Molecules 2012, 17, 11199-11215; doi:10.3390/molecules170911199 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Physicochemical and Antioxidant Properties of Algerian Honey Md. Ibrahim Khalil 1,2,*, Mohammed Moniruzzaman 1, Laïd Boukraâ 3, Mokhtar Benhanifia 3, Md. Asiful Islam 4, Md. Nazmul Islam 1, Siti Amrah Sulaiman 1 and Siew Hua Gan 4 1 Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia 2 Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh 3 Laboratory of Research on Local Animal Products, Ibn Khaldoun University, Tiaret 14000, Algeria 4 Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +8802-770-8478-85 (ext. 1379); Fax: +8802-770-8069. Received: 15 July 2012; in revised form: 25 August 2012 / Accepted: 2 September 2012 / Published: 20 September 2012 Abstract: The aim of the present study was to characterize the physical, biochemical and antioxidant properties of Algerian honey samples (n = 4). Physical parameters, such as pH, moisture content, electrical conductivity (EC), total dissolved solids (TDS), color intensity, total sugar and sucrose content were measured. Several biochemical and antioxidant tests were performed to determine the antioxidant properties of the honey samples. The mean pH was 3.84 ± 0.01, and moisture the content was 13.21 ± 0.16%. The mean EC was 0.636 ± 0.001, and the mean TDS was 316.92 ± 0.92. The mean color was 120.58 ± 0.64 mm Pfund, and the mean 5-hydroxymethylfurfural (HMF) content was 21.49 mg/kg. The mean total sugar and reducing sugar contents were 67.03 ± 0.68 g/mL and 64.72 ± 0.52 g/g, respectively. The mean sucrose content was 2.29 ± 0.65%. High mean values of phenolic (459.83 ± 1.92 mg gallic acid/kg), flavonoid (54.23 ± 0.62 mg catechin/kg), ascorbic acid (159.70 ± 0.78 mg/kg), AEAC (278.15 ± 4.34 mg/kg), protein (3381.83 ± 6.19 mg/kg) and proline (2131.47 ± 0.90) contents, as well as DPPH (39.57% ± 4.18) and FRAP activities [337.77 ± 1.01 µM Fe (II)/100 g], were also detected, indicating that Algerian honey has a high antioxidant potential. Strong positive correlations were found between flavonoid, proline and ascorbic acid contents and color intensity with DPPH and FRAP values. Thus, the present study revealed that Algerian honey is a good source of antioxidants. Molecules 2012, 17 11200 Keywords: Algerian honey; physicochemical properties; DPPH; FRAP; proline; ascorbic acid 1. Introduction Honey is a sweet and flavorful product that has been consumed over the years for its high nutritional values and beneficial effects on human health. According to a previous report [1], the chemical composition of honey is complex, containing approximately 181 substances including sugars, proteins, moisture, vitamins, minerals, 5-hydroxymethylfurfural (HMF), enzymes, flavonoids, phenolic acids and volatile compounds. The main constituents of honey are moisture, glucose, fructose, sucrose, minerals and proteins [2,3]. The quality of honey is mainly determined by its sensorial, chemical, physical and microbiological characteristics. The criteria for ensuring quality honey have been specified by the EC Directive 2001/110 [4]. The major criteria are moisture content, electrical conductivity, ash content, reducing and non-reducing sugars, free acidity, diastase activity and HMF content [2,5]. Honey contains a number of compounds and the antioxidant properties of honey are well known. The antioxidant properties of honey are derived from both enzymatic (e.g., catalase, glucose oxidase and peroxidase) and nonenzymatic substances (e.g., ascorbic acid, α-tocopherol, carotenoids, amino acids, proteins, Maillard reaction products, flavonoids and phenolic acids) [5–7]. The amount and type of these antioxidants are largely dependent on the floral source or honey variety, and a correlation between antioxidant activity with total phenolic content has been established [5,7]. Although honey is widely consumed in Algeria, there is still a lack of information on the physicochemical and antioxidant properties of Algerian honeys. To date, some physicochemical properties of Algerian honeys, such as pH, moisture content, electrical conductivity, total sugar content and proline have been reported [8,9], but other important physicochemical properties, such as HMF, sucrose and protein contents, as well as antioxidant properties (phenolics, flavonoids, ascorbic acid, DPPH, FRAP, AEAC), have never been reported. 