Procyanidin Concentrations and H-ORAC of Apples Cultivated in Japan

Abstract

Fruits are a major dietary source of phytochemicals with health benefits for humans. In particular, apples are an important source of dietary phytochemicals including procyanidins. Thus, in the present study, we investigated procyanidin concentrations in apples using normal-phase high-performance liquid chromatography with fluorescence detection and hydrophilic oxygen radical absorbance capacit. (H-ORAC) values. Apple procyanidin concentrations were significantly correlated with H-ORAC values (r = 0.8284, P < 0.0001) in 30 varieties of apple cultivars (dessert, processing and crab apples). These data suggest that the anti-oxidative activity of apples is mainly due to procyanidins.

Introduction

Oxidative stress is induced by lifestyle factors, including cigarette smoking, excessive stress, and/or an unbalanced diet, and has been associated with accelerated ageing and chronic diseases, including diabetes, obesity, coronary heart disease and cancer. Reactive oxygen species (ROS) and free radicals can damage biological molecules, such as proteins, lipids and DNA. The human body has protective systems that include anti-oxidative enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. However, these enzymes are not able to scavenge and remove ROS and free radicals completely. Epidemiological studies have suggested that the consumption of fruits and vegetables reduces the risk of developing lifestyle-related diseases (Liu et al., 2000; He et al., 2004; Hung et al., 2004; Muraki et al., 2013). The health benefits of fruits are partially due to the constituent dietary fiber and various phytochemicals. Polyphenols and carotenoids as phytochemicals have been known to scavenge ROS and free radicals as well as induce the expression of related anti-oxidant genes.

Apple consumption reduces the risk of coronary heart disease (Knekt et al., 2002), likely reflecting the presence of high concentrations of polyphenols in apples. Moreover, as a major source of hydrophilic anti-oxidants, apples are one of the most important fruits in Japan. The five major polyphenol classes, i.e., phenolcarboxylic acids (chlorogenic acid), anthocyanins (cyanidin glycosides), flavonols (quercetin glycosides), dihydrochalcones (phloretin glycosides) and flavan-3-ols/procyanidins, are found in various apple varieties. Among these, flavan-3-ols (catechins and procyanidins) are a major class of apple polyphenols (Vrhovsek et al., 2004). Polyphenols have received increasing interest due to their potential health benefits, and of these, procyanidins reportedly have the most significant physiological benefits (Eberhardt et al., 2000; Akiyama et al., 2005; Shoji et al., 2005; Sugiyama et al., 2007; Miura et al., 2008). Flavan-3-ols/procyanidins are major apple polyphenols and comprise (−)-epicatechin and (+)-catechin subunits that are linked through C4→C8 or sometimes C4→C6 bonds. Apple procyanidins have multiple isomers with varying degrees of polymerization (DP), combinations and types of flavanol units and linkage positions (Shoji et al., 2003).

Several reviews describe the use of reversed-phase and size-exclusion chromatography for analytical and preparative separation of flavan-3-ols/procyanidins (Hummer and Schreier, 2008). However, these methods offer limited separation of procyanidins from other polyphenols because many polyphenols and procyanidin isomers are present in almost all foods, causing many overlapping peaks of polyphenols and procyanidins on the chromatogram. Therefore, normal-phase chromatography has been performed for the separation of these compounds according to their DP (Hammerstone et al., 1999; Gu et al., 2002; Shoji et al., 2006).

In the present study, normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection was modified using a diol stationary phase, and procyanidin concentrations were determined in apple cultivars harvested in Japan, including commercial dessert, processing and crab apples. In subsequent experiments, anti-oxidant activities were assessed according to hydrophilic oxygen radical absorbance capacity (H-ORAC) values and were correlated with procyanidin concentrations in apples.

