Note Procyanidin Concentrations and H-ORAC of Apples Cultivated in Japan

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Note

Procyanidin Concentrations and H-ORAC of Apples Cultivated in Japan

1 1 1 2 1* Mina Obara , Saeko Masumoto , Yuko Ono , Yoshihiko Ozaki and Toshihiko Shoji

1National Institute of Fruit Tree Science, National Agriculture and Food Research Organization, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan 2Kinki University, Department of Science and Technology on Food Safety Faculty of Biology-Oriented Science and Technology, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan

Received December 15, 2015 ; Accepted March 29, 2016

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 capacity (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.

Keywords: apple, H-ORAC, procyanidins, normal-phase HPLC

Introduction Apple consumption reduces the risk of coronary heart disease Oxidative stress is induced by lifestyle factors, including (Knekt et al., 2002), likely reflecting the presence of high cigarette smoking, excessive stress, and/or an unbalanced diet, and concentrations of polyphenols in apples. Moreover, as a major has been associated with accelerated ageing and chronic diseases, source of hydrophilic anti-oxidants, apples are one of the most including diabetes, obesity, coronary heart disease and cancer. important fruits in Japan. The five major polyphenol classes, i.e., Reactive oxygen species (ROS) and free radicals can damage phenolcarboxylic acids (chlorogenic acid), anthocyanins (cyanidin biological molecules, such as proteins, lipids and DNA. The human glycosides), flavonols (quercetin glycosides), dihydrochalcones body has protective systems that include anti-oxidative enzymes, (phloretin glycosides) and flavan-3-ols/procyanidins, are found in such as superoxide dismutase, catalase and glutathione peroxidase. various apple varieties. Among these, flavan-3-ols (catechins and However, these enzymes are not able to scavenge and remove ROS procyanidins) are a major class of apple polyphenols (Vrhovsek et and free radicals completely. Epidemiological studies have al., 2004). Polyphenols have received increasing interest due to suggested that the consumption of fruits and vegetables reduces the their potential health benefits, and of these, procyanidins reportedly risk of developing lifestyle-related diseases (Liu et al., 2000; He et have the most significant physiological benefits (Eberhardt et al., al., 2004; Hung et al., 2004; Muraki et al., 2013). The health 2000; Akiyama et al., 2005; Shoji et al., 2005; Sugiyama et al., benefits of fruits are partially due to the constituent dietary fiber 2007; Miura et al., 2008). Flavan-3-ols/procyanidins are major and various phytochemicals. Polyphenols and carotenoids as apple polyphenols and comprise (−)-epicatechin and (+)-catechin phytochemicals have been known to scavenge ROS and free subunits that are linked through C4→C8 or sometimes C4→C6 radicals as well as induce the expression of related anti-oxidant bonds. Apple procyanidins have multiple isomers with varying genes. degrees of polymerization (DP), combinations and types of flavanol