2. Results and Discussion 2.1. Physical Analyses 2.1.1. pH of Honey Honey is naturally acidic irrespective of its geographical origin, which may be due to the presence of organic acids that contribute to its flavor and its stability against microbial spoilage. The pH of honey samples is important during the extraction process because it affects the texture of honey as well as its stability and shelf life [10]. All of the tested Algerian honey samples were acidic in nature, with pH values that varied between 3.70 and 4.00 (Table 1). These values were similar to those previously reported for other honey samples from India, Brazil, Spain and Turkey, which were reported to have pHs between 3.49 and 4.70 [11–13]. The pH values of Algerian honey samples have been previously reported to be Molecules 2012, 17 11201 3.49–4.43 [9] and 3.29–4.37 [8]. A highly acidic honey sample indicates the possible fermentation of sugars into organic acids. None of the investigated samples exceeded the allowed limit, which may be considered as an index of freshness of all honey samples. Table 1. Physical parameters (pH, moisture content, EC, TDS, color characteristics and HMF concentrations) of Algerian honey. Electrical Total Moisture Conductivity dissolved ABS 450 content HMF (mg/kg) Sample pH (EC) solids (TDS) (mAU; 50 w/v) (%) mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mS/cm ppm AH-1 3.70 ± 0.0 d 13.73 ± 0.12 b 0.417 ± 0.0006 d 208.0 ± 1.00 d 22.60 ± 0.02 c 724.00 ± 2.00 d AH-2 3.87 ± 0.06 b 11.59 ± 0.31 c 0.806 ± 0.0012 a 399.3 ± 1.53 a 24.21 ± 0.16 a 873.67 ± 2.52 c AH-3 4.00 ± 0.00 a 14.13 ± 0.12 a 0.764 ± 0.0023 b 381.7 ± 0.58 b 23.93 ± 0.29 b 1103.00 ± 3.61 b AH-4 3.80 ± 0.00 c 13.39 ± 0.12 b 0.558 ± 0.0006 c 278.7 ± 0.58 c 15.23 ±0.14 a 1188.00 ± 1.73 a Mean 3.84 ± 0.01 13.21 ± 0.16 0.636 ± 0.001 316.92 ± 0.92 21.49 ± 0.15 972.16 ± 2.46 Means are compared by using One way ANOVA-Post Hoc Multiple Comparisons; in each column, values with different letters (superscripts) indicate significant differences (p < 0.05). 2.1.2. Moisture Content The moisture content in the investigated honey samples was between 11.59 and 14.13%, which are within the limit (≤20%) recommended by the international quality regulations [4,14] (Table 1). Water content is very important for the shelf life of honey during storage [15] and can lead to undesirable honey fermentation due to osmotolerant yeasts, which form ethyl alcohol and carbon dioxide [16]. Generally, all of the investigated Algerian honey samples were of good quality, as indicated by the low moisture content. 2.1.3. Total Sugar Content The total sugar content of the honey tested was similar with the findings of other previously studied Algerian honeys [9]. None of the samples exceeded the highest limit set for total sugar content by the European community directive [4] (Table 2). Table 2. Reducing and non-reducing sugar content of Algerian honey. Sample Total sugar content Reducing sugar Sucrose mean ± SD% (g/mL) mean ± SD (%) g/g mean ± SD (%) AH-1 62.80 ± 1.06 a 60.19 ± 0.70 a 2.54 ± 0.71 a AH-2 65.73 ± 0.46 b 63.94 ± 0.67 b 1.80 ± 0.84 a AH-3 69.60 ± 0.40 a 67.08 ± 0.55 a 2.52 ± 0.37 a AH-4 70.00 ± 0.80 a 67.70 ± 0.18 a 2.30 ± 0.69 a Mean 67.03 ± 0.68 64.72 ± 0.52 2.29 ± 0.65 Means are compared by using One way ANOVA-Post Hoc Multiple Comparisons. In each column, values with different letters (superscripts) indicate significant differences (p< 0.05). Molecules 2012, 17 11202 2.1.4. Electrical Conductivity and Total Dissolved Solids EC is one of the most important factors for determining the physical characteristics of honey [17]. It is also an important physicochemical measurement for the authentication of unifloral honeys [18]. With the exception of a single sample (0.806 mS/cm), the EC values of samples were within the allowed parameters (lower than 0.8 mS/cm) (Table 1). The values of EC change when the amount of plant pollen decreases. According to Persano et al. [19], the nectars from some plants are ―stronger‖ than others, and even low contamination of honey with ―stronger‖ nectar can modify its sensory and physicochemical properties. The EC values of some Algerian honeys were reported to be 0.21–1.61 mS/cm in a previous study by Ouchemoukh et al. [9]. However, our results are similar to the findings previously reported by Saxena et al. [20] and Alvarez-Suarez et al. [21]. TDS is a measure of the combined content of all inorganic and organic substances in honey in the molecular, ionized or micro-granular (colloidal solution) suspended forms. Our results demonstrate that there is a good correlation between EC and TDS, indicating that both parameters can be used to determine honey purity. 