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INTRODUCTION
Honey bees are bees from the single genus Apis from the Apini tribe, where the most popular subspecies is Apis mellifera (Penk Kek et al., 2014). Nowadays, bees are at home in almost all habitats in the world, from the equatorial rainforests and tropical deserts to the Subarctic regions of Eurasia and North America (Bankova et al., 2018). According to the Codex Alimentarius (2001), honey is the natural sweet substance produced by honey bees from the nectar of plants or from secretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in the honeycomb to ripen and mature (Nordin et al., 2018).
Honey has been known for its nutritional and therapeutic values thanks to the benefits of its consumption as well as the use and enrichment of other products (Alqarni et al., 2016). China, Turkey, Argentina, Iran, Ukraine, the United States of America, India, the Russian Federation, Mexico, and Ethiopia were the main producers of honey in 2018. Mexico is the ninth-largest producer in the world with an annual production of 64,253.04 t of honey (FAOSTAT, 2018). The state of Zacatecas, one of the main producers of honey in Mexico, produced in 2016 1929 t, increasing this amount the following year (2078 t) (SIAP, 2019). In the state of Zacatecas, different types of honey are produced, including Mezquite honey (white), Multiflora honey (variable color), and Gatuño honey (dark color), however, there is little information about them.
There are about 320 different varieties of honey from various floral sources, so that the taste, color and smell of a specific type of honey depend on the various sources of liquid flowers and plants visited by the bee. The different types of honey are comparable in terms of temperature, rainfall, seasonal, and climatic changes, while the color of honey varies from light brown to dark brown depending on where the bees pollinated, differing in organoleptic properties as well as chemically from one country to another, even in different regions within the same country due to its floral origin, soil composition and other factors (Meo et al., 2017). Honey contains approximately 20 % moisture, monosaccharides (75 % glucose and fructose), disaccharides (3 - 10 % sucrose) and proteins, vitamins and minerals, as well as antioxidants (Alqarni et al., 2016).
Antioxidants are an element that can inhibit the oxidation of other molecules. Oxidation is a biochemical reaction that generates free radicals that may harm the cells, tissues, and ultimately the physiological functions. Antioxidants such as vitamin C terminate the chain reactions to protect the body from free radicals. To balance the oxidative status, the human body maintains complex systems of overlapping antioxidants. The food containing antioxidants has been shown to improve health (Meo et al., 2017).
The term ‘phenolic’ or ‘polyphenol’ can be defined chemically as a substance that possesses an aromatic ring bearing one or more hydroxyl substituents including functional derivatives (esters, methyl esters, and glycosides). These bioactive substances are widely distributed in the plant kingdom and are closely associated with the sensory and nutritional quality of fresh and processed plant food (Román-Cortés et al., 2018). Some phenolic compounds are extremely widespread, while others are specific to certain plant families or are found only in certain plant parts or at specific developmental stages (Hossen et al., 2017).
Different solvents and techniques of extraction could lead to different compositions of phenolic compounds in extracts because the solubility of each compound in a given solvent would be quite different, this implies that the phenolic compounds with more hydrophobic characteristics might occur in lower amounts than those with hydrophilic characteristics, consequently, the bioactivity of an extract might also be affected (Lou et al., 2014).
Due to the above and in the absence of information about the functional value of the different varieties of honey produced in Zacatecas, this research paper aims to determine the antioxidant capacity and the total phenolic content in the honey varieties multiflora, mesquite, gatuño flower and creamy gatuño flower using water (Kavanagh et al., 2019) and methanolic (Tomás-Barberán et al., 2001) extraction for bioactive compounds.
MATERIAL AND METHODS
Raw material
Multiflora, mesquite, gatuño, and creamy gatuño kinds of honey were obtained on the limits of the locality of Las Norias, in the municipality of Guadalupe, Zacatecas (22° 52’ 56.0” N, 102° 23’ 20.0” W). The creamy gatuño honey was mechanically mixed with a blender at room temperature until the desired consistency was obtained. Samples were analyzed for pH, °Brix, moisture content, as well as the total phenolic content and antioxidant activity. A pH meter (Ohaus, Parsippany-Troy Hills, NJ, EUA) was used to measure the pH of a 10 % (w/v) solution of honey prepared in deionized water (Kavanagh et al., 2019). °Brix and moisture content (from refraction index, using the Wedmore, 1955 Table) of all honey samples was measured at 20 °C in triplicate using an ATAGO® refractometer (NAR 3T, Tokyo, Japan).
