Notes
Antioxidant Activity of Different Grades of Maple Syrup as Determined by the Hydrophilic Oxygen Radical Absorbance Capacity Method
2015 Volume 21 Issue 3 Pages 495-498
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2015 Volume 21 Issue 3 Pages 495-498
Maple syrups are prepared by thermally concentrating maple sap. Based on their clarity, density and characteristic taste, they are divided into 5 grades: extralight, light, medium, amber and dark. This study aimed to evaluate differences in antioxidant activities among grades by the hydrophilic oxygen radical absorbance capacity (H-ORAC) method. The results demonstrated that H-ORAC values varied widely depending on the degree of brown coloration; darker-colored maple syrups showed stronger antioxidant activity. The darker-colored grades of maple syrup also contained more reducing sugars (fructose and glucose) than the lighter-colored ones; however, the grade had little impact on the content of free amino nitrogen in the syrup samples. The present study suggests that the brown pigments (melanoidins) produced by condensation of amines and reducing groups may contribute significantly to the antioxidant activity of maple syrups.
Maple syrups are consumed as a natural sweetener rich in flavor. Eighty-five percent of the annual worldwide production of maple syrup takes place in Canada (Perkins and van den Berg, 2009). Maple syrups are manufactured by thermally evaporating the sap of the sugar maple (Acer saccharum), which is collected during the early springtime and yields a total solid of approximately 66° brix. Syrups prepared with sap tapped at the beginning of the season are generally clearer and lighter in taste (Clément et al., 2010); as the season advances, they darken and become stronger in flavor. According to their clarity, density and characteristic taste, Canadian maple syrups are grouped into five different grades: grade AA (Canada No. 1 — Extra-light); grade A (Canada No. 1 — Light); grade B (Canada No. 1 — Medium); grade C (Canada No. 2 — Amber); and grade D (Canada No. 3 — Dark). In general, lighter-colored maple syrups have a more delicate flavor and darker-colored ones have a stronger taste. The flavor of maple syrup may also be influenced by where the sugar maple trees are grown.
Maple syrup is primarily composed of sugars, water, and minerals (Leaf, 1964). In addition, small amounts of other nutrients such as amino acids, proteins, and some organic acids are contained (Ball, 2007; Stuckel and Low, 1996). Recent investigations have paid an increasing amount of attention to the antioxidant potential of maple syrups. Phillips et al. (2009) reported that the total antioxidant activity of maple syrups, as measured by the ferric-reducing ability, was higher compared to honey and light-brown sugar. The use of maple syrups as an alternative to refined sugar might contribute to the antioxidant power of the diet. However, to date, detailed information regarding the effectiveness of the antioxidant activity of maple syrups among the different grades is limited. This study aimed to better assess the antioxidant activity of commercially available maple syrup samples by the hydrophilic oxygen radical absorbance capacity (H-ORAC) method, which has been widely accepted as a standard tool for measuring the antioxidant activity (Prior et al., 2005).
Chemicals and samples Fluorescein sodium salt was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), sucrose, fructose, glucose, and ninhydrin were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acetonitrile of HPLC quality was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Buffer salts and all other chemicals were of analytical grade.
In this study, 32 Canadian pure maple syrup samples (6 extra-light, 7 light, 7 medium, 5 amber, and 7 dark) collected during a 4-year production period, which were commercially available on the Japanese market, were analyzed.
Measurement of antioxidant potential by H-ORAC method The H-ORAC assay was carried out according to the method established in the literature (Watanabe et al., 2012). Analyses were conducted in the hydrophilic system composed of fluorescein, AAPH, and Trolox as a control standard. All reagents were prepared in 75 mM potassium phosphate buffer (pH 7.4). Thirty-five microliters of Trolox solutions (50, 25, 12.5, and 6.25 µmol/L for calibration), or test samples diluted in the assay buffer solution, were added to each well of the black and flat-bottomed 96-well microplate. The reaction was initiated by adding 115 µL of 110.7 nmol/L fluorescein and 50 µL of 31.7 mmol/L AAPH to the wells. The fluorescence intensity (excitation at 485 nm, emission at 528 nm) at a temperature of 37°C was monitored every 2 min for 90 min using a microplate reader (Infinite F200 PRO; Tecan Japan Co., Ltd., Kawasaki, Japan). The area under the curve (AUC) of fluorescence decay from 8 to 90 min after the addition of AAPH was calculated for each well. The net AUC was calculated by subtracting AUC for the sample or standard from that for the blank. A calibration curve was constructed from the net AUCs of Trolox standard solutions.