2.1.5. Color Characteristics The primary characteristic for honey classification is color, which is classified according to USDA-approved color standards [22]. Honey color varies naturally in a wide range of tones, ranging from light yellow to amber, dark amber and black, in extreme cases, and sometimes even green or red hues may occur [23]. The color of untreated honey depends on its botanical origins. For this reason, color is very important for the classification of monofloral honeys for commercial activities. Honey darkens with age, and other changes in color may result from the beekeeper’s interventions and different ways of conservation, such as the use of old honeycombs, contact with metals, and exposure to high temperatures or light. One of the Algerian honey samples (AH-3) was dark amber in color and has the highest Pfund value, whereas the other three honey samples were amber in color (Figure 1). Figure 1. Color characteristics of Algerian honey. Molecules 2012, 17 11203 2.1.6. Color intensity (ABS ) 450 ABS is related to the presence of pigments, such as carotenoids and flavonoids, which are known 450 to have antioxidant properties [24]. ABS values of the investigated samples ranged from 724 to 450 1188 mAU (Table 1). This is the first ABS report for Algerian honey. The reported ABS values 450 450 for some Italian, Slovenian and Indian honeys were reported to be 25–3413 mAU, 70–495 mAU and 524–1678 mAU, respectively [13,25,26]. 2.1.7. Determination of HMF Concentrations by HPLC Method HMF formation results from the acid-catalyzed dehydration of hexose sugars with fructose being particularly susceptible. In addition, HMF is only present in trace amounts in fresh honey, and its concentration has been reported to increase with storage and the prolonged heating of the honey. HMF is thus an essential parameter used to indicate honey purity. With the exception of a single sample (AH-4) that contained 15.23 mg/kg of HMF, the HMF concentrations of the remaining honey samples were similar, ranging from 22.60 to 24.21 mg/kg (Table 1). Notably, all HMF concentrations were within the recommended range set by the Codex Alimentarius [27] at 80 mg/kg. The values are also within the allowed maximum limit of 40 mg/kg, as recommended by the Turkish Alimentarus Codex [28] for honey samples from tropical countries. We compared our results with honey samples from different countries. Persano et al. [29] reported low HMF concentrations of two unprocessed Australian honey samples, Grey box and Banksia (1.35 and 1.12 mg/kg, respectively). The HMF concentrations of some Australian honeys, such as rainforest, Homebrand and Mallee honey, were reported to be 2.2, 17.7 and 34.0 mg/kg, respectively [29,30]. High HMF formation may occur due to overheating, exposure to high temperatures [29] or the type of sugar present in the honey, as well as the fructose/glucose ratio [31]. Overall, the low HMF concentrations of the tested Algerian honey confirm that these samples are of good quality. 2.2. Antioxidant Analyses 2.2.1. Polyphenol Content The antioxidant activity of natural honeys depends largely on their chemical composition, such as phenolics, flavonoids, enzymes, organic acids, amino acids, Maillard reaction products, ascorbic acid, carotenoids, as well as their origins [32,33]. Thus, phenolics or polyphenols are one of the most important classes of compounds found in honey. The total concentration of phenols in honey is highly dependent on its plant source. High concentrations of polyphenols were found in all of our honey samples (Table 3). The highest concentration of polyphenols was determined to be 498.16 mg/kg for sample AH-4, indicating its high antioxidant potential. Because the content of phenolic compounds is usually lower in light-colored honey compared to that of dark honeys, the high levels of polyphenols in all honeys may contribute to its darker color [34]. All of our honey samples were amber to dark amber in color. The total polyphenol content of the tested Algerian honey is higher than those of two reported Malaysian honey samples, as well as that of Gelam and Coconut honeys [35], which are lighter in color. In addition, the phenolic content of Molecules 2012, 17 11204 the four honey samples analyzed were higher than those of natural honeys [1,26,32,36,37]. The high level of polyphenols in the studied Algerian honeys further indicates their high antioxidant properties. 