Materials and Methods

Apple samples were collected during the crop production years 2011–2013, and a total of 30 cultivars were examined. Commercially available dessert apple cultivars (‘Fuji’ (n = 57), ‘Jonagold’ (n = 60), ‘Ohrin’ (n = 58), ‘Tsugaru’ (n = 45), ‘Shinanosweat’ (n = 5), ‘Shinanogold’ (n = 5) and ‘Akibae’ (n = 5)) were purchased from the four major apple-producing regions (Aomori, Nagano, Iwate and Yamagata) in Japan. All other apple cultivars were harvested at the experimental orchard of the National Institute of Fruit Tree Science (NIFTS), Apple Research Division (Morioka, Iwate, Japan). These included the dessert apples ‘Morinokagayaki’ (n = 14), ‘Kitarou’ (n = 15), ‘Koutarou’ (n = 12), ‘Santarou’ (n = 12), ‘Waltz’ (n = 14), ‘Polka’ (n = 11) and ‘Burgundy’ (n = 10), the processing apples ‘Yarrington Mill’ (n = 6), ‘Harry Master Jersey’ (n = 8), ‘Bramley's Seedling’ (n = 3), ‘Chisel Jersey’ (n = 6), ‘Sweet Alford’ (n = 7), ‘Sweet Coppin’ (n = 4) and ‘Maypole’ (n = 3) and the crab apples ‘Dolgo seedling’ (n = 5), ‘Geneva’ (n = 5), ‘Hu Bei Hi Tang’ (n = 5), ‘Hyslop Crab’ (n = 2), ‘Mary Potter Crab’, ‘Niedzwetzkyana’ (n = 9), ‘Pink Pearl’ (n = 9), ‘Red Field’ (n = 6), ‘Sentinel Crab’ (n = 2) and ‘Mary Porter Crab’ (n = 1). Apple samples were cut meridionally into eight pieces of equal size and four diagonally positioned parts were selected for analytical sample preparation per one apple. Subsequently, skins and cores were removed as non-edible parts and the remaining edible parts were weighed. Samples of approximately 200 g were immediately frozen in liquid nitrogen and were stored at −80°C until lyophilisation. Frozen samples were lyophilised using a vacuum freeze drier (FDU-2110, EYELA, Tokyo, Japan) for 5 days. Lyophilised samples were ground in a mechanical mill (Waring blender 7011HS, Osaka Chemical Co. Ltd., Osaka, Japan) and the resulting fine powders were stored at −30°C until analysis.

For HPLC analyses of procyanidins, extraction from lyophilised apple powders (1 g each) was performed by shaking for 15 min in 8 mL of acetone-water-acetic acid (70:29.5:0.5, v/v/v) under ambient conditions. Extracted solutions were collected by centrifugation (1500 × g for 10 min) at 25°C. Extraction procedures were performed twice and extracts were collected to a total volume of 25 mL. Apple extracts were filtered through a 0.45 µm PTFE syringe filter prior to injection into a Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a RF-20AXS fluorescence detector (Shimadzu) and an Inertsil WP300 Diol (GL Sciences Inc., Tokyo, Japan) column (i.d. 4.6 × 250 mm; 5 µm) at 30°C. Mixtures of acetonitrile, water and acetic acid (mobile phase A, CH₃CN:H₂O:HOAc = 98:0:2) and methanol, water and acetic acid (mobile phase B, MeOH:H₂O:HOAc = 95:3:2) were used as the mobile phases. Elution was performed using a linear gradient of 0 – 7% B for 0 – 3.0 min, followed by a linear gradient of 7 – 30% B for 57.0 min. Subsequently, mobile phase B was increased from 30% to 100% over 60.0 – 70.0 min. The mobile phase was subsequently returned to initial conditions (0% B) to re-equilibrate for 10.0 min. The injection volume was 5 µL, the flow rate was set at 1.0 mL/min and fluorescence detection of flavan-3-ols/procyanidins was performed with excitation and emission wavelengths of 230 and 321 nm, respectively. The photomultiplier tube gain was set to x4 from 29 to 65 min. Apple procyanidin standards from monomer to heptamer were prepared according to their DP using previously modified methods (Shoji et al., 2006). Clear relationships between each procyanidin concentration and peak area were observed with extremely high regression coefficients for standards covering a calibration range (monomer, 2.5 – 36.8 µg/mL; dimer, 2.1 – 31.3 µg/mL; trimer, 2.5 – 38.0 µg/mL; tetramer, 1.4 – 20.6 µg/mL; pentamer, 2.1 – 31.9 µg/mL; hexamer, 2.0 – 29.6 µg/mL; heptamer, 1.5 – 22.8 µg/mL) of DP (r² = 0.9987 – 0.9999).

H-ORAC assays were performed after automated extraction from lyophilised samples using an ASE-200 accelerated solvent extraction apparatus (Dionex, San Jose, CA, USA) according to previously described methods (Watanabe et al., 2014). H-ORAC values were determined as described previously (Watanabe et al., 2012). H-ORAC values were determined by calculating the net area under the curve of Trolox^® standard and the data were expressed as moles of Trolox^® equivalent (TE) per 100 g of fresh weight (FW).

Data are expressed as means ± standard deviations (SD). Statistical analyses were performed using Graph Pad Prism^® version 6 for Macintosh (San Diego, CA, USA).

