Physicochemical and Antioxidant Properties of Honeys from the Sundarbans Mangrove Forest of Bangladesh

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

This study evaluated the physicochemical, nutritional, antioxidant, and phenolic properties of ten honey samples from the Sundarbans mangrove forest, Bangladesh. The average pH, electrical conductivity, total dissolved solid, ash, moisture, hydroxymethyl furfural, titrable acidity, and absorbance were 4.3, 0.38 mS/cm, 187.5 ppm, 0.14%, 17.88%, 4.4 mg/kg, 37.7 meq/kg, and 483 mAU, respectively. In the honeys, the average contents of Ca, Cu, Fe, K, Mg, Mn, and Na were 95.5, 0.19, 6.4, 302, 39.9, 3.4, and 597 ppm, respectively, whereas Cd, Cr, Pb, and Ni were not found. The average contents of total sugar, protein, lipid, vitamin C, polyphenols, flavonoids, and anthocyanins in the honeys were 69.3%, 0.8%, 0.29%, 107.3 mg/kg, 757.2 mg gallic acid equivalent/kg, 43.1 mg chatechin equivalent/kg, and 5.4 mg/kg, respectively. The honeys had strong 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, reducing power and total antioxidant capacity. High-performance liquid chromatography analysis of the honey fractions revealed the quantification of six polyphenols namely, (+)-catechin, (−)-epicatechin, p-caumeric acid, syringic acid, trans-cinnamic acid, and vanillic acid at 194.98, 330.34, 74.64, 218.97, 49.55, and 118.84 mg/kg, respectively. Therefore, the honeys in the Sundarbans are of excellent quality and a prospective source of polyphenols, and antioxidants.

INTRODUCTION

Sundarbans, the world’s largest contiguous tract of mangrove forest, is located in the South-Western regions of Bangladesh. This mangrove ecosystem produces about 50% of the total production of honey in the country (1). Harvesting the honey from the Sundarbans is open to the public from April to June. Among the various plant species in the Sundarbans, the flowering periods of 12~13 plant species are synchronizing with the time of honey collection. At that time, the giant honey bee, Apis dorsata, collects nectars mainly from the flowers of Acanthus ilicifolius, Aegicerus majus, Avicennia alba, Avicennia officinalis, Brugiera gymnorrhiza, Ceriops decandra, Cynometra ramiflora, Excoecaria agallocha, Heritiera fomes, Rhizophora mucronata, Sonneratia apetala, Sonneratia caseolaris, and Xylocarpus mekingensis, and store the honey in the combs built in an open place on the branches of the trees (2). The physicochemical characteristics of these multi-floral honeys are possibly different from those of other honeys around the world due to a unique floral composition, geographical origin, and environmental conditions.

Simple sugars, such as glucose (31%) and fructose (38 %), are the major components in honeys, whereas proteins, phenolic compounds, free amino acids, carotenoids, organic acids, minerals, enzymes, vitamins, and aroma compounds constitute the minor components (3–5). Reportedly, honey has more than 500 active components and is considered as part of many traditional medicines and cultures. These components contribute to anti-bacterial, anti-oxidant, anti-inflammatory, anti-browning, anti-allergic, anti-parasitory, anti-ulcer, anti-tumor, and anti-viral activities (5,6). Vitamins such as phyllochinon (K), thiamin (B₁), riboflavin (B₂), niacin (B₃), panthothenic acid (B₅), pyridoxin (B₆), folic acid (B₉), ascorbic acid (C), and α-tocopherol (E) are present in small amounts in honey, and their contribution to the recommended daily intake is marginal (4,5). The physicochemical characteristics of honeys from different regions of the world have been studied in Malaysia (7), Algeria (8), Portugal (9), and India (10). These characteristics include moisture content, electrical conductivity, reducing and non-reducing sugars, free acidity, and hydroxymethylfurfural (HMF) which refer to the quality criteria of honey as specified in the EC Directive 2001/110 (11). At present, the antioxidant potential of honey is also being considered as a useful quality criterion. Honey with high antioxidant potential must have high amounts of functional components. The content of polyphenols and flavonoids contribute to the antioxidant capacity of honey (6). Amino acids, ascorbic acid, carotenes, flavonols, organic acids, protein, selenium, α-tocopherol, glucose oxidase, catalase, and peroxidase are also antioxidants in honey (4–6, 12). It was reported that compared to rats fed with fructose, honey-fed rats had higher plasma α-tocopherol levels, higher α-tocopherol/triacylglycerol ratios, lower plasma nitrate levels, and lower susceptibility of the heart to lipid peroxidation (13). Selenium is an essential trace element especially for 1 to 15 years old children (5). Incorporating into selenoproteins, selenium is involved in various cellular processes such as removal of peroxides, reduction of oxidized proteins and membranes, and regulation of redox signaling (14). Antioxidant compounds inhibit the pathogenesis of various diseases including cataract, cancer, diabetes, inflammation, artherosclerosis, cardiovascular, and neurodegenerative diseases (15). Recently, studies on the physicochemical and antioxidant properties of both monofloral and multifloral honeys from different parts of Bangladesh have been conducted except for the honeys of the Sundarbans (16,17). Every year, natural honey is collected from the Sundarbans, and it is popularly consumed in South-Asian countries, especially in Bangladesh and India, whereas no reports showed detailed study of the physical, nutritional, mineral, antioxidant properties as well as polyphenolic compounds in the honeys.