The extraction for the quantification of the total phenols (CFT) and determination of the antioxidant capacity were carried out by an adaptation of the method described by Tomás-Barberán et al. (2001). For this, 1 g of honey was mixed with 25 mL of a solution of MeOH and 6 N HCl 80:20 (v/v), dissolved with continuous agitation for 30 min at room temperature, and then filtered on Wathman 1 paper (methanolic extraction). A second extraction was performed according to Kavanagh et al. (2019) diluting 1g of honey in 20 mL of deionized H2O (water extraction) with continuous agitation for 30 min at room temperature, it was then filtered on Wathman paper 1.
Total phenolics (TPC)
Total phenolic content (TPC) was quantified using the Folin-Ciocalteu test (Li et al., 2006); 250 μL of the extract was mixed with 15 mL deionized water and 1.25 mL of Folin- Ciocalteu phenol reagent (Sigma-Aldrich, MO, USA). After 5 min 3.75 mL of Na2CO3 (7.5 %) was added and leveled to 25 mL with deionized water. Absorbance was measured at 765 nm in a spectrophotometer UVVis (Thermo Scientific 10S, Thermo Fisher Scientific Inc, Waltham, MA, USA) after a 2.5 min reaction at 20 °C. The results were reported as mg of gallic acid (mg GAE 100 g-1).
Antioxidant capacity (AC)
Regarding the antioxidant capacity (CA), this was quantified by the spectrophotometric technique of ABTS•+ (Re et al., 1999), DPPH (Brand-Williams et al., 1995) and FRAP. In all cases, the results were expressed in micromoles of Trolox (TEAC) in 100 g of sample.
ABTS•+ radical scavenging ability
The same extract obtained for TPC quantification was used to evaluate AC. The AC was determined through a modification of the spectrophotometric technique developed by Re et al. (1999), using 7 mM ABTS•+ radical (Sigma) generated by 2.45 mM potassium persulfate (K2S2O8). The mixture of the honey remained in the dark at room temperature (~20 °C) for 16 h before use, and then the ABTS•+ solution was diluted to give an absorbance of 0.7 ± 0.1 at 734 nm. Afterwards, 100 μL of honey extract were mixed with 900 μL of the ABTS•+ diluted solution, incubated 2.5 min at 20 °C the absorbance was measured at 734 nm. The results were expressed as antioxidant activity equivalent to μmol units of Trolox (TEAC) 100 g-1. All the experiments were replicated thrice.
DPPH radical scavenging activity
Also, a slightly modified version of the method described by Brand-Williams et al. (1995) to analyze the AC of the samples was used: 100 μL of honey extract was added to 1 mL of 2,2-diphenyl- 1-picrylhydrazyl (DPPH) mM (3 mg 100 mL-1 in methanolic solution). The scavenging activity of the free radicals, using the free radical reaction DPPH, was evaluated by measuring the absorbance at 515 nm, after a 2.5 min reaction at 20 °C, in a spectrophotometer. Results were expressed in μmol equivalents of Trolox (TEAC) 100 g-1.
FRAP (ferric reducing antioxidant power)
The FRAP assay was carried out based on the procedure described in the literature (Benzie and Strain, 1996). Briefly, the FRAP reagent was prepared with 2.5 mL of sodium acetate buffer (300 mmol L-1, pH 3.6), 2.5 mL of 10 mmol L-1 TPTZ solution (40 mmol L-1 HCl as solvent) and 25 mL of 20 mmol L-1 iron (III). The FRAP reagent was prepared freshly every day and warmed to 37 °C in a water bath prior to use. One hundred μL of the diluted sample were added to 1 mL of the FRAP reagent. The absorbance of the mixture was measured at 593 nm after 30 min. The results were expressed as antioxidant activity equivalent to μmol units of Trolox (TEAC) 100 g-1. All the experiments were replicated thrice.
The experimental design was completely randomized with three replications. All analyses were carried out in triplicate and the results expressed as mean and standard deviation (X ± SD). To determine statistically significant differences between the data of the variables in the honey varieties, a one-way ANOVA was carried out and, if significant, a Tukey test was applied (P ≤ 0.05). The values of antioxidant activity and total phenol content were analyzed using the Pearson correlation. All statistical analyses were performed using Statgraphics® Centurion XV (Statpoint Technologies Inc., Warrenton, VA, USA).
