthocyanidins, catechins, phenolic acids, essential oils and
terpenoids ( Bahorum et al., 1994; Edwards et al., 2012; García-Mateos et al., 2012)
explain their use as natural therapy for the treatment of
neurodegenerative diseases, in some types of cancer, in the affectation
of the immunological system and cardiovascular disorders ( Craig, 1999; Chang et al., 2002; Cui et al., 2006).
Hawthorn extracts exert a wide range of pharmaceutical properties,
especially on the cardiovascular system, including cardiotonic,
antiarrhythmic, hypotensive, hypolipidemic, and antioxidant activities (
Craig, 1999; Barceloux, 2008; Arrieta et al., 2010).
The
wide diversity and genotypic variability that exists in the Mexican
hawthorn species demands the characterization of its fruits and the
determination of its antioxidant properties to be recommended as a food
of high nutraceutical value that permits a more efficient
agro-industrial use and provides new economic alternatives for the
producer. The nutraceutical content in products (jellies, preserves and
candied fruit) made from the fruit is unknown. The objective of the
present study was to evaluate the content of phenolic compounds,
flavonoids, vitamin C and the antioxidant activity in a selection of
Mexican hawthorn species.
Materials and methods
Collection of plant material.
The present investigation was carried out with hawthorn fruits from the
central plateau and the south of Mexico located in the hawthorn
germplasm bank of the Universidad Autónoma Chapingo, located at 19°29'
N, 98°53' W, at 2 240 m ( García, 1981).
The climate is C(Wo) (w)b (I')g, rainy moderate temperate and the
driest of sub-humid climates, with rains in summer; the mean annual
temperature is 17.8 ° C and rainfall is approximately 644.8 mm annually.
Ten grams of physiologically mature fruits were randomly selected from
each genotype. A total of 20 genotypes were sampled; they were
originally collected from 3 states of the center and south of Mexico
(Puebla, State of Mexico and Chiapas). Preparation of the extract.
One gram of fresh fruit pulp was weighed; each sample was dissolved in
25 ml of ethanol at 95% v/v. After 24 h the volume was adjusted to 25 ml
with ethanol at 80% v/v, and the mixture was centrifuged at 1 409 g.
Quantification of total phenols. Quantification was made according to the method proposed by Waterman and Mole (1994).
A mixture of phosphowolframic and phosphomolybdic acids in basic medium
is used as reactive, and reduced by oxidizing the phenolic compounds,
originating blue oxides of wolframic and of molybdenum. For the
analysis, 0.5 ml of ethanolic extract were taken, 10 ml of a solution of
Na2CO3 was added at 10% p/v, was homogenized and
the mixture was incubated in darkness at 38 ° C for 15 min. One ml of
the mixture was taken, 3 ml of water was added along with 1 ml of the
reactive of Folin Ciocalteu:water (1:1). The mixture was left to set for
15 min in darkness. Finally, the absorbance reading was taken at 600 nm
in a Genesys 10s spectrophotometer. The concentration was obtained from
a standard curve (y = 0.0014x; R2 = 0.997) prepared with
gallic acid. Total phenol values are expressed in mg equivalent of
gallic acid per 100 g of fresh weight. Each analysis was done in
triplicate.
Quantification of flavonoids.
One aliquot of 0.5 ml of the supernatant of the ethanolic extract was
previously prepared; 1.5 ml of ethanol at 95% v/v was added, along with
0.1 ml of a solution of AlCl3 at 10% p/v, 0.1 ml of solution
of 1 M of potassium acetate and 2.8 ml of distilled water. The mixture
was incubated in darkness for 30 min. Absorbance was read in a Genesy
10s spectrophotometer at a wave length of 415 nm. For the
quantification, a standard curve was made (y = 0.0122x-0.0067; R2 = 0.965) based on the flavonol quercetin ( Chang et al., 2002). Flavonoid values are expressed in mg equivalent of quercetin per 100 g of fresh weight.
Quantification of vitamin C.