Color measurement The color intensity of maple syrup samples after diluting 10-times with distilled water was recorded by measuring the absorption at a wavelength of 420 nm, using a spectrophotometer (UV-VIS 1400; Shimadzu Co., Kyoto, Japan) with a 1 cm-path length cell. Distilled water was used as a blank.
Analysis of sucrose, glucose and fructose High performance liquid chromatographic determination of sugar content in different grades of maple syrup was carried out in a system equipped with an LC-10AD pump, a DGU-14A degasser, a CTO-10AS VP column oven, and an RID-10A refractive index detector. Separation of the sugars was conducted on a 3-µm silica-based aminopropyl column (Unison UK-amino; Imtakt Co., Kyoto, Japan), with a mobile phase of acetonitrile/water (80/20, v/v) at a flow rate of 0.8 mL/min. For glucose and fructose, maple syrup samples were diluted by a factor of 10 with 50% (v/v) aqueous acetonitrile, and for sucrose, the samples were diluted by a factor of 100. Standard solutions and diluted maple syrup samples were passed through a 0.45-µm membrane filter (Ekicrodisc type 13; Pall Japan Co., Tokyo, Japan). Ten microliters of the resulting filtrates were injected into the HPLC system. From the chromatograms, standard curves were constructed according to the peak areas and sugar concentrations of the standard solutions.
Analysis of free amino nitrogen by ninhydrin reaction The ninhydrin method was applied to measure the levels of free amino nitrogen (Coghe et al., 2005). Exactly 0.5 mL of color reagent (100 g/L Na2HPO4·12H2O, 60 g/L KH2PO4, 5 g/L ninhydrin and 3 g/L fructose) was added to 1.0 mL of the 10-times aqueous diluted maple syrup sample in a glass tube. The sample was placed in a water bath (100°C) for 16 min. After cooling in an water bath (20°C) for 20 min, 2.5 mL of a dilution reagent (2 g KIO3 in 1 L of 60% (v/v) aqueous ethanol) was added to the reaction mixture. Samples were centrifuged at 10,000×g for 10 min, and then the colorimetric absorbance was measured at 570 nm. A sample blank was prepared by adding the color reagent without ninhydrin to the diluted maple syrup samples to be analyzed. Spectrophotometric quantification was made using a glycine standard solution.
Statistical analysis Results are shown as the mean ± standard deviation. Analysis of variance (ANOVA) was performed using a software package (GraphPad Prism 5 for Windows, GraphPad Software, Inc., CA, USA). Pearson's correlation coefficient was used to characterize the association between antioxidant activity and absorbance value at 420 nm. Differences were considered significant at p < 0.05.
The antioxidant activity of different grades of maple syrup, as determined by the H-ORAC method, is presented in Fig. 1A. Antioxidant activity (expressed as µmol Trolox Equivalents (TE)/100 g) of samples was in the following rank order: extra-light (576 ± 185) < light (673 ± 143) < medium (820 ± 126) < amber (1136 ± 182) < dark (1502 ± 310). The grade of maple syrup was observed to be closely associated with antioxidant activity. This result is supported by Singh et al. (2014), who revealed that very dark syrup possessed the most effective activity against scavenging 2,2-diphenyl-1-picrylhydrazyl radicals among 3 grades of maple syrup (amber, dark and very dark). This is the first study to demonstrate the inhibitory effect of 5 grades of maple syrup against peroxyl radical-induced AAPH oxidation. Further measurement via spectrometric absorbance at 420 nm (analytically used to assess the degree of browning) shows the strong positive correlation with H-ORAC values (correlation coefficient value of 0.902 (p < 0.0001)), indicating that darker maple syrup possessed higher antioxidant content (Fig. 1B).
Antioxidant activity of maple syrup as determined by H-ORAC methods. A, Differential antioxidant activity among the grades of maple syrup; a significant difference (p < 0.0001) by ANOVA. B, positive correlation between antioxidant activity and absorbance value at 420 nm (p < 0.0001).
It is recognized that the color of maple syrup mainly results from the degree of thermal evaporation of maple sap. The duration and intensity of heating strongly impact the color development. This may be due to the nonenzymatic reaction (often called the Maillard reaction) of reducing sugars in combination with certain components having amino bases, which forms brownish polymeric components (melanoidins). The present study also measured the concentrations of reducing sugars in the respective grades of maple syrup by HPLC (Table 1). Of the 32 samples tested, total sugar content (the sum of sucrose, fructose and glucose) accounted for up to 65.0 ± 1.3 g/100 g of maple syrup. Sucrose occupied the largest part of total sugar content in maple syrup. Both fructose and glucose were less common; the level of fructose (1.2 ± 0.4 g/100 g of maple syrup) was approximately twice that of glucose (0.7 ± 0.5 g/100 g), but were significantly increased with further browning. Lighter maple syrup contained significantly more sucrose, and less fructose and glucose. Table 1 also shows the glycine-equivalent amino nitrogen concentrations in different grades of maple syrup, as determined by the ninhydrin reaction. Compared to the total sugar content, maple syrups had considerably smaller amounts of amino nitrogen, with large intra-group variance. The ANOVA revealed that there was no significant difference among the grades of maple syrup (p = 0.51). As previously mentioned, the reaction of components having amino bases with reducing sugars during the thermal evaporation process may influence the degree of browning of maple syrup. Since the level of amino bases did not differ substantially among the respective grades of maple syrups, differences in the level of reducing sugars in the original maple sap may have the greatest impact on the Maillard reaction.