2.2.2. Flavonoid Content Flavonoids are low molecular weight phenolic compounds that are vital components for the aroma and antioxidant properties of honey. Flavonoid content is expressed as mg of catechin per kg of honey. Similar to polyphenol content, the AH-4 sample contained the highest amount (71.78 mg/kg) of flavonoids (Table 3). This result is similar to a previous study in which honey samples with high polyphenol concentrations also contained high flavonoid levels [38]. The flavonoid content of these honeys is higher than that of Turkish [39] and Malaysian honeys, which range from 4.80 to 22.80 mg/kg and 11.52–25.31 mg/kg, respectively [38], indicating that Algerian honey has a higher antioxidant potential. Table 3. Biochemical and antioxidant properties of Algerian honey. Total Flavonoids FRAP values Proline Protein polyphenols Sample mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD (mg /kg) (µM Fe (II)/100 g) (mg/kg) (mg/kg) (mg /kg) catechin gallic acid AH-1 411.10 ± 1.55 d 27.07 ± 0.35 d 287.45 ± 0.92 d 1692.18 ± 1.00 d 3007.33 ± 3.54 d AH-2 483.01 ± 2.15 b 52.24 ± 0.03 c 306.60 ± 1.16 c 1946.01 ± 0.84 3031.67 ± 10.61 c AH-3 447.06 ± 2.67 c 65.85 ± 1.31 b 353.50 ± 0.65 b 2175.31 ± 0.78 b 3393.33 ± 7.07 b AH-4 498.16 ± 1.32 a 71.78 ± 0.84 a 403.54 ± 1.31 a 2712.39 ± 0.98 a 4095.00 ± 3.54 a Mean 459.83 ± 1.92 54.23 ± 0.62 337.77 ± 1.01 2131.47 ± 0.90 3381.83 ± 6.19 Means are compared by using One way ANOVA-Post Hoc Multiple Comparisons. In each column, values with different letters (superscripts) indicate significant differences (p < 0.05). 2.2.3. DPPH free Radical-Scavenging Activity The radical scavenging activities of honey samples were measured using the DPPH radical scavenging assay. DPPH is a stable nitrogen-centered radical that has been extensively used to test the free radical scavenging ability of various samples. In evaluating the radical-scavenging potential of honeys, the DPPH assay is frequently used because the antioxidant potential of honey has been shown to be directly associated with its phenolic and flavonoid contents [25]. High DPPH scavenging activity confers the superior antioxidant activity of the sample. The DPPH radical scavenging activities of all honey samples were measured at the following concentrations: 10, 20, 40, 60 and 120 mg/mL. The highest percentage of inhibition was observed at 120 mg/mL for all honey samples. The highest percentage of inhibition was exhibited by the AH-4 sample (44.57%), thus indicating its high antioxidant potential (Figure 2). The percentage of inhibition exhibited by Algerian honey is similar to that of some Malaysian [40] and Indian honey samples [13]. 2.2.4. Determination of Total Antioxidant Content by FRAP Assay To determine the antioxidant capacity of the honeys studied, a FRAP assay, which is a simple direct test widely used for the determination of antioxidant activity in many different samples, including Molecules 2012, 17 11205 honey [25,35,37,41–43], was used. Again, the AH-4 sample exhibited the highest FRAP values (403.54 ± 1.31 µM Fe (II)/100 g), confirming its high antioxidant properties (Table 3). These are the first FRAP values published for Algerian honey samples. High FRAP values indicate a greater reduction of ferric ions to ferrous ions. Samples with a higher reducing power increased in absorbance at 700 nm. Figure 2. Percentage of inhibition of DPPH radical scavenging activity at different concentrations of Algerian honey. 2.2.5. Proline Content Proline is an important amino acid that originates mostly from the salivary secretions of Apis mellifera during the conversion of nectar into honey [44]. Proline content is an indication of honey ripeness and, in some cases, sugar adulteration. Proline was detected in high concentrations (1692–2712 mg/kg) in all of Algerian honey samples tested. Some authors have reported that high concentrations of proline are also typical for honeydew honeys [9,23,32]. 