*To whom correspondence should be addressed. E-mail: [email protected] 564 M. Obara et al. units and linkage positions (Shoji et al., 2003). _30℃ until analysis. Several reviews describe the use of reversed-phase and size- For HPLC analyses of procyanidins, extraction from exclusion chromatography for analytical and preparative separation lyophilised apple powders (1 g each) was performed by shaking for of flavan-3-ols/procyanidins (Hummer and Schreier, 2008). 15 min in 8 mL of acetone-water-acetic acid (70:29.5:0.5, v/v/v) However, these methods offer limited separation of procyanidins under ambient conditions. Extracted solutions were collected by from other polyphenols because many polyphenols and procyanidin centrifugation (1500 × g for 10 min) at 25℃. Extraction procedures isomers are present in almost all foods, causing many overlapping were performed twice and extracts were collected to a total volume peaks of polyphenols and procyanidins on the chromatogram. of 25 mL. Apple extracts were filtered through a 0.45 µm PTFE Therefore, normal-phase chromatography has been performed for syringe filter prior to injection into a Prominence HPLC system the separation of these compounds according to their DP (Shimadzu Corporation, Kyoto, Japan) equipped with a RF-20AXS (Hammerstone et al., 1999; Gu et al., 2002; Shoji et al., 2006). fluorescence detector (Shimadzu) and an Inertsil WP300 Diol (GL In the present study, normal-phase high-performance liquid Sciences Inc., Tokyo, Japan) column (i.d. 4.6 × 250 mm; 5 µm) at chromatography (HPLC) with fluorescence detection was modified 30℃. Mixtures of acetonitrile, water and acetic acid (mobile phase using a diol stationary phase, and procyanidin concentrations were A, CH3CN:H2O:HOAc = 98:0:2) and methanol, water and acetic determined in apple cultivars harvested in Japan, including acid (mobile phase B, MeOH:H2O:HOAc = 95:3:2) were used as commercial dessert, processing and crab apples. In subsequent the mobile phases. Elution was performed using a linear gradient experiments, anti-oxidant activities were assessed according to of 0 _ 7% B for 0 _ 3.0 min, followed by a linear gradient of hydrophilic oxygen radical absorbance capacity (H-ORAC) values 7 _ 30% B for 57.0 min. Subsequently, mobile phase B was and were correlated with procyanidin concentrations in apples. increased from 30% to 100% over 60.0 _ 70.0 min. The mobile phase was subsequently returned to initial conditions (0% B) to re- Materials and Methods equilibrate for 10.0 min. The injection volume was 5 μL, the flow Apple samples were collected during the crop production years rate was set at 1.0 mL/min and fluorescence detection of flavan-3- 2011–2013, and a total of 30 cultivars were examined. ols/procyanidins was performed with excitation and emission Commercially available dessert apple cultivars (‘Fuji’ (n = 57), wavelengths of 230 and 321 nm, respectively. The photomultiplier ‘Jonagold’ (n = 60), ‘Ohrin’ (n = 58), ‘Tsugaru’ (n = 45), tube gain was set to x4 from 29 to 65 min. Apple procyanidin ‘Shinanosweat’ (n = 5), ‘Shinanogold’ (n = 5) and ‘Akibae’ (n = 5)) standards from monomer to heptamer were prepared according to were purchased from the four major apple-producing regions their DP using previously modified methods (Shoji et al., 2006). (Aomori, Nagano, Iwate and Yamagata) in Japan. All other apple Clear relationships between each procyanidin concentration and cultivars were harvested at the experimental orchard of the peak area were observed with extremely high regression National Institute of Fruit Tree Science (NIFTS), Apple Research coefficients for standards covering a calibration range (monomer, Division (Morioka, Iwate, Japan). These included the dessert 2.5 _ 36.8 µg/mL; dimer, 2.1 _ 31.3 µg/mL; trimer, 2.5 _ 38.0 µg/ apples ‘Morinokagayaki’ (n = 14), ‘Kitarou’ (n = 15), ‘Koutarou’ (n mL; tetramer, 1.4 _ 20.6 µg/mL; pentamer, 2.1 _ 31.9 µg/mL; = 12), ‘Santarou’ (n = 12), ‘Waltz’ (n = 14), ‘Polka’ (n = 11) and hexamer, 2.0 _ 29.6 µg/mL; heptamer, 1.5 _ 22.8 µg/mL) of DP (r2 ‘Burgundy’ (n = 10), the processing apples ‘Yarrington Mill’ (n = = 0.9987 _ 0.9999). 6), ‘Harry Master Jersey’ (n = 8), ‘Bramley’s Seedling’ (n = 3), H-ORAC assays were performed after automated extraction ‘Chisel Jersey’ (n = 6), ‘Sweet Alford’ (n = 7), ‘Sweet Coppin’ (n from lyophilised samples using an ASE-200 accelerated solvent = 4) and ‘Maypole’ (n = 3) and the crab apples ‘Dolgo seedling’ (n extraction apparatus (Dionex, San Jose, CA, USA) according to = 5), ‘Geneva’ (n = 5), ‘Hu Bei Hi Tang’ (n = 5), ‘Hyslop Crab’ (n previously described methods (Watanabe et al., 2014). H-ORAC = 2), ‘Mary Potter Crab’, ‘Niedzwetzkyana’ (n = 9), ‘Pink Pearl’ (n values were determined as described previously (Watanabe et al., = 9), ‘Red Field’ (n = 6), ‘Sentinel Crab’ (n = 2) and ‘Mary Porter 2012). H-ORAC values were determined by calculating the net Crab’ (n = 1). Apple samples were cut meridionally into eight area under the curve of Trolox® standard and the data were pieces of equal size and four diagonally positioned parts were expressed as moles of Trolox® equivalent (TE) per 100 g of fresh selected for analytical sample preparation per one apple. weight (FW). Subsequently, skins and cores were removed as non-edible parts Data are expressed as means ± standard deviations (SD). and the remaining edible parts were weighed. Samples of Statistical analyses were performed using Graph Pad Prism® approximately 200 g were immediately frozen in liquid nitrogen version 6 for Macintosh (San Diego, CA, USA). and were stored at _80℃ until lyophilisation. Frozen samples were lyophilised using a vacuum freeze drier (FDU-2110, EYELA, Results and Discussion Tokyo, Japan) for 5 days. Lyophilised samples were ground in a Normal-phase chromatography is commonly performed using mechanical mill (Waring blender 7011HS, Osaka Chemical Co. silica columns and has achieved significant improvements in the Ltd., Osaka, Japan) and the resulting fine powders were stored at separation and resolution of procyanidins. However, normal-phase Procyanidins and H-ORAC in Apples 565