Results and Discussion

Normal-phase chromatography is commonly performed using silica columns and has achieved significant improvements in the separation and resolution of procyanidins. However, normal-phase chromatography using silica columns remain limited in their analyses of aqueous samples including juices. Therefore, we employed a diol stationary phase and a HPLC column that is compatible with a wide range of solvents including water. Figure 1 shows a typical chromatogram for ‘Fuji’ apple using diol normal-phase HPLC with fluorescence detection. Here, we analyzed flavan-3-ols/procyanidins (up to heptamers) in apples using the purified standards according to their DP.

Fig. 1.

The chromatogram of ‘Fuji’ using diol normal-phase HPLC with fluorescence detection. HPLC conditions are described in the Materials and Methods section. The photomultiplier tube gain was set to x4 from 29 to 65 min.

Flavan-3-ol/procyanidin concentrations in 30 varieties of apple cultivars (dessert, processing and crab apples) are shown in Fig. 2 (a–d). Flavan-3-ol/procyanidin concentrations were 3-fold lower in dessert apples (33.2 ± 12.0 mg/100 g FW) than in processing apples (101.1 ± 64.3 mg/100 g FW) and were 6-fold lower than in crab apples (198.3 ± 159.9 mg/100 g FW) (Fig. 2a–c). Guyot et al. determined the procyanidin concentrations of dessert and cider apple varieties in France using thiolysis methods and showed concentration ranges of 37.8 – 75.3 and 122.8 – 345.0 mg/100 g FW, respectively (Guyot et al., 2002; Guyot et al., 2003). Using normal-phase HPLC, Gu et al. showed flavan-3-ol/procyanidin concentrations ranging from 69.6 to 136.0 mg/100 g fresh apple (Gu et al., 2004). These data studied in the present study are in agreement with the findings of other authors. ‘Hu Bei Hai Tang’ and ‘Sentinel Crab’ (crab apples) had the highest concentrations of procyanidins, whereas ‘Polka’, ‘Burgandy’, ‘Maypole’, and ‘Pink Pearl’ had the lowest concentrations of procyanidins. And, some varieties (‘Burgundy’, ‘Yarlington Mill’, ‘Dolgo seedling’, ‘Pink Pearl’) showed relatively high proportions of flavan-3-ol monomers composition. As reported by Wojdylo et al., polyphenol compositions vary significantly between apple varieties and growing regions and may vary with fruit maturity, cultivar condition, harvest year and storage condition (Wojdylo et al., 2008). Moreover, we studied dessert apples from the four major apple-producing regions (Aomori, Nagano, Iwate and Yamagata) in Japan during 2011–2013. ‘Tsugaru’ apples had significantly lower flavan-3-ol/procyanidin concentrations (30.4 ± 6.5 mg/100 g FW) than ‘Ohrin’ (37.4 ± 8.5 mg/100 g FW), ‘Jonagold’ (37.9 ± 7.4 mg/100 g FW) and ‘Fuji’ (40.9 ± 8.4 mg/100 g FW) apples among the four major apple cultivars (Fig. 2d) using analysis of variance and Tukey's test. In a previous study, Vrhovsek et al. reported procyanidin concentrations of 52.2 ± 16.9 mg/100 g FW in ‘Fuji’ apples using normal-phase chromatography with detection at 280 nm and epicatechin as a standard (Vrhovsek et al., 2004). Moreover, Gu et al., reported flavan-3-ol/procyanidin concentrations of 69.6 ± 15.8 mg/100 g FW in ‘Fuji’ apples with peels, which contained ≥ octamer procyanidins (Gu et al., 2004).

Fig. 2.

Flavan-3-ol/procyanidin concentrations of dessert (a), processing (b), crab (c), and the major Japanese apple varieties (d). Thirty varieties of apple cultivars (dessert, processing and crab apples) were obtained between the harvest years 2011–2013. Procyanidin concentrations are presented as mg of monomer–heptamer per 100 g FW.

Various methods have been developed to evaluate the anti-oxidant capacities of dietary components. For example, ORAC (Cao et al., 1993; Wu et al., 2004), ferric-reducing ability of plasma, 2,2-di (4-tert-octylphenyl-)-1-picrylhydrazyl and Trolox^® equivalent anti-oxidant capacity are popular means of measuring the anti-oxidant capacities of foods, and differences between these methods have been discussed in several reviews (Huang et al., 2005; Prior et al., 2005). The ORAC method is the most widely used method for evaluating anti-oxidant capacities (Prior et al., 2005) and hydrophilic-ORAC (H-ORAC) is used to determine hydrophilic anti-oxidants including vitamin C and polyphenols (Wu et al., 2004). However, previously used H-ORAC methods are compromised by poor reproducibility. Hence, we applied a modified H-ORAC method, with improved intermediate precision and reproducibility in inter-laboratory studies, to determine H-ORAC values in apples cultivated in Japan (Watanabe et al., 2012).