2.2.6. Ascorbic Acid and AEAC Assay The AEAC content of Algerian honey samples was measured in mg of AEAC/100 g of honey using an ascorbic acid standard curve (r2 = 0.9447). Algerian honey samples exhibited AEAC values ranging from 236.80 to 315.90 mg of AEAC/kg (Figure 3). These values are similar to those of honeys from Burkina Fasan [32], whereas Indian honey samples exhibited lower values (between 151 and 295 mg of AEAC/kg) [13]. To our knowledge, this is the first report on the ascorbic acid content and AEAC values of Algerian honeys. A negative correlation was observed between ascorbic acid and AEAC values in honey samples that contained higher ascorbic acid and had lower AEAC values. Thus, the high ascorbic acid content contributes to the reduction in DPPH free radical activity. Molecules 2012, 17 11206 Figure 3. Ascorbic acid and AEAC contents of Algerian honey. Values with different letters indicate significant differences (p < 0.05). 2.3. Biochemical Analyses 2.3.1. Reducing Sugar Content The total reducing sugar content in honey samples ranged from 62.80 to 70.00% (Table 2). Our data indicate that reducing sugars are the major soluble sugars present in Algerian honey samples. With respect to reducing sugars (fructose and glucose), the EC Directive 2001/110 imposes that the amount of reducing sugars should be ≥60 g/100 g, with the exception of honeydew honey, which has a lower limit (≥45 g/100 g). Our results meet this standard and are similar to other published levels for reducing sugars [13,45]. The amount of non-reducing sugars, such as sucrose content (%), was measured by subtracting the reducing sugar content from total sugar content, which is expressed by the following equation: Sucrose content (%) = Total sugar content − Reducing sugar. The sucrose content in Algerian honey samples ranged from 1.80 to 2.54%, which is below 5% or the maximum prescribed limit set by the Codex standard [14]. 2.3.2. Protein Content The concentrations of proteins and amino acids in honeys vary depending on their botanical or geographical origin and storage time. Protein content in honey samples are reported to consist of mainly enzymes [3]. Different enzymes are also added by bees during the process of honey ripening, indicating that the colorimetric determination of the protein content of honey samples using the method of Lowry is suitable. High protein content (4,097.00 ± 3.54 mg/kg) was found in AH-4 when compared to the mean protein content of other analyzed honeys. Protein content in honey generally ranges from 2,000 to 5,000 mg/kg [46]. Molecules 2012, 17 11207 2.4. Correlation amongst Biochemical Parameters and Antioxidant Properties The correlation matrix (Table 4) indicates a significant correlation between biochemical and antioxidant parameters. A strong correlation was found between the color intensity of honey samples and the antioxidant parameters, flavonoid and proline contents, as well as the DPPH and FRAP values, at 0.968, 0.934, 0.964, 0.963, respectively. Thus, color pigments may have a role in the observed antioxidant activities of honey samples. The color intensity also increases with increases in the phenolic and flavonoid contents of honey. A strong correlation between ABS , DPPH and FRAP values suggest the involvement 450 of pigments that confer antioxidant properties to honey. A study conducted by Terrab et al. [26] also found a strong correlation between ABS and FRAP values (r = 0.85) in Slovenian honeys. In Indian honeys, 450 the correlation between ABS and FRAP values was 0.83 [13]. Thus, the higher correlation in our study 450 indicates that Algerian honeys have a stronger antioxidant capacity compared to Indian honeys. Table 4. Correlation matrix showing the interrelation among phenolics, flavonoids, DPPH scavenging, FRAP, ascorbic acid, proline, ABS and protein. 