Blueberry leaves from 73 different cultivars in southeastern China as nutraceutical supplements rich in antioxidants

Food Research International 122 (2019) 548–560

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Blueberry leaves from 73 different cultivars in southeastern China as nutraceutical supplements rich in antioxidants

T

Han Wua, Zhi Chaia, Ruth Paulina Hutabarata, Qilong Zengb, Liying Niua, Dajing Lia, Hong Yub, , ⁎⁎ Wuyang Huanga,c,d, ⁎

a

Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, PR China c Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China d School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China b

ARTICLE INFO

ABSTRACT

Keywords: Blueberry leaves Different cultivars Phenolic composition Antioxidant activity Correlation analysis

Blueberries (Vaccinium spp.) have great beneficial effects, and their leaves are rich in phenolics. In the present study, the total phenolic, total flavonoid, and proanthocyanidin contents in the leaf extracts from 73 different blueberry cultivars among five categories were investigated. The phenolic composition was analyzed, and the antioxidants were also evaluated. Here, a total of 23 individual phenolic constituents were identified, among which eight predominant phenolics were quantified, including five caffeoylquinic acids, two quercetin glycosides, and one kaempferol glycoside. The different cultivars could be well clustered according to their phenolic compositions and antioxidant capacities. The correlations among the quantified phenolic constituents and the antioxidant capacities were determined using principal component analysis. The results indicated that blueberry leaves may be a potential resource of antioxidant phenolics, and the differences among the cultivars should be considered when blueberry leaves are further developed.

1. Introduction

The composition, concentration, availability, and bioactivity of phenolic substances impact the application of blueberry leaves to a large extent. Commonly, genetic and environmental diversity are two important factors that regulate the physicochemical and biological properties of the phytochemicals in plants. Specifically, the major environmental factors include the soil type, water stress, climatic conditions, applied manure, fertilization practices, and harvest time (Routray & Orsat, 2011). As previously reported, the total phenolic content and the total antioxidant capacity of blueberries were from moderately to highly heritable (Scalzo, Miller, Edwards, Meekings, & Alspach, 2008). The genotype has a stronger effect on the phenolic composition than the environment (Connor, Luby, Tong, Finn, & Hancock, 2002). For blueberry leaves, the phenolic compounds probably vary greatly among the different species or cultivars. Thus, surveying the phenolic variation in the leaves of different blueberry cultivars is necessary for the use of blueberry leaves. The colorimetric methodology is nonspecific but convenient and global, and the high performance liquid chromatographic (HPLC) profiling approach is separative. Therefore, these two methods were used for the phenolic quantification of different blueberry leaves, and their “total content” and the “individual content”

Blueberries are a perennial shrub of the genus Vaccinium belonging to the family Ericaceae. Numerous studies on the chemical composition and therapeutic applications have led to an increase in the propagation of blueberries worldwide (Norberto et al., 2013). In addition to the fruits, blueberry leaves are also a rich source of phenolic compounds (Cyboran, Oszmianski, & Kleszczynska, 2013). Blueberries (V. bracteatum Thunb.) have acted as a traditional medicinal herb in China. Seeram et al. (2006) reported the wide use of blueberry leaves, and described their preventive effects against cataracts, premature aging, and anemia. It has been determined that highbush blueberry leaves possess higher antioxidant activity than their fruits (Ehlenfeldt & Prior, 2001). However, in many countries, the leaves are discarded after being pruned and cause a large amount of waste. Thus, the application of the phenolic compounds from the discarded part of blueberry plants is ecofriendly and adds to the consumption of compounds that are beneficial for health. With the increasing interest in the complete utilization of blueberry plants, more scientists have explored the nutritional contents and industrial applications of blueberry leaves.

⁎

Corresponding author. Corresponding author at: Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China E-mail addresses: [email protected] (H. Yu), [email protected] (W. Huang).

⁎⁎

https://doi.org/10.1016/j.foodres.2019.05.015 Received 27 December 2018; Received in revised form 23 April 2019; Accepted 12 May 2019 Available online 13 May 2019 0963-9969/ © 2019 Published by Elsevier Ltd.