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.

chromatography using silica columns remain limited in their condition, harvest year and storage condition (Wojdylo et al., analyses of aqueous samples including juices. Therefore, we 2008). Moreover, we studied dessert apples from the four major employed a diol stationary phase and a HPLC column that is apple-producing regions (Aomori, Nagano, Iwate and Yamagata) compatible with a wide range of solvents including water. Figure 1 in Japan during 2011–2013. ‘Tsugaru’ apples had significantly shows a typical chromatogram for ‘Fuji’ apple using diol normal- lower flavan-3-ol/procyanidin concentrations (30.4 ± 6.5 mg/100 g phase HPLC with fluorescence detection. Here, we analyzed FW) than ‘Ohrin’ (37.4 ± 8.5 mg/100 g FW), ‘Jonagold’ (37.9 ± flavan-3-ols/procyanidins (up to heptamers) in apples using the 7.4 mg/100 g FW) and ‘Fuji’ (40.9 ± 8.4 mg/100 g FW) apples purified standards according to their DP. among the four major apple cultivars (Fig. 2d) using analysis of Flavan-3-ol/procyanidin concentrations in 30 varieties of apple variance and Tukey’s test. In a previous study, Vrhovsek et al. cultivars (dessert, processing and crab apples) are shown in Fig. 2 reported procyanidin concentrations of 52.2 ± 16.9 mg/100 g FW in (a-d). Flavan-3-ol/procyanidin concentrations were 3-fold lower in ‘Fuji’ apples using normal-phase chromatography with detection at dessert apples (33.2 ± 12.0 mg/100 g FW) than in processing apples 280 nm and epicatechin as a standard (Vrhovsek et al., 2004). (101.1 ± 64.3 mg/100 g FW) and were 6-fold lower than in crab Moreover, Gu et al., reported flavan-3-ol/procyanidin apples (198.3 ± 159.9 mg/100 g FW) (Fig. 2a-c). Guyot et al. concentrations of 69.6 ± 15.8 mg/100 g FW in ‘Fuji’ apples with determined the procyanidin concentrations of dessert and cider peels, which contained ≥ octamer procyanidins (Gu et al., 2004). apple varieties in France using thiolysis methods and showed Various methods have been developed to evaluate the anti- concentration ranges of 37.8 _ 75.3 and 122.8 _ 345.0 mg/100 g oxidant capacities of dietary components. For example, ORAC FW, respectively (Guyot et al., 2002; Guyot et al., 2003). Using (Cao et al., 1993; Wu et al., 2004), ferric-reducing ability of normal-phase HPLC, Gu et al. showed flavan-3-ol/procyanidin plasma, 2,2-di (4-tert-octylphenyl-)-1-picrylhydrazyl and Trolox® concentrations ranging from 69.6 to 136.0 mg/100 g fresh apple (Gu equivalent anti-oxidant capacity are popular means of measuring et al., 2004). These data studied in the present study are in the anti-oxidant capacities of foods, and differences between these agreement with the findings of other authors. ‘Hu Bei Hai Tang’ methods have been discussed in several reviews (Huang et al., and ‘Sentinel Crab’ (crab apples) had the highest concentrations of 2005; Prior et al., 2005). The ORAC method is the most widely procyanidins, whereas ‘Polka’, ‘Burgandy’, ‘Maypole’, and ‘Pink used method for evaluating anti-oxidant capacities (Prior et al., Pearl’ had the lowest concentrations of procyanidins. And, some 2005) and hydrophilic-ORAC (H-ORAC) is used to determine varieties (‘Burgundy’, ‘Yarlington Mill’, ‘Dolgo seedling’, ‘Pink hydrophilic anti-oxidants including vitamin C and polyphenols (Wu Pearl’) showed relatively high proportions of flavan-3-ol monomers et al., 2004). However, previously used H-ORAC methods are composition. As reported by Wojdylo et al., polyphenol compromised by poor reproducibility. Hence, we applied a compositions vary significantly between apple varieties and modified H-ORAC method, with improved intermediate precision growing regions and may vary with fruit maturity, cultivar and reproducibility in inter-laboratory studies, to determine 566 M. Obara et al.