RESULTS AND DISCUSSION
Table 1 shows the results of the physicochemical analysis of the four honey varieties analyzed, where it can be observed that there were significant differences between them, in °Brix, pH, and moisture content.
Table 1 Mean values and standard deviation (n = 3) in °Brix, pH and moisture content (% Xw) of honey samples.
| Honey variety | °Brix | pH | % Xw |
|---|---|---|---|
| Multiflora | 79.46 (0.06) a | 3.426 (0.005) a | 18.96 (0.02) d |
| Mezquite | 80.00 (0.05) b | 3.443 (0.005) a | 18.23 (0) c |
| Gatuño | 82.66 (0.28) c | 4.486 (0.011) b | 15.60 (0.30) b |
| Creamy gatuño | 83.53 (0.06) d | 4.493 (0.005) b | 14.65 (0.02) a |
Means with equal letters in the same column are not statistically different according to the ANOVA (Tukey test, P ≤ 0.05).
The data obtained concurs with that reported by Alqarni et al. (2016) who found values of 70 to 85 °Brix in 23 samples of honey, from countries such as Germany, Malaysia, Egypt, Australia, New Zealand, Yemen, and Saudi Arabia. With regard to water content, Alqarni et al. (2016) reported from 12.2 to 27.4 % as well as values of 3.03 to 4.76 in pH, while Leyva-Daniel et al. (2017) found 18.4 % moisture and a pH of 3.8. Kavanagh et al. (2019) reported a pH range of 3.24 to 4.84, as well as 65.42 to 85.42 in °Brix and a moisture content of 12.9 to 24.2 %.
The soluble solids serve as an indicator parameter of the rate in solution solids such as sugars, organic acids, and minerals, nonetheless directly related to sugars and the water levels in the samples, being related to higher water content with lower sugar content (Biluca et al., 2016). According to the standards of the Codex Alimentarius Committee on Sugars (2001), the minimum amount of reducing sugars is 60 g 100 g-1 for floral honey. The sugar composition of honey is influenced by the types of flowers used by the bees, as well as regions and climate conditions (Da Silva et al., 2016).
The pH value in honey is not directly related to free acidity because of the buffering action of various acids and minerals present. The pH of honey varied from 3.42 to 6.10 (Alqarni et al. 2016). The pH is a useful criterium of possible microbial growth. Most bacteria grow in a neutral and mildly alkaline environment, while yeasts and molds grow in acidic ones (Alqarni et al., 2016). Also, pH is used for discrimination between honeydew (high pH values) and blossom honeys (low ones). There is no standard in terms of pH for honey (Vit et al., 2004).
Water content in honey is responsible for its stability against fermentation and granulation. Normally ripe honey has a moisture content below 18.6 % (Alqarni et al., 2016). In 2001, the Codex Alimentarius was revised and the parameters for honey quality were set (International Honey Commission, 2009). Good quality honey should have a moisture content that is no more than 20 g 100 g-1 (Nordin et al., 2018). Honey with a high-water content translates to a greater fermentation potential, resulting in difficult preservation and storage (Nordin et al., 2018). Water content is the second largest constituent of honey and is closely related to several factors, such as the floral and geographical origin of the nectar, soil characteristics, climatic conditions, conditions during harvesting, degree of maturation, manipulation by beekeepers during harvest, extraction, processing methods and storage conditions (Bobis et al., 2020).
Mexico has a wide variety of honey due to its diverse climates and flora, which depend directly on the geographic origin, resulting in honey with particular physicochemical parameters for each blooming process. These parameters are dependent on pollen grains, allowing for honey classification in unifloral and multifloral (Frausto-Reyes et al., 2017). Zacatecas is located in the northern region of Mexico, this region is characterized by xerophytic vegetation and large areas of grassland, thorny and coniferous forests. It also includes irrigated agricultural areas for export products, which has facilitated the development of pollination with bees. Honey production in this region is considered to be of excellent quality and takes place from March to May and from August to October (Martínez-Pérez et al., 2017). Three-fourths of the territory of the state of Zacatecas (56,463 km2) correspond to arid zones and shrubs (Mata-Páez et al., 2016).
Figure 1 shows the values in total phenols, where a very marked difference from the honey of gatuño and gatuño creamy can be seen. In addition, a better extraction of the phenolic compounds was observed using the methanolic extraction, especially in Gatuño honey. The values were 56.5 mg of GAE 100 g-1 in Mezquite honey, 67.9 mg of GAE 100 g-1 in multiflora honey, as well as 141 and 144 mg of GAE 100 g-1 in honey of gatuño and creamy gatuño, respectively, when the methanolic extraction was applied, while with water extraction values of 50.47 were obtained in the multiflora honey, 55.8 in Mezquite honey, as well as 108 and 102 mg of GAE 100 g-1 in gatuño flower and creamy gatuño flower.