Quantification of vitamin C was carried out through the determination
of ascorbic acid (vitamin C). To a 1 g of sample, 3 ml of metaphosphoric
acid at 3% v/v was added, and then the mixture was macerated for 3 min
and was filtered. One ml of the filtrate was
taken and the volume brought to 10 ml with the solution of
metaphosphoric acid at 3% v/v. Two ml of the extract was taken and 2 ml
of the buffer solution, pH = 4 (glacial acetic acid: sodium acetate 5%,
1:1) was added, along with 3 ml of dichloroindophenol and 15 ml of
xylene, and the mixture was agitated vigorously. The organic phase was
separated and dried with the addition of anhydrous Na2SO4,
the mixture was filtered and absorbance was read in a Genesis 10s
spectrophotometer at a wavelength of 520 nm. From the standard curve
(y = −2.666x + 0.567; R2 = 0.995) the concentration of
ascorbic acid present in each sample was obtained by means of the
following equation: mg total ascorbic acid/mg= (C × V x100) / (A × P);
where: C= ascorbic acid in the sample, V= final volume, A= ml of aliquot
of the solution taken, P= weight or volume of the sample. The
concentration of vitamin C was expressed in mg equivalent of ascorbic
acid per 100 g of fresh weight.
Evaluation of antioxidant activity. The analysis was performed using the free radical DPPH method (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich), described by Amico et al. (2008).
The antioxidant capacity of the samples was observed at 516 nm from the
gradual color change of the DPPH (purple) to reduced-DPPH (yellow) ( Cotelle et al., 1996).
For the analysis, 10 g of sample were macerated (hawthorn fruit pulp)
in methanol (GR) for 48 h, the mixture was decanted and the plant
residue was placed once more in methanol for the same period of time.
The extracts were reunited and the dissolvent was evaporated in a Büchi
evaporator. From the highest concentration of the methanolic extract
(stock solution) the following dissolutions were prepared in methanol to
obtain the concentrations of 0.2, 0.15, 0.1, 0.05 mg ml−1. As references the concentrations 0.1, 0.001 and 0.0001 mg ml−1
of quercetin and ascorbic acid were prepared separately. To each
concentration of the extracts (1 ml) and of the references 3 ml were
added separately of the solution of DPPH (0.1 mM). The mixtures were
left at room temperature during 30 min and later the absorbance readings
were made at 516 nm. The percentage of DPPH was determined through the
formula: % DPPH= (Acontrol Asample)*100/AbControl; where Acontrol is the absorbance of the control (DPPH 0.1 mM) and Asample
is the absorbance obtained after 30 min of each sample with DPPH
0.1 mM. The antioxidant activity of the samples was determined through
the calculation of the mean inhibitory concentration (IC50),
which is the concentration required by the sample to decrease the
absorbance of DPPH to 50%. The low absorbance of the reaction mixture
indicated high antioxidant activity.
To construct the standard curve (y = 9.393 ×+ 0.006; R2 = 0.999)
of DPPH, 3.93 mg were weighed and dissolved in 100 ml of methanol to
obtain a concentration of 0.1 mM. From this solution the different
concentrations were prepared: 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 mM of
DPPH. Absorbance was measured at 516 nm in a Genesys 10s
spectrophotometer; the readings were taken by triplicate and methanol
(GR) was used as reagent blank.
Statistical analysis. A Pearson's correlation and analysis of variance (Anova) were carried out, along with the comparison of means of Tukey (p ≤ 0.05),
using the program Statistical Analysis System (SAS, version 8.0)
according to a completely randomized experimental design, where each
selected genotype was considered as treatment of which 3 replicates were
made.
Results
Statistical analysis showed significant differences (p ≤ 0.05) in the concentration of phenolic compounds among the 20 genotypes of fruits of different species and origin ( Fig. 1); the same tendency was observed in the content of flavonoids and vitamin C (ascorbic acid) ( Figs. 2, 3).
Genotypes 27, 51 and 55 were the ones that presented the highest
concentration of phenols, genotypes 27 and 51 have a common geographic
origin (Chiapas) in contrast to genotype 55 (Puebla). The lowest
concentration was found in genotypes 2 (Crataegus rosei) and 5 (Crataegus aurescens),
both have as origin the state of Puebla. Genotypes 25 and 27 presented
the highest content of flavonoids, similarly, the content of these
metabolites was also lower in genotypes 2 and 5. Results showed that the
content of total phenols and flavonoids can not be associated with the
origin and species of the samples ( Fig. 1). Genotypes 2, 26 and 66 presented the highest concentration of vitamin C ( Fig. 3).