In the present study, mean H-ORAC values of selected dessert apples, processing apples and crab apples from 2011–2013 (Fig. 3a–c) were 1662.5 ± 488.6, 7167.5 ± 4431.9 and 9775.0 ± 6438.2 µmol TE/100 g FW, respectively, and were lower in dessert apples than in the other apple types. In particular, the crab apple varieties ‘Sentinel Crab’ and ‘Hu Bei Hi Tang’ showed much higher H-ORAC values than all the other varieties. Among the major Japanese dessert apples ‘Fuji’, ‘Jonagold’, ‘Ohrin’ and ‘Tsugaru’, ‘Tsugaru’ had significantly lower H-ORAC values (1301.0 ± 271.4 µmol TE/100 g FW) (p < 0.001), followed by ‘Ohrin’ (1857.5 ± 469.1 µmol TE/100 g FW), ‘Jonagold’ (1858.2 ± 342.1 µmol TE/100 g FW) and ‘Fuji’ (1956.2 ± 412.4 µmol TE/100 g FW) (Fig. 3d).

Fig. 3.

H-ORAC values of dessert (a), processing (b), crab (c), and the major Japanese apple varieties (d). H-ORAC values were measured as described in the Materials and Methods section and are expressed as µmol TE/100 g FW.

Relationships between H-ORAC values and flavan-3-ol/procyanidin concentrations of dessert, processing and crab apple varieties were determined using Pearson correlation analyses (Fig. 4). In accordance with previous studies (Cho et al., 2007; Prior et al., 2005), H-ORAC values and procyanidin concentrations from 30 apple varieties were significantly correlated (n = 30, r = 0.8284, p < 0.0001). Because flavan-3-ols/procyanidins are a major class of apple polyphenols, the present data strongly suggest that flavan-3-ols/procyanidins are central contributors to the anti-oxidant activities of apples. And, the correlation between procyanidin concentrations and H-ORAC values was not observed in some varieties (‘Sentinel crab’ and ‘Mary Potter crab’). The second polyphenol classes, i.e., phenolcarboxylic acids (e.g. chlorogenic acid) and dihydrochalcones (phloretin glycosides), are found in apple depending upon the variety. Normal-phase chromatography used in the current study was not able to identify phenolcarboxylic acids and dihydrochalcones. Phenolcarboxylic acids accounted for 1.2 – 31.2% in some apple varieties (Wojdylo et al., 2008). Thus, the second polyphenol classes may influence H-ORAC value in these apple varieties.

Fig. 4.

Correlation between flavan-3-ol/procyanidin concentrations and H-ORAC values in 30 apple cultivars. Flavan-3-ol/procyanidin concentrations are presented as mg/100 g FW and H-ORAC values are expressed as µmol TE/100 g FW.

Because dietary fruit is a major source of anti-oxidants and phytochemicals, determination of polyphenol concentrations is critical to assessments of dietary anti-oxidant intake. Accordingly, the anti-oxidant capacities of cultural foods have been reported in several countries (Rothwell et al., 2013), and the anti-oxidant capacities of fruits, vegetables and cereals have been demonstrated in the United States (Wu et al., 2004), Italy (Pellegrini et al., 2003), France (Brat et al., 2006) and Korea (Cho et al., 2007). Fruit is a major source of hydrophilic anti-oxidants such as polyphenols, and corresponding databases are important resources for estimates of daily dietary polyphenol intake in epidemiological and cohort studies and for investigations of the relationships between dietary polyphenols and disease prevalence. Although the physiological functions of foods have been widely investigated in Japan, the anti-oxidant capacities of Japanese daily diets have not been reported using standardised methods. Thus, in the present study the H-ORAC values and procyanidin concentrations of apples were determined. Furthermore, ORAC values of various fruits have been reported during ripening (Kalt et al., 2003), storage (Serrano et al., 2009) and manufacture (Rababah et al., 2005). Further studies will be required to determine the anti-oxidant concentrations and activities of other fruits and of overall daily diets in Japan for application to epidemiological and cohort studies.

Acknowledgements    We wish to thank Dr. Y. Takano-Ishikawa, National Food Research Institute, National Agriculture and Food Research Organization, for the provision of the H-ORAC experimental system. We also acknowledge Dr. K. Abe, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization, for providing the large number of apple samples. This work was supported in part by a grant-in-aid for research on ‘new demand creation of agricultural products’ by the Ministry of Agriculture, Forestry and Fisheries.

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