MATERIALS AND METHODS

Chemicals and reagents

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Arbutin, benzoic acid, bovine serum albumin, caffeic acid, (+)-catechin hydrate, trans-cinnamic acid, p-coumaric acid, ellagic acid, (−)-epicatechin, trans-ferulic acid, Folin-Ciocalteu’s phenol reagent, gallic acid, hydroquinone, kaempferol, myricetin, quercetin, rosmarinic acid, rutin hydrate, syringic acid, vanillic acid, and vanillin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Acetonitrile, acetic acid, ascorbic acid, diethyl ether, dimethyl sulfoxide (DMSO), ethanol, HCl, H₂SO₄, and methanol were obtained from Merck (Darmstadt, Germany).

Honey samples

Ten composite samples of honeys namely S1, S2, S3, S4, S5, S6, S7, S8, S9, and S10 were collected from the honey collectors in the Sundarbans from March to July in 2015. Two composite samples were collected each month from different parts of the Sundarbans. The collected honey samples were taken in the laboratory and kept in a refrigerator at 4°C in air tight glass containers.

Fractionation of honey

Five grams of each sample was placed in a beaker to make 50 g, and the honey was mixed thoroughly. Then, 20 g of honey was placed in an airtight container. It was then extracted by adding 200 mL of 100% diethyl ether and vigorous shaking the mixture for 30 min. Hereafter, the mixture was filtered through Whatman filter paper no. 1. The filtrate was air-dried, and the extract was stored at 4°C in a refrigerator as the diethylether fraction. Similarly, ethanol, methanol, and distilled water fractions were successively prepared following the same procedure using the residues on the filter paper. The diethyl ether, ethanol, methanol, and water fractions were designated as DEH, ETH, MEH, and DWH, respectively. Finally, 10 mg of the solid was dissolved in 1 mL DMSO (10 mg/mL) to determine the total antioxidant capacity and amounts of different polyphenols.

Determination of the physicochemical properties of honeys

The pH of the honeys was determined according to the method described by the International Honey Commission (18). Electrical conductivity (EC) and total dissolved solid (TDS) were measured according to the harmonized methods of the European Honey Commission (19). The ash content of the honeys was determined as described by Piazza et al. (20). The moisture content of the honeys was determined according to the method followed by Association of Official Analytical Chemists (AOAC) (21). HMF content in the honeys was determined according to White (22). Titrable acidity (TA) of the honeys was determined according to the method of AOAC (23). The color intensity of honeys was determined using the method of Beretta et al. (24). The absorbance (ABS) was taken at 450 and 720 nm, and intensity was calculated using the formula, ABS₄₅₀=(ABS₄₅₀−ABS₇₂₀)×1,000 mAU.

Determination of the nutritional properties of honeys

The total carbohydrate of the honeys was determined by the titrimetric method (25). Protein contents were calculated by the Lowry et al. (26) method. Total lipids were determined by extracting the honey with chloroform: methanol (1:2) (27). The vitamin C content was determined as described by Plummer (28) using 2,6-dichlorophenolindophenol with minor modifications, and was expressed as mg ascorbic acid/kg honey.