Figure 1 Total phenolic content of the different honey samples analyzed according to the extraction method. Means with equal letters between the bars for the same extraction are not statistically different according to the ANOVA (Tukey test, P ≤ 0.05).
Phytochemical extraction yield and antioxidant activity not only depend on the extraction method but also on the solvent used for extraction. The presence of different antioxidant compounds with diverse chemical characteristics and polarities may or may not be soluble in a particular solvent. Polar solvents are frequently used for recovering polyphenols from plant matrices. The most suitable solvents are aqueous mixtures containing ethanol, methanol, acetone, or ethyl acetate. Methanol has been generally found to be more efficient in the extraction of lower molecular weight polyphenols. The combined use of water and organic solvent may facilitates the extraction of chemicals that are soluble in water and/or organic solvent (Do et al., 2014).
On the other hand, the result of the Folin-Ciocalteau reaction in honey samples should be interpreted as a quantitative estimation of total phenols because the reducing sugars of honey can also react with the FolinCiocalteau reagent (Alves et al., 2013). Reductive sugars (such as fructose, glucose, and sucrose), organic acids (such as ascorbic, citric, and tartaric acids), ferrous sulfate, and sodium sulfite are potentially capable of interfering with the assessment of phenolic compounds in food matrices by the FC assay (Bridi et al., 2017). However, the cleaning process during Amberlite XAD-2 or PVPP (Polyvinylpolypyrrolidone) extraction can remove sugars and other polar compounds, including glycosylated phenolic compounds, which can also contribute to the antioxidant capacity of honey, sugars and other compounds in the entire honey may have a good reducing capacity (Ferreira et al., 2009).
Leyva-Daniel et al. (2017) found 29.89 mg of GAE 100 g-1, while Alqarni et al. (2016) from 42 to 84 mg of GAE 100 g-1 in honeys from different countries, Kavanagh et al. (2019) reported a range from 2.59 to 81.10 mg of GAE 100 g-1 in Irish multifloral honey, while Al-Mamary et al. (2002) found a range between 56.3 and 246.2 mg of GAE 100 g-1 in nine varieties of honey produced in Yemen. Peng-Kek et al. (2014) found a range from 51.04 to 105.88 mg of GAE 100 g-1 in Malaysian honey. A higher phenolic content was observed in gatuño flower honey, which has a darker color than the mezquite and multiflora varieties which are pale yellow, agreeing with those found by Alqarni et al. (2016) who observed that darker honeys, e.g. Acacia, Manuka and Tualang seemed to have more phenolic compounds than light ones.
Many phenolic compounds are found in honey with different quality and quantity according to the floral source. Total phenolic content is a good criterion to determine the quality and curative properties of honey (Al-Mamary et al., 2002). Among the phytochemical compounds present in honey, the phenolic compounds play a major role in the antioxidant activity. The phenolic compounds found in honey are free phenols, phenolic acids, polyphenols (usually in the form of flavonoids), anthocyanins, procyanidins, and pigments (Oroian and Ropciuc, 2017). Polyphenols mainly exert their antioxidant activity by neutralizing free radicals, which is accomplished by donating an electron or hydrogen atom (Hossen et al., 2017).
Table 2 shows the values of the antioxidant capacity of the honey samples analyzed, where significant differences can be observed between the samples. There were also differences between the extraction methods. The methanolic extraction showed a higher yield in the gatuño flower samples when the antioxidant capacity was analyzed with the ABTS•+ method; on the contrary, the extraction did not suppose differences between the multiflora honey samples, neither between the mesquite honey samples analyzed by the ABTS•+. In contrast, when the antioxidant capacity was measured with the DPPH and FRAP methods, greater antioxidant power was observed when water extraction was used.
