Antioxidant Activity of Artisanal Honey From Tabasco, Mexico

Abstract

Potential claims for honey, floral variety and their health properties are relevant for small farmers and artisan producers. The antioxidant activity of honey samples from cacao farms, mangrove, citrus, and coconut groves from Mexico was established by applying a multiple-method approach, which included determination of the level of total phenolic compounds and total flavonoids. Total phenolics and total flavonoids ranged from 51 to 134 mg gallic acid equivalents (GAE)/100 g honey and from 29.6 to 187.1 mg rutin equivalents/100 g honey respectively. Methanolic and aqueous solutions had similar profiles for inhibition of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical (range from 33 to 85%) but the aqueous solutions tended to show lower radical scavenging properties. Linear correlations were established between flavonoids contents and percentage inhibition of DPPH, but phenolics contents were not well correlated (R² = 0.68) to the ferric reducing antioxidant power (FRAP) values which showed wide ranges from 48 to 152 mg Trolox/100 g honey. No patterns could be found in relation to antioxidant activities and the agrifood system of origin, floral availability, collection location, or season. Artisan honey samples from Tabasco were highly variable in their antioxidant properties, possibly because of the biodiversity and seasonal variations, which contribute to their unique nature. The antioxidant tests used in this study could be useful to verify the antioxidant function of honey.

Keywords:

• Honey
• Antioxidant
• Phenolic compounds
• Flavonoids
• Rancimat
• DPPH
• FRAP

INTRODUCTION

Oxidative deterioration is one of the main culprits in the reduction of the quality and acceptability of food products. This process is initiated by heat, ionising radiation, light, metal ions, exposure to the enzyme lypoxygenase and/or metallo-protein catalysts.[1] Lipid hydroperoxides, and their breakdown products have been implicated to have a detrimental effect on foods, including a significant loss of nutritional value, since it involves a loss of vitamins and essential fatty acids. Oxidation also affects sensory quality by changing its color, texture and taste, which shortens its shelf life and can result in rejection on the part of consumers.[2] To control the oxidation processes of foods, food technologists have adopted a range of strategies that involve an appropriate choice of raw material, processing technologies, packing materials and storage conditions, and it is often necessary to use antioxidants. These inhibitory substances, when added to the product, may preserve the lipid fraction from oxidation without reducing the food quality.[3] Researchers at Plymouth have successfully applied honey-like antioxidant systems to effectively control oxidative deterioration of meat products.[4]

Antioxidants also have an important role in preventing a variety of lifestyle related diseases and ageing because these are closely related to active oxygen and lipid peroxidation.[5] Widely used synthetic antioxidants could be unstable; most of them are heat-sensitive and volatile in steam, so their safety and efficiency have been a cause of concern.[6] Consequently, there has been a growing interest in the search for natural antioxidants to replace synthetic substances.

Besides the most popular use of honey as a natural edulcorant, it is also used in traditional medicine, as it is know to have antimicrobial, antioxidant, antiviral, antitumor and anti-inflammatory properties.[7] Honey and other bee products, such as royal jelly and propolis may also be used as natural antioxidants.[7] Honey could have over 180 compounds, but it is essentially a solution supersaturated in sugars, with fructose (38%) and glucose (31%) being the most important[8] while sucrose and maltose are present in minor concentrations. The moisture content of honey is about 17.7 g/100 g honey, its total acidity 0.08 g/100 g honey and ashes 0.18 g/100 g honey.[9] The great variety of minor components include phenolic acids and flavonoids, enzymes such as glucose oxidase and catalase, ascorbic acid, carotenoids, organic acids, amino acids, proteins, and α-tocopherol[10,11] which may provide an indication of honey origin, variety, color, and flavor. The actual composition of honey is variable and depends on factors such as the pollen source, climate, environmental conditions, and the processing that it undergoes.[8,12–14] Mexico is the world's fifth largest honey producer after China, Argentina, USA, and Turkey, with an annual production of 57,000 tonnes, of which approximately 44% is exported. Mexico is the third largest exporter after only China and Argentina. The state of Tabasco in Mexico produces over 200 tonnes, and local artisan producers differentiate their products according to the collection area or type of farm where it is collected (i.e., citrus, cacao, coconut, multifloral). Honey production could be an additional source of income for farmers and residents of agricultural systems such as cocoa, coconut, or citrus fruit farms. In these enterprises, product differentiation, based on links to traditional farming systems or possibly health claims, would benefit small producers. Some of these products are not labelled, some are labelled to indicate the area or origin (honey from the mountains, honey from the mangroves/coconut grove), and most are considered to be multifloral. The aim of this work was to determine the phenolics content (phenols and flavonoids) of eleven honey samples collected by small producers during two seasons on different agrifood ecosystems from Tabasco State (Mexico). Three testing methods for antioxidant activity were applied and compared in relation to the antioxidant activity of the honey samples.