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were determined, respectively. Regarding the basic function of leaves, some studies have evaluated the plants' health and monitored the nutritional deficiencies in plants during different growth stages. The overall bioactive variation in North American highbush blueberry leaves at different harvest times has been assessed (Routray & Orsat, 2014). Some studies on the chemicals and bioactivities of blueberry leaves, such as their phenolic compounds and antioxidant capacities, have been attempted; however, few studies have systematically reported on more than two varieties (Hicks et al., 2012; Matsuo et al., 2010; Routray & Orsat, 2014). No existing report has investigated the interactions among the different cultivars, phenolic components, and oxidation resistances of blueberry leaves. The effects of genetic variations on the phenolic composition and antioxidant capacity of blueberry leaves is still unknown. Therefore, in the present study, the phenolic compositions of the leaves from 73 different blueberry cultivars among five categories that were collected from the same region in southeastern China, including rabbiteye, southern highbush, northern highbush, half-high, and lowbush blueberries, were systematically investigated using qualitative and quantitative research. Both the MS fragmentation spectra and DAD detection were used to identify the phenolic compounds in the leaf extracts. In addition to conventional measurements, such as those of the DPPH radical scavenging activity, the ABTS radical cation scavenging activity, and the ferric reducing antioxidant power (FRAP) assay, the oxygen radical absorbance capacity (ORAC) assay, which is the only method that combines both the inhibition time and the degree of inhibition into a single measure, was also conducted in order to assess the antioxidant activity (Janiuk, Najda, Gantner, & Blazewicz-Wozniak, 2013; Prior & Gao, 1999; Wang et al., 2015). Furthermore, the correlations between the phenolic constituents and antioxidant capacities of different cultivar leaves were analyzed. In this way, the comprehensive utilization of blueberry cultivars could be extensively explored, and the excellent leaf-derived nutraceutical ingredients could be feasibly developed.

2,4,6-tris (2-pyridyl)-S-triazine (TPTZ) were purchased from SigmaAldrich (St. Louis, MO, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchromate-2-carboxylic acid) was obtained from Acros Organics (Morris Plains, NJ, USA). Fluorescein disodium was purchased from Chemical Industrial of East China Normal University (Shanghai, China). Folin-Ciocalteu's reagent, AAPH (2,2′-azobis(2-methylpropionamide)dihydrochloride), catechin, gallic acid, and rutin were obtained from J& K Chemical Ltd. (Beijing, China). Eight HPLC standards, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, quercetin-3-O-galactoside, quercetin-3-O-glucoside, and kaempferol-3-O-glucoside were bought from Yuanye Chemical Ltd. (Shanghai, China). HPLC solvents methanol and acetonitrile were purchased from TEDIA (Ohio, USA), and formic acid was from Sigma-Aldrich (St. Louis, MO, USA). Purify of chemicals and solvents used for HPLC was > 95.00%, and all the other chemicals and reagents used were of analytical grade. 2.3. Extraction of the phenolics from the samples The samples were extracted in batches using the method of Wang et al. (2015) with a few modifications. Approximately 1.000 g of dried powder was extracted twice with 10.00 mL of 85.00% methanol containing 0.500% formic acid at room temperature (RT), followed by 5.000 mL of the same solvent for a third extraction. Ultrasound assisted extraction was carried out by sonicating the samples in a DKZ-450B water bath ultrasonic system (Sengxin Ultrasonic Instruments, Shanghai, China) at RT for 20.00 min. The slurry containing the phenolics was then centrifuged at 5000g for 10.00 min using a Sigma 3 K15 centrifuge (Sigma-Laborzentrifugen, Osterode am Harz, Germany), and the supernatants of each sample were collected and combined in order to get the extract. The prepared extracts in each batch were filtered using a Millipore filter with a 0.450 μm nylon membrane and were used for the assays as soon as possible. The extracts were wrapped with aluminum foil in order to protect them from light exposure.

2. Materials and methods

2.4. Determination of the total phenolic, flavonoid, and proanthocyanidin contents

2.1. Plant materials and sample preparation The fresh blueberry leaves from 73 different cultivars, including 13 cultivars of rabbiteye blueberries (Vaccinium ashei), 20 cultivars of southern highbush blueberries (V. corymbosum L interspecific hybrid), 33 cultivars of northern highbush blueberries (V. corymbosum L.), 5 cultivars of half-high blueberries (V. corymbosum L. × V. angustifolium Ait.), and 2 cultivars of lowbush blueberries (V. angustifolium Ait.), were collected from Zhuji, Zhejiang, China, in July 2017. Zhuji is located in southeastern China at a latitude of 29°72′ and a longitude of 120°23′ within the north subtropical monsoon climate zone. The different cultivars were originally introduced from various places worldwide. In addition, due to the large number of samples, each cultivar was given a sample code such as “R/S/N/H/L + an arabic numeral” during the sampling process, where the capital letters represented the respective blueberry categories that were given above. The information on the sampled cultivars is listed in Table 1. For each cultivar, three trees were randomly selected and we handpicked approximately 30 mature, damage-free leaves from at least 10 shoots across all whole plants. The collected blueberry leaves were packed in plastic bags and brought back to the lab. The leaves were dried using an Eyela FDU-1200 freeze-dryer (Rikakikai, Tokyo, Japan) and then ground using a pulverizer. Flour with a uniform particle size was obtained by passing the ground leaves through a 0.200 mm sieve. All the samples in the form of dry powder were stored at −20.00 °C in a refrigerator until further analysis.