Mineral contents in the honey samples were estimated as described by Hoenig and de Kersabiec (29) with slight modifications. The concentrations of Ca, Cd, Cr, Cu, Fe, Pb, Mg, Mn, Ni, and Zn were determined by flame atomic absorption spectrophotometry. One g of honey was placed in a 50 mL flask and 15 mL of HNO₃ and HClO₄ as a ratio of 2:1 was added. The mixture was heated in a fume hood (Esco Frontier Acid Digestion, ESCO Pte. Ltd., Singapore) on a hot plate (model VWR, VELP Scientifical, Frankfurt, Germany). Generation of white fumes from the flasks indicated the completion of digestion, and the flasks were allowed to cool. These digested samples were transferred into 100 mL volumetric flasks, and the volume was adjusted to 100 mL by adding distilled water. Then, the extract was filtered with filter paper (Whatman no. 42), and the filtrate was collected in labeled plastic bottles. The solutions were analyzed for the content of elements using an atomic absorption spectrophotometer (Shimadzu AA-7000, Shimadzu Corporation, Kyoto, Japan) with suitable hollow cathode lamps. The concentrations of different elements in honeys were determined by the corresponding standard calibration curves obtained by using standard analytical reagent grade solutions of the elements, Ca, Cd, Cr, Cu, Fe, Pb, Mg, Mn, Ni, and Zn. A 0.5 M chloride solution containing 20% trichloroacetic acid and 10% lanthanum chloride (w/v) was added to the sample used for Ca measurement to prevent interference by coexisting elements. A 0.5 M chloride solution containing 10% lanthanum chloride was added to the sample used for Mg measurement. Digested honeys were used to determine the concentration of Na and K using a flame photometer.

Determination of total polyphenols (TPH), flavonoids (TF), and anthocyanins

The concentration of TPH in the honeys was determined according to the Folin-Ciocalteu method (30) with gallic acid (GA) as the standard and expressed as gallic acid equivalents (mg GAE). The TF content in the honeys was determined by the colorimetric assay described by Zhishen et al. (31). The results were expressed as (+)-catechin equivalents (mg CE). Total anthocyanin was estimated using the method described by Fuleki and Francis (32), and the results were expressed as μg/g honey.

DPPH free radical scavenging activity

The reaction mixture (total volume, 3 mL), consisting of 0.5 mL of a 0.5 M acetic acid buffer solution at pH 5.5, 1 mL of 0.2 mM DPPH in ethanol, and 1.5 mL of a 50% (v/v) ethanol aqueous solution, was shaken vigorously with the honey according to Blois (33). After incubation at room temperature for 30 min, the amount of remaining DPPH was determined by measuring the absorbance at 517 nm. Mean values were obtained from triplicate experiments.

Reducing power capacity

The reducing power of the honeys was determined according to the method of Oyaizu (34). Briefly, different concentrations of the honeys were mixed with 2.5 mL of 0.2 M phosphate buffer, pH 6.6 and 2.5 mL of 1% potassioum ferricyanide solution. After incubation at 50°C for 20 min, the mixtures were mixed with 2.5 mL of 10% trichloroacetic acid followed by centrifugation at 650 g for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride. The absorbance of this solution was measured at 700 nm. Ascorbic acid served as the positive control.

Total antioxidant capacity (TAC)

The TAC assay was done according to the method described by Prieto et al. (35). The tubes containing honey or a honey fraction and reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) were incubated at 90°C for 90 min. After cooling at room temperature, the absorbance was measured at 695 nm against a blank. The antioxidant capacity was expressed as mg ascorbic acid equivalents (AAE/g honey or fraction) and GAE/g honey or fraction.