MATERIALS AND METHODS

Honey Samples

For this study, eleven honey samples from Tabasco (Mexico) were analyzed. Samples of artisanal honey were collected in locations where the local Agrifood production ecosystems and the season determine the flora near the collection point.[13,14] All the samples were collected in either high season (Feb–April) or low season (December) 2005–2006, classification that coincides with the expected flowering patterns of plants. Most samples are considered multifloral. Collection location, description, and collection times are indicated on Table 1.

Table 1 Collection location, description and time of collection, (High Season, HS; Low Season, LS) of artisanal honey samples from Tabasco State, Mexico

Sample code	Description	Collection season	Location
A	Cacao honey	HS, 2005	Finca Cholula, Comalcalco
B	Cacao honey	LS, 2005	Finca Cholula, Comalcalco
C	Coconut honey	LS, 2006	Comalcalco
D	Multifloral	LS, 2005	Ejido Victoria, Tenosique
E	Citrus	HS, 2006	Huimanguillo
F	Multifloral	LS, 2005	Ejido Victoria, Tenosique
G	Multifloral	HS, 2005	Ejido Estapilla, Tenosique
H	Multifloral	HS, 2005	Ejido Esperanza, Tenosique
I	Multifloral	HS, 2006	El Jacalito, Centro Tular, Comalcalco
J	Cacao, Coconut	HS, 2006	Colpos, Cardenas
K	Multifloral	LS, 2005	Tacotalpa

Chemical Reagents

Folin–Ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH·), aluminum chloride, gallic acid, iron(III) chloride, trichloroacetic acid (TCA) and Trolox were obtained from Sigma Chemical Co. Sodium chloride, sodium carbonate, sodium nitrite (II), sodium hydroxide chloride acid and methanol of HPLC ultra-gradient grade were obtained from Merck (Darmstadt, Germany) and potassium hexacyanoferrate from Fluka BioChemika (Germany).

Total Phenolics Content

The total phenolics content (TPC) was determined using the Folin-Ciocalteu reagent.[15] A volume of 0.3 mL of a methanolic solution of honey (0.2 g/mL) was introduced into the test tubes followed by 2.5 mL of Folin Ciocalteu's reagent (diluted 10 times with water) and 2 mL of sodium carbonate (7.5% w/v). The tubes were vortex-mixed, covered with parafilm and incubated at 50°C for 5 min. Absorption at 760 nm was measured with an HP 8451 spectrophotometer (Hewlett-Packard, Cambridge, UK) and compared to a gallic acid calibration curve.

Total Flavonoids Content

The total flavonoid content (TFC) was determined by the method based on Blasa et al.[16] with some modifications for the honey. Each sample (2 g) was mixed with 10 mL acid water (pH 2 with HCl, 0.02 M). The solution was stirred with a vortex mixer until the honey was totally fluid. Solutions were centrifuged at 5000 rpm (8 min). Briefly, 1 ml of honey solution was mixed with 0.3 ml NaNO₂ (5%), and after 5 min 0.3 ml AlCl₃ (10%) were added. The honey samples were mixed and held for six minutes, and then neutralised with 2 ml NaOH (1 M). For all the samples, the absorbance was read at 510 nm and the quantification was carried out using a calibration curve. Different concentrations of rutin (8.5–170 μg/mL) were used for calibration, giving a linearity of 0.997 (R²). The results were expressed in mg rutin equivalents (RE)/100 g of honey as mean of 3 replicates.

Determination of Antioxidant Activity Using DPPH Radical Scavenging Method

The antioxidant activity of honey was measured in terms of hydrogen-donating or radical-scavenging ability, using the stable radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH).[17] Two grams of honey samples were dissolved in 5 ml of methanol. Then, 200 μL of this methanolic solution were placed in a cuvette, and 2 mL of 6·10⁻⁵ mol L⁻¹ methanolic solution of DPPH was added. The mixtures were thoroughly stirred in a vortex (2500 rpm for 1 min) and then placed in a dark room for 1 h. The decrease in absorbance at 517 nm was determined with an HP 8451 spectrophotometer (Hewlett-Packard, Cambridge, UK) after 1 h, with methanol as a blank. The absorbance of the radical without antioxidant was used as a control sample. To prepare water extracts, 2 g of honey sample were dissolved into 5 ml of water. The antioxidant activity of these water extracts against DPPH·was determined as described above for methanolic extracts. The inhibition (in %) was plotted against the sample concentration in the reaction system. The inhibition percentage of the DPPH radical was calculated according to the formula given by Yen and Duh[18]:

1

where % I is the proportion of DPPH inhibition; A_B is the absorbance of the control sample (t = 0); and A_S is the absorbance of tested sample at the end of the reaction (t = 1 h).

