Article pubs.acs.org/JAFC
Chemical Compositional, Biological, and Safety Studies of a Novel Maple Syrup Derived Extract for Nutraceutical Applications Yan Zhang, Tao Yuan, Liya Li, Pragati Nahar, Angela Slitt, and Navindra P. Seeram* Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States ABSTRACT: Maple syrup has nutraceutical potential given the macronutrients (carbohydrates, primarily sucrose), micronutrients (minerals and vitamins), and phytochemicals (primarily phenolics) found in this natural sweetener. We conducted compositional (ash, fiber, carbohydrates, minerals, amino acids, organic acids, vitamins, phytochemicals), in vitro biological, and in vivo safety (animal toxicity) studies on maple syrup extracts (MSX-1 and MSX-2) derived from two declassified maple syrup samples. Along with macronutrient and micronutrient quantification, thirty-three phytochemicals were identified (by HPLC-DAD), and nine phytochemicals, including two new compounds, were isolated and identified (by NMR) from MSX. At doses of up to 1000 mg/kg/day, MSX was well tolerated with no signs of overt toxicity in rats. MSX showed antioxidant (2,2diphenyl-1-picrylhydrazyl (DPPH) assay) and anti-inflammatory (in RAW 264.7 macrophages) effects and inhibited glucose consumption (by HepG2 cells) in vitro. Thus, MSX should be further investigated for potential nutraceutical applications given its similarity in chemical composition to pure maple syrup. KEYWORDS: maple syrup, extract, nutraceutical, phytochemicals, biological, safety
1. INTRODUCTION Maple syrup is a natural sweetener produced by boiling sap collected from the sugar maple (Acer saccharum L.) tree and certain other maple species.1 The large-scale commercial production of maple syrup occurs primarily in eastern North America, in Canada and the United States, with the province of Quebec (in Canada) responsible for the majority of the world’s supply (ca. 80%). Maple syrup has different grades based on light transmittance (e.g., five grades in Canada are no. 1 extra light, no. 1 light, no. 1 medium, no. 2 amber, and no. 3 dark) and meets strict food quality standards that are regulated by state, provincial, and/or federal agencies in Canada and the United States. The maple syrup industry is of significant economic importance to this region of the world, with millions of gallons of syrup produced every year with price ranging from ca. $40.59 per gallon in 2013 in Canada alone (data available from Statistics Canada, Table 001-0008: Production and farm value of maple products annually). During the production of maple syrup, declassified maple syrup samples are generated which are economical raw materials for the generation of extracts with functional food and nutraceutical applications. However, to date, there have been no chemical compositional, biological, and safety studies conducted on food-grade extracts derived from maple syrup. Maple syrup is a widely consumed food product, and its macronutrient and micronutrient constituents are well established. The major carbohydrate found in maple syrup is sucrose (range of ca. 60−66%) along with lesser amounts of glucose and fructose and complex carbohydrates, including high molecular weight polysaccharides.2 Maple syrup also contains minerals (K, Ca, Mg, Na, Mn, Al, Zn, Fe, etc.), vitamins (riboflavin, niacin, thiamine, etc.), amino acids (arginine, threonine, proline, etc.), organic acids (fumaric acid, malic acid, etc.), and phytohormones (abscisic acid and phaseic acid and their metabolites).1,3 A wide © 2014 American Chemical Society