Determination of phenolic compounds in the fractions

Detection and quantification of selected phenolic compounds in the fractions were determined by high-performance liquid chromatography (HPLC)-diode-array detection (DAD) analysis as described by Jahan et al. (36) with some modifications. The analysis was carried out on a Dionex UltiMate 3000 system equipped with a quaternary rapid separation pump (LPG-3400RS, Thermo Fisher Scientific, Waltham, MA, USA) and photodiode array detector (DAD-3000RS, Thermo Fisher Scientific). Separation was performed using an Acclaim ® C₁₈ (5 μm) Dionex column (4.6×250 mm, Thermo Fisher Scientific) at 30°C with a flow rate of 1 mL/min and an injection volume of 20 μL. The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C) with the gradient elution program of 5%A/95%B (0~5 min), 10%A/90%B (6~9 min), 15%A/75%B/10%C (11~15), 20%A/65%B/15%C (16~19 min), 30%A/50%B/20%C (20~29 min), 40%A/30%B/30%C (30~35 min), and 100%A (36~40 min). The UV detector was set to 280 nm for 22 min, changed to 320 nm for 28 min, again changed to 280 nm for 35 min, and finally to 380 nm for 36 min and held for the rest of the analysis period while the diode array detector was set at an acquisition range from 200 nm to 700 nm. For the preparation of the calibration curve, a standard stock solution was prepared in methanol containing arbutin, (−)-epicatechin (5 μg/mL each), gallic acid, hydroquinone, vanillic acid, rosmarinic acid, myricetin (4 μg/mL each), caffeic acid, syringic acid, vanillin, transferulic acid (3 μg/mL each), p-coumaric acid, quercetin, kaempferol (2 μg/mL each), (+)-catechin hydrate, ellagic acid (10 μg/mL each), trans-cinnamic acid (1 μg/mL), rutin hydrate (6 μg/mL), and benzoic acid (8 μg/mL). A solution of the fraction was prepared a concentration of 10 mg/mL. Prior to HPLC analysis, all the solutions (mixed standards, sample, and spiked solutions) were filtered through a 0.20 μm syringe filter (Sartorius AG, Göttingen, Germany) and then degassed in an ultrasonic bath (Hwashin, Seoul, Korea) for 15 min. Data acquisition, peak integration, and calibrations were calculated with the Dionex Chromeleon software (version 6.80 RS 10, Dionex, Sunnyvale, CA, USA).

Statistical analysis

Statistical analysis was performed using SPSS (version 16, SPSS Inc., Chicago, IL, USA). Results were expressed as mean±standard deviation (SD) for a given number of observations, n=3~10. One way analysis of variance (ANOVA) followed by least significant difference (LSD) multiple comparison post-hoc tests were used to analyze the statistical difference. Differences with P-values

RESULTS AND DISCUSSION

Physicochemical properties

The physicochemical properties of honey are attributed to its characteristics, tests, quality, and functional parameters. The average pH, EC, TDS, ash, moisture, HMF, TA, and ABS₄₅₀ of the honeys were 4.3, 0.38 mS/cm, 187.5 ppm, 0.14%, 17.88%, 4.4 mg/kg, 37.7 meq/kg, and 483 mAU, respectively ( Table 1 ). The Codex Alimentarius (37) set up the standard quality criteria of honeys for authenticity, and that includes physical, nutritional, and chemical properties. The pH values of the analyzed honeys ranged from 3.9 to 4.4, and none of them exceeded the allowed limit of 3.2 to 5.0 set by the Codex Alimentarius (37). The Codex Alimentarius is an index of freshness of the honeys, and reflects the ability to inhibit the growth of microorganisms. These values were similar to previously reported honeys from Bangladesh, pH 3.2~4.5 (16), and also from other countries such as Brazil (38) and India (10). The honeys showed smaller EC values than the maximum limit of 0.8 mS/cm from the Codex Alimentarius (37), suggesting that they were nectar honeys. Our study also showed that honey samples with the highest EC had the highest TDS. Ranging from 0.09 to 0.18%, the ash content were lower than the allowed limit of 0.6% for floral honeys, indicating the honeys were clear and free from adulteration. The moisture content in the analyzed honeys ranged from 13.5 to 19.9%, which was less than the maximum limit of 20% (37). Moisture is one of the most important factors that determine the quality of honeys. Moisture content determines the growth of osmotolerant microorganisms in honeys. Low moisture prevents the growth of microorganisms resulting in the protection of quality and increases the shelf-life of honey, whereas high moisture shows adverse effects. Therefore, honeys of the Sundarbans exhibited low moisture content and thus were of good quality. The HMF content was determined to know the freshness and quality of the honeys. All the analyzed honeys showed HMF levels within the allowed limits of 40 mg/kg (37) that demonstrated their freshness and good quality. Reportedly, HMF is absent in fresh honey whereas various factors such as aging, processing, temperature, pH, and floral source influence its levels. Free acidity in honey is caused by the presence of organic acids in equilibrium with their corresponding lactones or internal esters, and some inorganic ions, such as phosphate, sulphate, and chloride (39). Free acidity should be within the limits of

Table 1

Physicochemical characteristics of the honeys

EC, electrical conductivity; TDS, total dissolved solid; HMF, hydroxymethylfurfural; TA, titrable acidity; ABS₄₅₀, absorbance at 450 nm.

Values represent the means±SD (n=3~10).