Ferric Reducing Antioxidant Power

The ferric reducing power (FRAP) of honey was determined by using the potassium ferricyanide–ferric chloride method.[19] For this, 1 mL of honey solutions (0.4 g/mL) was added to 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (1%, w/v). The mixtures were incubated at 50°C for 20 min, after which 2.5 mL trichloroacetic acid (10% v/v) was added. An aliquot of the mixture (2.5 mL) was taken and mixed with 2.5 mL water and 0.5 mL 1% FeCl₃. The absorbance at 700 nm was measured after allowing the solution to stand for 30 min. The FRAP was estimated in terms of Trolox equivalent antioxidant capacity (TEAC) in mg Trolox/100 g honey. Each assay was carried out in triplicate.

Determination of Oxidative Stability of Fat (Rancimat Assay)

An instrumental method based on conductivity changes with a Rancimat 743 (Methrohm, Switzerland) was used to determine the antioxidant activity of honey on fat. Previously melted lard (2.5 g), was mixed with 0.2; 0.1 and 0.05 g of honey giving a final concentration of 8, 4, and 2 g of honey/100 g of lard in the reacting system. A blank without honey was prepared. Samples were heated at 110°C and an air flow of 20 L/h was constantly bubbled into the mixture. The end of the induction period (IP) was characterized by the sudden increase of water conductivity, due to the dissociation of volatile carboxylic acids.[20] The antioxidant activity index (AAI) was calculated from the induction times, according to the following formula provided by Forster et al.[21]: AAI = (induction period of lard with antioxidant/Induction period of pure lard). An antioxidant activity index greater than 1 indicates inhibition of lipid oxidation, and the higher the value, the better the antioxidant activity.[22]

Statistical Analysis

Means and standard deviations of three simultaneous assays carried out with the different methods were computed. Statistical analysis (ANOVA) was applied to the data to determine differences (p < 0.05). Between means, Tukey's test was used to establish whether there were significant differences between the levels of the main factor.[23] For the DPPH activity and phenolic compounds, with honey (A to K), one factor ANOVA tests were used, and for Rancimat results, the choice was ANOVA with two factors (honey: A to K; Concentration: 8, 4, and 2%). Statistical analyses were computed using Statgraphics 5.1 for Windows. Correlations between total phenols and total flavonoids were established using the function CORREL from Microsoft Excel software.

RESULTS AND DISCUSSION

Total Phenolic Content

The total phenolic content (TPC) values of the honey methanolic solutions are presented in Fig. 1. TPC of the honey samples, expressed as gallic acid equivalent, ranged from 51.32 mg/100 g for sample E to 134.02 mg/100 g for sample F thus falling in general within the broad range reported in the literature.[24–26] According to several authors,[24,27,28] the concentration and type of phenolic substances in honey depended on several factors, such as flower source of the nectar, season and environmental factors, such as soil type and climate, genetic factors and processing methods. The color of natural honeys is often an indication of their polyphenols composition. In particular, Meda et al.[29] and Socha et al.[30] found that dark-colored honeys have a higher phenolic and flavonoids contents that light-colored honeys, but the links were not clear for these set of Tabasco honey samples, possibly because they are linked to a wide range of botanical sources.

Figure 1 Total phenolic content of artisanal Tabasco honey samples (Refer to Table 1 for sample identification [A–K].) expressed as gallic acid equivalent.

The phenolic compound content could be used as an indicator of the antioxidant capacity, and therefore, serve as a preliminarily screen for honeys intended for use as natural sources of antioxidants in functional foods.[31] Many authors[32,33] have described the potential antioxidant properties of polyphenols, which act by donating a hydrogen atom, as an acceptor of free radicals, by interrupting chain oxidation reactions, or by chelating metals.[34]

Total Flavonoids Content

Similar to the total phenolic content, the variation of the total flavonoid content (TFC) of honey samples was also significant. TFC of honey samples (mg rutin/100 g honey) ranged from 29.58 ± 0.49 mg in sample G to 187.08 ± 0.59 mg in sample F. The values obtained for all samples are presented in Fig. 2.

Figure 2 Total flavonoid content of artisanal Tabasco honey (Refer to Table 1 for sample identification [A–K].) expressed as mg rutin equivalent.