Table 2 Mean values and standard deviation (n = 3) in the antioxidant capacity of honey [μmol equivalents of Trolox (TEAC) 100 g-1].
| Extraction | Honey variety | ABTS•+ | DPPH | FRAP |
|---|---|---|---|---|
| Methanolic extraction | Multiflora | 16.67 (2.61) a | 387.07 (53.03) b | 70.4 (22) a |
| Mezquite | 16.03 (3.41) a | 282.11 (26.62) a | 71.5 (14.2) a | |
| Gatuño | 308.75 (9.32) b | 306.3 (18.8) ab | 624.1 (44.7) b | |
| Gatuño Creamy | 325.03 (11.73) b | 333.3 (11.2) ab | 647.80 (8.03) b | |
| Water extraction | Multiflora | 16.56 (3.68) a | 1097.3 (83.8) a | 215.7 (28.8) a |
| Mezquite | 31.71 (14.04) a | 1063.1 (61.3) a | 205.9 (12.9) a | |
| Gatuño | 183.09 (22.91) b | 1118.3 (55.6) a | 680.4 (49.9) b | |
| Gatuño Creamy | 179.96 (9.85) b | 1113.1 (41.3) a | 774.8 (18.7) c |
Means with equal letters in the same column under the same extraction are not statistically different according to the ANOVA (Tukey test, P ≤ 0.05).
The ferric reducing antioxidant power (FRAP) assay is a measure of overall antioxidant capacity, while 2,2-Diphenyl1-picrylhydrazyl (DPPH) and high Trolox (a vitamin E analog) equivalent antioxidant capacity (TEAC) assays measure capacity to scavenge radicals. All are recognized as valid methods used to determine the antioxidant activity of food and beverages.
The composition of a particular honey sample greatly depends on the composition of nectar, whence it originates. Polyphenols have been recognized as the main responsible for the antioxidant activity of honey that is mainly associated with the ability of free radical scavengers, through the formation of more stable and less toxic molecules. Phenolic compounds stabilize free radicals when they give off hydrogen from one of their hydroxyl group; the degree of activity is related to the number of their hydroxyl groups (Cianciosi et al., 2018). The natural antioxidants, especially flavonoids, exhibit a wide range of biological effects, including antibacterial, anti-inflammatory, anti-allergic, anti-thrombotic, and vasodilatory actions.
Phenols are very efficient scavengers of peroxyl radicals because of their molecular structures which include an aromatic ring with hydroxyl groups containing mobile hydrogens. Moreover, the action of phenolic compounds can be related to their capacity to reduce and chelate ferric ions which catalyzes lipid peroxidation. The differences in activities of antioxidants depend on structural dissimilarities, primarily the degree of hydroxylation and methylation of the compounds (Al-Mamary et al., 2002). The AC of honey is given primarily by phenolic compounds, but enzymes, amino acids and carotenoids also contribute to this ability. Radical scavenging and protection against the lipid peroxidation of honey can reduce and prevent diseases and physiological situations where oxidative stress plays an important role (Cianciosi et al., 2018).
Analysis of the correlation between the antioxidant capacities and total phenolic contents of the honey samples showed a positive correlation (R = 0.9692 ABTS•+) (R = 0.8488 FRAP). High TPC honeys have been shown to exhibit significant antioxidant capacity using the FRAP, DPPH, and TEAC assays (Alvarez-Suarez et al., 2013). Literature suggests that honey contains potent antioxidative agents. The role of honey also depends on its concentration and its geographical origin. As an antioxidant, honey has numerous preemptive properties against many clinical conditions such as inflammatory disorders, coronary artery diseases, neurological worsening, aging and cancer. The increase in phenolic compounds in honey provides antioxidant properties (Meo et al., 2017). The composition of honey is difficult to define since it is a mixture of different active compounds and it depends on several factors. They contain almost the same phenolic profile, including p-coumaric acid, eugenol, ferulic acid, caffeic acid and flavonoids like pinobanksin, pinocembrin, chrysin, quercetin, apigenin and naringin in different concentration (Badolato et al., 2017). As honey samples are different and may not contain all of the polyphenols described and because not all polyphenols exert the same protective effects, consumption of a wide variety of honey samples at any one time is recommended (Hossen et al., 2017).
CONCLUSIONS
Gatuño flower and creamy gatuño flower honey have a greater total phenolic content compared to Mezquite and multiflora varieties. The total phenolic content of honey correlated positively with its antioxidant capacity. Methanolic extraction showed better performance in total phenols and antioxidant capacity (ABTS•+) while water extraction did when DPPH and FRAP methods were applied. Climatological, geographical factors, as well as the type of flora, influence and favor the sensory, physicalchemical and functional diversity of honey. In this sense, honey produced in the state of Zacatecas can compete commercially with other national or even international honeys and at the same time contribute to the ecological balance through the practice of beekeeping.