range of phytochemicals, most of which are phenolics (belonging to lignan, phenolic acid, stilbene, coumarin, and flavonoid subclasses) have also been identified in maple syrup.4−9 Apart from these chemical compositional studies on maple syrup, both in vitro and in vivo biological studies have been reported on this natural sweetener. For instance, animal studies suggest that pure maple syrup may have liver-protective effects10 and the ability to reduce plasma glucose levels compared to a sucrose solution alone.11 In addition, in vitro biological studies of phenolicenriched maple syrup extracts suggest potential anticancer, antioxidant, α-glucosidase enzyme inhibitory, and anti-inflammatory effects.12−14 Therefore, the chemical composition and biological effects attributed to this natural sweetener warrant further investigation into its derived extracts for functional food ingredient and nutraceutical applications. Our laboratory has been involved in the isolation and structure elucidation (by NMR) of phytochemicals from maple food products (sap and syrup)4−7 and also from maple plant parts.15−18 This overall program of study has resulted in the isolation and structure elucidation of more than 100 phytochemicals from maple. Given our laboratory’s extensive experience with maple phytochemicals, and our unique access to these chemical standards, we have established HPLC-DAD methods to identify compounds (on the basis of comparison of retention times and UV spectra) in a wide variety of maple materials. Also, our laboratory has established standard operating protocols for the detection and quantification of carbohydrates (sucrose, glucose, fructose, and complex carbohydrates), minerals (K, Ca, Mg, Na, Mn, Al, Zn, Fe, etc.), amino acids Received: Revised: Accepted: Published: 6687
April 22, 2014 June 30, 2014 July 1, 2014 July 1, 2014 dx.doi.org/10.1021/jf501924y | J. Agric. Food Chem. 2014, 62, 6687−6698
Journal of Agricultural and Food Chemistry
Article
Table 1. Compounds Identified in MSX and Their UV Absorbance peak
UV (nm)
(6R)-6-hydroxy-3-(hydroxymethyl)-2-cyclohexenone 3,4-dihydro-5-(hydroxymethyl)pyran-2-one 4,4′-dihydroxy-3,3′,5,5′-tetramethoxystilbene 4,4′-dihydroxy-3,3′,5′-trimethoxystilbene 4-hydroxy-2-(hydroxymethyl)-5-methyl-3(2H)- furanone benzenemethanol 5-(hydroxymethyl)furfural 4-methyl-1,2-venzenediol 4-(hydroxymethyl)-1,2-benzenediol 2-hydroxy-3,4-dihydroxyacetophenone catechol C-veratroylglycol threo,threo-1-[4-(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy)-3-methoxyphenyl]-1,2,3-propanetriol 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone 4-acetylcatechol tyrosol catechaldehyde 1,2-diguaiacyl-1,3-propanediol
10 11
3′,5′-dimethoxy-4′-hydroxy-2-hydroxyacetophenone leptolepisol D
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
3,4-dihydroxy-2-methylbenzadehyde vanillin fraxetin syringaldehyde syringenin scopoletin threo-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol erythro-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol 3-[[4-[(6-deoxy-α-L-mannopyranosyl)oxy]-3-methoxyphenyl]methyl]-5-(3,4-dimethoxyphenyl)dihydro-3-hydroxy-4-(hydroxymethyl)2(3H)-furanone 5-(3″,4″-dimethoxyphenyl)-3-hydroxy-3-(4′-hydroxy-3′-methoxybenzyl)-4-(hydroxymethyl)dihydrofuran-2-one 1-(2,3,4-trihydroxy-5-methylphenyl)ethanone erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2,6-dimethoxyphenoxy]-1,3-propanediol icariside E4 3′,4′,5′-trihydroxyacetophenone dehydroconiferyl alcohol
27
sakuraresinol
28 29 30
secoisolariciresinol acernikol (1S,2R)-2-[2,6-dimethoxy-4-[(1S,3aR,4S,6aR)-tetrahydro-4-(4-hydroxy- 3,5-dimethoxyphenyl)-1H,3H-furo[3,4-c]furan-1-yl]phenoxy]-1-(4hydroxy-3-methoxyphenyl)-1,3-propanediol buddlenol E
31 32 33 a
compd
S1 S2 S3 S4 S5 S6 S7 S8 S9 1 2 3 4 5 6 7 8 9
2-[4-[2,3-dihydro-3-(hydroxymethyl)-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenoxy]-1-(4-hydroxy-3methoxyphenyl)-1,3-propanediol (E)-3,3′-dimethoxy-4,4′-dihydroxystilbene
238 228, 284 222, 331 222, 331 271 229, 284 229, 284 210, 288 228, 276, 308 229, 276, 307 210, 276 231, 280, 310 230, 279 216, 306 231, 280, 312 231, 280, 312 231, 280, 312 210, 226(s),a 280 228, 300 210, 229(s),a 280 233, 285 229, 280, 309 229, 338 216, 307 224, 273 229, 345 228, 279 229, 279 231, 278 232, 280 230, 295 228, 279 225, 280 231, 284 211, 230(s),a 282 210, 236(s),a 280 231, 281 210, 231, 280 210, 232(s),a 280 210, 229(s),a 280 211, 232(s),a 281 222, 331
“s” indicates a shoulder peak.