A high correlation (R² = 0.92) between TFC and antioxidant activity in the DPPH reaction was found, indicating that flavonoids may be among the main components responsible for the antioxidant effects of honey. However, other factors may be involved, including phenolic content, and the presence of non-phenolic antioxidants such as ascorbic acid, α-tocopherol, β-carotene, peptides, organic acids, enzymes and Maillard reaction products.[35,36] Some reports have pointed to possible correlations between floral origin and flavonoid profiles.[24,37] The predominance of an individual component or groups of compounds in honey is a promising marker for the determination of the botanical origin of the honeys.[11]

DPPH Assay

The antioxidant activity of honey has been widely demonstrated.[26,30,38] The radical scavenging capacity of the samples was tested using the “stable” free radical, DPPH. The DPPH assay measures the ability of the sample to donate hydrogen to the DPPH radical, which results in a quantitative discoloration of the DPPH reagent, which is related to the antioxidant activity.

Figure 3a compares the antioxidant activity of aqueous solutions of the honey samples with their scavenging values shown as inhibition percentage. The honey solutions exhibited varying degrees of scavenging capacity ranging from 82.8% for sample C to 32.6% for sample G. No statistically significant differences (p > 0.05) were found between season (samples A and B), but differences were found on samples from the same location and agro-food system (D and F). Samples E and H were not significantly different either.

Figure 3 Antioxidant activity of (a) ■ water solutions and (b) □ methanolic solutions of Tabasco honey samples in reaction with DPPH radical. (Refer to Table 1 for sample identification [A–K].)

Scavenging capacities of methanolic sample solutions (Fig. 3b) ranged from 85.5% for sample F to 33.4% for sample G. All the samples were significantly different (p < 0.05). It is interesting to note that the methanolic honey solutions showed stronger radical scavenging properties than the honey water solutions. In plants and tissues, this solvent would extract a different range of compounds than water.

Generally, the antioxidant activity values obtained for Tabasco honey were lower than those obtained for honey from Lithuania, Romania, Portugal, Italy and Argentina.[11,26,37,39,40] An accurate estimation of the antioxidant activity of honeys of different floral origins requires the evaluation of this optimal concentration. On the other hand, the differences found between the honeys may, be explained by the relative amounts of minor compounds to a certain extent, which may play an important role in the antioxidant effect. It is interesting to note that linear correlations were found between TPC and % inhibition of DPPH for both aqueous and methanolic solutions (y = 6.9 + 0.55x, R² = 0.91; y = 11.7 + 0.45x, R² = 0.75, respectively). Other authors[41,11,25] demonstrated a linear correlation between the total phenolic compounds content and antioxidant capacity on plant extracts.

The antioxidant activity is essentially due to the presence of phenolic compounds and flavonoids, although their mechanism of action is not fully understood.[7] Several explanations have been provided, for example Mathew and Abraham[42] suggested that activity of cinnamon extracts is linked to the sequestration of free radicals, hydrogen donation, metallic ion chelation, or even to a role as substrate for superoxide or hydroxyl radicals.[42] These bioactive compounds with their antioxidant properties also interfere with propagation reactions.[43] The antioxidant activity of honey depends on a variety of factors such as concentration, temperature, light, type of substrate, and the physical state of the system, in addition to the presence of micro-components acting as pro-oxidants or synergists.[44] It has also been suggested that the organic acids such as gluconic, malic, and citric acids, which are present in honey, contribute to the antioxidant activity through metal chelation, increasing the effect of flavonoids by synergic effects.[7] The enzymes glucose oxidase and catalase contribute to the antioxidant activity through their ability to eliminate oxygen from the media.[45]

Antioxidant Activity by FRAP Assay

For determination of the antioxidant capacity the FRAP assay (Ferric reducing antioxidant power) was used, because it is a simple direct test that is widely used for antioxidant determination in a variety of samples, including honey.[10,24] Figure 4 shows significant differences (p < 0.05) between the honey samples. The antioxidant activity for different types of honey increased in the following order: G < H < E < B < A < D < K < C < J < I < F. Sample G had an average FRAP value of only 48.34 mg Trolox/100 g honey, while the highest FRAP values of 151.64 mg Trolox/100 g honey were obtained from multifloral sample F.

Figure 4 Antioxidant activity of aqueous solutions of honey samples by FRAP assay. (Refer to Table 1 for sample identification [A–K].)

Some studies showed high linear correlation coefficients between TFC and FRAP,[10,36,40] which was not the case for this set of samples, where comparisons between the total antioxidant activity (FRAP), and total phenolics content only poor correlations were found. However, a positive linear correlation was found between the total antioxidant activity and total flavonoids content (y = 0.74x + 29.11, R² = 0.92). This indicates that flavonoids may be one of the main components responsible for the reduction capacity of the honey samples, which, in addition to other components in the honey such as glucose and fructose, may also contribute, to the reducing power.