450 Ascorbic Phenolics Flavonoids DPPH FRAP Proline ABS Protein acid 450 Phenolics 1.000 0.776 ** 0.615 * 0.668 * 0.165 0.764 ** 0.662 * 0.629 * Flavonoids 0.776 ** 1.000 0.888 ** 0.893 ** 0.730 ** 0.887 ** 0.968 ** 0.778 ** DPPH 0.615 * 0.888 ** 1.000 0.982 ** 0.785 ** 0.956 ** 0.964 ** 0.940 ** FRAP 0.668 * 0.893 ** 0.982 ** 1.000 0.749 ** 0.987 ** 0.963 ** 0.973 ** Ascorbic acid 0.165 0.730 ** 0.785 * 0.749 ** 1.000 0.644 * 0.828 ** 0.646 * Proline 0.764 ** 0.887 ** 0.956 ** 0.987 ** 0.644 * 1.000 0.934 ** 0.974 ** ABS 0.662 * 0.968 ** 0.964 ** 0.963 ** 0.828 ** 0.934 ** 1.000 0.876 ** 450 Protein 0.629 * 0.778** 0.940 ** 0.973** 0.646 * 0.974 ** 0.876 ** 1.000 ** Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed). A positive significant linear correlation was observed between the following antioxidant parameters: flavonoid content with DPPH RSA (r = 0.888) and FRAP values (r = 0.893). Similar correlations between DPPH RSA and flavonoid content (r = 0.888) were observed in some Malaysian honey samples [38]. However, the correlations between phenolics with DPPH radical scavenging activity (r = 0.615) and FRAP values (r = 0.668) were lower than with flavonoids. The correlation between DPPH RSA and total phenolic content suggest that phenolics were the strongest contributing factor to the RSA of these honeys compared to FRAP. Proline is an important amino acid that confers antioxidant properties to honey and strongly correlates with DPPH, FRAP and ABS . The most significant correlation was observed between proline 450 content and FRAP values (r = 0.987), which is higher than those values reported by Diez et al. [32], indicating that proline content also contributes to the antioxidant potential of Algerian honey. The correlation between DPPH scavenging activity and proline content was 0.956. However, in another report, the correlation between proline content and DPPH RSA was lower (0.75) [32], even though the correlation in Indian honey samples (r = 0.94) was similar to our results [13]. Thus, as with Indian honey samples, the total proline content in the honey samples in this study may be a critical factor responsible for the antioxidant activity of Algerian honey. However, in future, it will be good to confirm these findings with larger number of samples. Molecules 2012, 17 11208 To summarize, proline content may be a significant determinant of the antioxidant capacity of Algerian honey samples as well as their reducing ability and radical scavenging potential. Protein content was also strongly correlated with proline content (r = 0.974), FRAP (r = 0.973) and DPPH RSA (r = 0.940) values. Ascorbic acid is an important vitamin that is well known for its antioxidant properties. It was observed to significantly correlate with flavonoids (r = 0.730), DPPH RSA (r = 0.785) and FRAP (r = 0.749). The best correlation between ascorbic acid and ABS was 450 observed at r = 0.828, indicating that similar to ascorbic acid content, color pigments are good indicators of antioxidant potential. These correlations demonstrate that the overall antioxidant property in the investigated Algerian honeys can be attributed to various factors, such as proline, phenolic, flavonoid and ascorbic acid contents, as well as color pigments. Furthermore, proline content appears to be highly important for antioxidant activity as shown by the correlation values. 3. Experimental 3.1. Honey Samples Four local Algerian honey samples (AH1, AH2, AH3 and AH4) were purchased from supermarket shelves in the capital of Algeria between May 2010 and July 2010. All of the honey samples were stored at room temperature (22–24 °C) in airtight plastic containers until analysis. 3.2. Chemicals and Reagents Ascorbic acid, bovine serum albumin (BSA), catechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(1-pyridyl)-1,3,5-triazine (TPTZ), HMF, Folin–Ciocalteu’s reagent, gallic acid and proline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate (Na CO ), aluminum 2 3 chloride (AlCl ), sodium nitrite (NaNO ) and sodium hydroxide (NaOH) were purchased from Merck 3 2 (Darmstadt, Germany). All chemicals used were of analytical grade. 3.3. Physical Analysis 3.3.1. pH A pH meter (HI 98127, Hanna instruments, Mauritius) was used to measure the pH of a 10% (w/v) solution of honey prepared in milli-Q water (Millipore Corporation, Billerica, MA, USA). 3.3.2. Moisture Content The moisture content was determined using a refractometric method. In general, the refractive index increases with increases in the solid content of a sample. The refractive indices of honey samples were measured at ambient temperature using an Atago handheld refractometer (KRUSS, HRH30, Hamburg, Germany), and measurements were further corrected for the standard temperature of 20 °C by the addition of the 0.00023/°C correction factor. The moisture content was measured in triplicate, and the percentage of moisture content, which corresponds to the corrected refractive index, was calculated using Wedmore’s table [47].