HPLC-DAD methods as well as by isolation and subsequent structure elucidation by NMR), (3) in vitro biological (antioxidant, anti-inflammatory, and glucose consumption bioassays), and (4) in vivo safety (acute animal toxicity) studies on MSX. This is the first chemical and biological study of a foodgrade extract derived from maple syrup.
(arginine, threonine, proline, etc.), vitamins (riboflavin, niacin, thiamine, etc.), and organic acids (fumaric acid, malic acid, etc.) in maple materials.7 Therefore, given our ongoing research interest in maple products, the primary objective of this project was to investigate the nutraceutical potential of a novel maple syrup derived extract (named MSX) produced under food-grade conditions starting from declassified maple syrup generated by the industry. Here we report (1) chemical compositional (quantification of sugars, minerals, amino acids, organic acids, vitamins, fiber, and ash), (2) phytochemical identification (by
2. MATERIALS AND METHODS 2.1. General Experimental Procedures. All 1D nuclear magnetic resonance (1H and 13C NMR) and 2D NMR [1H−1H correlation 6688
dx.doi.org/10.1021/jf501924y | J. Agric. Food Chem. 2014, 62, 6687−6698
Journal of Agricultural and Food Chemistry
Article
2.4. Identification of Compounds 1−33 (by HPLC-DAD) in MSX. We have established HPLC-DAD methods to identify compounds (on the basis of comparison of retention times and UV spectra) in a variety of maple-derived materials using authentic standards previously isolated from maple sap and maple syrup by our group.4−7 Therefore, on the basis of comparison of the HPLC-DAD profiles to that of pure maple syrup (chromatogram shown in Figure 2A), we were able to identify 33 compounds (1−33; compound identities shown in Table 1 and chemical structures shown in Figure 1) in phenolic-enriched extracts of each of the MSX samples (see Figure 2B,C). To accomplish this, phenolic-enriched extracts of the MSX samples (1.5 g each) were prepared as previously reported for pure maple syrup by liquid−liquid partitioning with ethyl acetate (40 mL × 3).5,7 The combined ethyl acetate extracts were dried under reduced pressure in vacuo (water bath kept at 40 °C), and accurate masses were obtained as follows: 282 mg (for MSX-1) and 310 mg (for MSX-2). All samples were dissolved in dimethyl sulfoxide (DMSO), standardized to solid content (50 mg/mL), and then subjected to HPLC-DAD analyses using an Alltima C18 column (250 × 4.6 mm i.d., 5 μM; Alltech) with a flow rate of 0.75 mL/min and an injection volume of 20 μL. A linear gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol) at room temperature was used as follows: 0−30 min, from 5% to 33.4% B; 30−80 min, from 33.4% to 71% B; 80−85 min, from 71% to 100% B; 85−86 min, from 100% to 5% B; 86−94 min, 5% B. 2.5. Isolation and Identification (by NMR) of Compounds S1− S9 from MSX. We pursued the isolation and structure elucidation (by NMR) of nine compounds (S1−S9; see Table 2) from the MSX samples which, despite their presence in pure maple syrup as evident from the HPLC-DAD profile (see Figure 2), were not obtained from our previous isolation studies.4−6 Given the similarities in the HPLC-DAD profiles of the MSX-1 and MSX-2 samples (see parts B and C, respectively, of Figure 2), we sought to increase the quantity of initial starting material for our planned isolation studies by combining the MSX samples (1:1, w/w; total of 140.0 g). The combined MSX sample was subjected to liquid−liquid partitioning with ethyl acetate (700 mL × 3) to yield a dried MSX ethyl acetate extract (MSX−EtOAc; 29.0 g) after solvent removal in vacuo. The MSX−EtOAc (29.0 g) extract was subjected to C18 MPLC eluting with a gradient system of MeOH/H2O (5:95 to 100:0, v/v) to afford eight major fractions (A1−A8). Fraction A1 (1.2 g) was chromatographed over a Sephadex LH-20 column (4 × 65 cm) with MeOH/H2O (1:1, v/v) and was further purified by reversed-phase semipreparative HPLC with MeOH/0.1% TFA in water (0.8:99.2, v/v) to afford compound S1 (9.2 mg, tR = 19.5 min). Fraction A2 (1.5 g) was chromatographed over a Sephadex LH-20 column (4 × 65 cm) with MeOH/H2O (1:1, v/v) and was further purified by reversed-phase semipreparative HPLC with MeOH/0.1% TFA in water (4:96, v/v) to afford compounds S2 (2 mg, tR = 29.8 min) and S5 (10.1 mg, tR = 19.4 min) and then with MeOH/0.1% TFA in water (2.6:97.4, v/v) to afford compound S7 (9.8 mg, tR = 37.1 min). Fraction A3 (0.5 g) was chromatographed over a Sephadex LH-20 column (4 × 65 cm) with MeOH/H2O (1:1, v/v) and was then further purified by reversed-phase semipreparative HPLC with MeOH/0.1% TFA in water (10:90, v/v) to afford compounds S6 (4 mg, tR = 43.0 min) and S8 (3.8 mg, tR = 26.8 min) and with MeOH/0.1% TFA in water (5:95, v/v) to afford compound S9 (5.1 mg, tR = 34.7 min). Similarly, fraction A7 (0.9 g) was chromatographed over a Sephadex LH-20 column (4 × 65 cm) with MeOH/H2O (1:1, v/v) and was then further purified by reversed-phase semipreparative HPLC with MeOH/0.1% TFA in water (41:59, v/v) to afford compounds S3 (3 mg, tR = 75.3 min) and S4 (12.6 mg, tR = 80.2 min). 2.5.1. Data for (6R)-6-hydroxy-3-(hydroxymethyl)-2-cyclohexenone (S1): yellow oil; [α]20 D +27.8 (c 0.9, MeOH); CD (MeOH) 232 (Δε −4.62), 247 (Δε −10.49), 323 (Δε −0.39) nm; HR-ESIMS m/z 141.0557 [M − H]− (calcd for C7H9O3, 141.0552); 1H and 13C NMR data shown in Table 3. 2.5.2. Data for 3,4-dihydro-5-(hydroxymethyl)pyran-2-one (S2): white amorphous powder; HR-ESIMS m/z 127.0396 [M − H]− (calcd for C6H7O3, 127.0395); 1H and 13C NMR data shown in Table 3.
spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond coherence (HMBC)] experiments were acquired either on a Bruker 300 MHz or a Varian 500 MHz instrument. Unless otherwise stated, deuterated methanol (CD3OD) was used as the solvent for all of the NMR experiments. HR-ESIMS data were acquired using a Waters SYNAPT G2-S QTOFMS system. Highperformance liquid chromatography (HPLC) was performed on a Hitachi Elite LaChrom system (Pleasanton, CA) consisting of an L2130 pump, L-2200 autosampler with L-2455 diode array detector (DAD), and L-2490 refractive index (RI) and L-2485 fluorescence (FL) detectors, all operated by EZChrom Elite software. Medium-pressure liquid chromatography (MPLC) was carried out on prepacked C18 columns. Optical rotation was performed on an AutoPol III automatic polarimeter (Rudolph Research, Flanders, NJ) with samples dissolved in methanol at room temperature. CD spectra were recorded on a JASCO J-810 spectropolarimeter. 2.2. Chemicals and Reagents. All solvents were either ACS or HPLC grade and were obtained from Pharmco-AAPER through Wilkem Scientific (Pawcatuck, RI). Sephadex LH-20, XAD-16 Amberlite resin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), the Folin− Ciocalteau reagent, and all of the cell culture supplies were purchased from Sigma-Aldrich (St. Louis, MO). Standards of compounds used to aid in the phytochemical characterization of MSX were previously isolated and identified (by NMR) from maple syrup and maple sap by our laboratory.4−7 2.3. Production of MSX. In collaboration with the Federation of Maple Syrup Producers of Quebec (FPAQ), our laboratory has previously investigated the chemical constituents present in maple sap and syrup4−7 and maple plant parts,15−18 which has resulted in the isolation and identification (by NMR) of more than 100 compounds. Maple syrup is classified into different grades (light to dark) on the basis of light transmission. Notably, maple syrup with light transmission of