Antioxidant Activity Index Using The Rancimat Method

The Rancimat test is an easy and inexpensive method with achieves reproducible results, and it is widely used. Table 2 lists the antioxidant activity index (AAI) of lard with honey added as antioxidant. Higher induction periods of the lard with added honey, when compared against a lard control, indicate higher antioxidant activity of that sample.[46] The AAI was concentration-dependent, and decreased in the following order: I > B > D > J > A > E > K > H > G > C > F, with values between 1.0 and 1.8. At the maximum concentration (8%), multifloral sample I showed the highest (p < 0.05) antioxidant activity index (1.80) of all honeys analyzed. At 4% and 2% concentrations no differences were found (p > 0.05) between samples C, D, E, F and K. At each concentration (2, 4 and 8%) sample C showed the lowest antioxidant activity.

Table 2 Antioxidant activity of Tabasco honey samples when applied to lard (2, 4, and 8 g/100 g) measured by the Rancimat method

	Antioxidant activity Index (AAI)
	Honey concentration (g sample/100 g lard)**
Sample code*	8	4	2
A	1.60 ± 0.01^a	1.50 ± 0.01^a	1.23 ± 0.05^a
B	1.73 ± 0.00^b	1.20 ± 0.01^b	1.13 ± 0.01^b
C	1.24 ± 0.01^c	1.12 ± 0.03^c	1.00 ± 0.01^c
D	1.69 ± 0.01^d	1.19 ± 0.04^bc	1.03 ± 0.06^c
E	1.57 ± 0.16^a	1.14 ± 0.06^bc	1.08 ± 0.08^c
F	1.27 ± 0.01^e	1.15 ± 0.02^cd	1.02 ± 0.01^c
G	1.29 ± 0.01^c	1.21 ± 0.01^b	1.17 ± 0.02^a
H	1.32 ± 0.00^f	1.20 ± 0.02^b	1.15 ± 0.02^ad
I	1.80 ± 0.17^g	1.47 ± 0.05^a	1.35 ± 0.00^e
J	1.65 ± 0.04^a	1.23 ± 0.01^b	1.12 ± 0.01^d
K	1.35 ± 0.06^f	1.17 ± 0.06^c	1.06 ± 0.0.3^c
*Within columns, values followed by the same superscript are not significantly different (p > 0.05) from other honey samples according to Tukey's multiple range test.
** There was no significant differences (p > 0.05) between honey sample extracts at a fixed concentration (between columns).

The mechanisms for the antioxidant activity of honey appear to be complex, because they are linked to a number of compounds which include enzymes, sugars and plant substrates.[47] When applied to foods, additional factors may play a role, for example in the creation of physical barriers. In this study, most honey samples are considered multifloral, but additional work has been carried out by the authors to identify monofloral products using palynological analysis. However, it is evident that in most cases, the diversity of plants is wide and bees will use a variety of resources during the season, which together with the biodiversity of the region, yields products with an individual character, and a wide range of antioxidant activities. Therefore, the tests applied could be used to verify the antioxidant activity where origin by itself cannot assure functionality.

CONCLUSIONS

Different methods (DPPH, FRAP and Rancimat) were successfully applied in order to establish the antioxidant activity of honey samples. The results showed that honey could be considered a good source of natural phenolic and flavonoids compounds. With levels ranging from 51 to 134 mg gallic acid equivalents (GAE)/100 g honey for the former and from 29.6 to 187.1 mg rutin equivalents/100 g honey for the latter, it is clear that they have significant antioxidant activity. When compared, methanolic and aqueous solutions showed similar profiles for inhibition of DPPH radical (range from 33 to 85%), but the aqueous honey solutions tended to show lower radical scavenging properties. The ferric reducing antioxidant power (FRAP) of honey was also quite variable with values on a wide range from 48 to 152 mg Trolox/100 g honey. The antioxidant activity of honey can be attributed to the presence of antioxidant compounds, and to possible synergies between additional food constituents. There is potential for natural antioxidants to replace synthetic compounds in food systems to improve consumer perception. The study of antioxidant properties of honey would continue to yield information that contributes to re-valuing natural products from artisan producers and informing the development of novel applications.

ACKNOWLEDGMENTS

Thanks to Dr. Cesar Vázquez-Navarrete (Colegio de Posgraduados, Cardenas, Tabasco) for facilitating collection of honey samples by establishing links with honey and cocoa producers.