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.

H-ORAC values in apples cultivated in Japan (Watanabe et al., polyphenol classes, i.e., phenolcarboxylic acids (e.g. chlorogenic 2012). acid) and dihydrochalcones (phloretin glycosides), are found in In the present study, mean H-ORAC values of selected dessert apple depending upon the variety. Normal-phase chromatography apples, processing apples and crab apples from 2011–2013 (Fig. used in the current study was not able to identify phenolcarboxylic 3a-c) were 1662.5 ± 488.6, 7167.5 ± 4431.9 and 9775.0 ± 6438.2 acids and dihydrochalcones. Phenolcarboxylic acids accounted for µmol TE/100 g FW, respectively, and were lower in dessert apples 1.2 _ 31.2% in some apple varieties (Wojdylo et al., 2008). Thus, than in the other apple types. In particular, the crab apple varieties the second polyphenol classes may influence H-ORAC value in ‘Sentinel Crab’ and ‘Hu Bei Hi Tang’ showed much higher these apple varieties. H-ORAC values than all the other varieties. Among the major Because dietary fruit is a major source of anti-oxidants and Japanese dessert apples ‘Fuji’, ‘Jonagold’, ‘Ohrin’ and ‘Tsugaru’, phytochemicals, determination of polyphenol concentrations is ‘Tsugaru’ had significantly lower H-ORAC values (1301.0 ± critical to assessments of dietary anti-oxidant intake. Accordingly, 271.4 µmol TE/100 g FW) ( p < 0.001), followed by ‘Ohrin’ the anti-oxidant capacities of cultural foods have been reported in (1857.5 ± 469.1 µmol TE/100 g FW), ‘Jonagold’ (1858.2 ± several countries (Rothwell et al., 2013), and the anti-oxidant 342.1 µmol TE/100 g FW) and ‘Fuji’ (1956.2 ± 412.4 µmol capacities of fruits, vegetables and cereals have been demonstrated TE/100 g FW) (Fig. 3d). in the United States (Wu et al., 2004), Italy (Pellegrini et al., 2003), Relationships between H-ORAC values and flavan-3-ol/ France (Brat et al., 2006) and Korea (Cho et al., 2007). Fruit is a procyanidin concentrations of dessert, processing and crab apple major source of hydrophilic anti-oxidants such as polyphenols, and varieties were determined using Pearson correlation analyses (Fig. corresponding databases are important resources for estimates of 4). In accordance with previous studies (Cho et al., 2007; Prior et daily dietary polyphenol intake in epidemiological and cohort al., 2005), H-ORAC values and procyanidin concentrations from studies and for investigations of the relationships between dietary 30 apple varieties were significantly correlated (n = 30, r = 0.8284, polyphenols and disease prevalence. Although the physiological p < 0.0001). Because flavan-3-ols/procyanidins are a major class of functions of foods have been widely investigated in Japan, the anti- apple polyphenols, the present data strongly suggest that flavan-3- oxidant capacities of Japanese daily diets have not been reported ols/procyanidins are central contributors to the anti-oxidant using standardised methods. Thus, in the present study the activities of apples. And, the correlation between procyanidin H-ORAC values and procyanidin concentrations of apples were concentrations and H-ORAC values was not observed in some determined. Furthermore, ORAC values of various fruits have been varieties (‘Sentinel crab’ and ‘Mary Potter crab’). The second reported during ripening (Kalt et al., 2003), storage (Serrano et al., Procyanidins and H-ORAC in Apples 567

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.

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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