| Grades of maple syrup2 | ANOVA | |||||
|---|---|---|---|---|---|---|
| Extralight | Light | Medium | Amber | Dark | ||
| Sugars (g/100g of maple syrup) | ||||||
| Sucrose | 64.9 ± 1.8 | 63.8 ± 1.0 | 63.8 ± 1.1 | 62.0 ± 1.7 | 61.3 ± 1.0 | p = 0.0002 |
| Fructose | 0.8 ± 0.4 | 0.9 ± 0.1 | 1.1 ± 0.2 | 1.2 ± 0.2 | 1.7 ± 0.3 | p < 0.0001 |
| Glucose | 0.4 ± 0.4 | 0.3 ± 0.1 | 0.5 ± 0.3 | 0.9 ± 0.3 | 1.2 ± 0.5 | p < 0.0001 |
| Glycine equivalent (mg/100 g of maple syrup) | ||||||
| Amino nitrogen | 4.9 ± 2.8 | 7.1 ± 3.6 | 4.9 ± 2.5 | 4.7 ± 0.8 | 5.1 ± 3.0 | p = 0.51 |
Melanoidins are polymeric-structured brown pigments formed in the last stage of the Maillard reaction, and function as antioxidants (Friedman, 1996). A linear correlation between the intensity of color and the antioxidant potential in model systems has been previously reported (Chen and Kitts, 2008; Vhangani and Van Wyk, 2013). Melanoidins with stronger absorbance at 420 nm were more efficient at inhibiting the AAPH-induced oxidation of aqueous dispersions of linoleate (Morales and Jiménez-Pérez, 2004). In the present study, the difference in antioxidant potential among respective grades of maple syrups are likely associated to some extent with the relative abundance of melanoidins. Meanwhile, maple syrups are recognized as a source of antioxidant phenolic compounds. The presence of phenolic compounds in maple syrup samples has been reported previously (Abou-Zaid et al., 2008; Sadiki and Martin, 2013). Some of these phenolic compounds possess antioxidant activity (Legault et al., 2010; Li and Seeram, 2010; Thériaulta et al., 2006). The amounts of these beneficial phenolic compounds have yet to be fully determined, but they might be partially responsible for the antioxidant activity of maple syrups. In order to estimate the contribution of melanoidins and/or phenolic compounds to the overall antioxidant potential of maple syrup, quantitative analyses of these compounds are required.
Aside from its use as a sweetener, the medical functionality of maple syrups has been gaining increasing attention. Legault et al. (2010) indicated that pure maple syrup inhibited the in vitro growth of cancer cells, particularly against prostate and lung cancer cells. Genomic investigation showed that the expression of genes for ammonia-forming enzymes were down-regulated in the liver of rats fed a maple syrup diet, indicating a potential hepatic protection function (Watanabe et al., 2011). A butanol extract of maple syrup exerted α-glucosidase-inhibitory activity in vitro, indicating suppression of rapid increases in blood glucose (Apostolidis et al., 2011). The present study provides evidence that maple syrup is a source of dietary antioxidants, and that a possible connection lies between its antioxidant potential and the degree of (brown) coloration. This result is noteworthy since the antioxidant potential of maple syrup is not taken into account during product grading, in which lighter maple syrup is typically given a higher grade. These beneficial effects indicate that maple syrup is a healthful choice as a sweetener.
In conclusion, this study revealed significant differences in the antioxidant potential of different grades of maple syrup. H-ORAC values varied widely depending on the degree of brown coloration: darker-colored maple syrups showed greater antioxidant activity. These results suggest that the brown pigments (melanoidins), produced by condensation of the reducing sugars and amino bases, may contribute significantly to the antioxidant activity of maple syrups.
Acknowledgments The author thanks Royal Yuki Inc. (Tokyo, Japan) for their financial support and helpful suggestions on the characteristics of maple syrup. The author also thanks Dr. J. Takebayashi of National Institute of Health and Nutrition for his technical advice on the H-ORAC measurements.