2.4.1. Determination of the total phenolic content (TPC) The total phenolic contents were determined using Folin-Ciocalteu's method (Zhang, Huang, Chen, & Zhang, 2014). Briefly, 0.400 mL of sample was oxidized with 2.000 mL of 0.500 mol/L Folin-Ciocalteu reagent at RT for 4.000 min. Then, the reaction was neutralized by adding 2.000 mL of 75.00 g/L saturated sodium carbonate. After 2.000 h of incubation in the dark, the absorbance at 760.0 nm was recorded using a Mapada UV-1600PC spectrophotometer (Meipuda Instrument Co. LTD, Shanghai, China). The quantification was based on the standard curve of gallic acid. The TPC results were expressed in gallic acid equivalent (GAE), i.e., mg GAE/g dry weight (DW).

2.2. Chemicals and reagents

2.4.3. Determination of the proanthocyanidin content (PAC) The proanthocyanidin contents were determined by the vanillinhydrochloric acid method (Nakamura, Tsuji, & Tonogai, 2003). A 1.500 mL of sample and 3.000 mL of 4.000% vanillin methanol solution

2.4.2. Determination of the total flavonoid content (TFC) The contents were spectrophotometrically measured based on the formation of a flavonoid–aluminum complex (Huang, Zhang, Liu, & Li, 2012). Briefly, 1.000 mL of sample was mixed with 0.100 mL of 5.000% NaNO2 for 6.000 min. Then, 0.100 mL of 10.00% AlCl3ˑ6H2O solution was added to the mixture for another 5.000 min. After adding 1.000 mL of 1.000 mol/L NaOH, the reaction solution was mixed well and allowed to stand for 15.00 min. The absorbance was measured at 510.0 nm. Rutin was used as a standard in order to establish the calibration curve. The TFCs were calculated and expressed in rutin equivalent (RTE), i.e., mg RTE/g DW.

Ferrozine, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 549

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Table 1 TPC, TFC, PAC, and antioxidant capacities of 73 different blueberry leaves. Sample code

blueberry cultivar

TPC

TFC

PAC

DPPH

ABTS

FRAP

ORAC

(mg GAE/g DW)

(mg RTE/g DW)

(mg CTE/g DW)

(μmol TEAC /g DW)

(μmol TEAC /g DW)

(μmol FEAC/g DW)

(μmol TEAC/g DW)

204.3 403.9 224.6 202.9 380.4 119.7 304.7 222.1 175.2 247.5 203.3 311.6 248.2

168.1 399.2 404.7 290.4 323.0 168.3 508.4 326.8 252.2 205.0 323.0 244.7 429.2

± ± ± ± ± ± ± ± ± ± ± ± ±

9.7 8.9 5.8 13.6 15.7 1.4 5.5 17.2 2.6 9.2 14.9 2.1 14.3

241.6 272.0 247.5 208.4 255.3 301.8 275.7 267.6 231.8 225.6 248.3 255.7 282.4

± ± ± ± ± ± ± ± ± ± ± ± ±

21.3 21.0 3.2 12.1 20.6 13.5 6.5 8.7 5.2 34.0 4.5 15.4 15.2

321.7 426.3 225.3 343.2 244.8 450.8 447.6 402.4 282.3 338.8 341.8 146.8 424.9

± ± ± ± ± ± ± ± ± ± ± ± ±

18.4 3.1 28.4 61.9 44.2 0.9 0.9 7.2 14.8 34.9 31.4 1.0 12.6

855.9 ± 56.7 916.6 ± 78.7 901.9 ± 103.8 1143 ± 55 934.0 ± 65.4 1177 ± 39 588.0 ± 19.4 1531 ± 30 1095 ± 91 1173 ± 60 1179 ± 24 576.3 ± 2.4 1209 ± 65

482.9 ± 35.3 822.0 ± 38.7 500.0 ± 64.7 596.9 ± 41.8 714.6 ± 79.4 391.9 ± 33.8 631.1 ± 40.1 1040 ± 69 719.1 ± 14.9 642.2 ± 21.3 1348 ± 50 892.2 ± 11.6 642.1 ± 82.0

Southern highbush blueberry Ericaceae, V. corymbosum L interspecific hybrid S1 Anna 77.80 ± 0.01 112.7 ± 0.2 45.97 S2 Magnolia 117.7 ± 0.3 271.8 ± 9.7 199.7 S3 Sunshineblue 96.90 ± 1.28 182.5 ± 0.3 104.4 S4 Georgiagem 68.55 ± 0.01 86.41 ± 1.76 59.03 S5 Bladen 133.7 ± 6.9 173.5 ± 1.8 154.8 S6 Gulfcoast 112.7 ± 0.1 89.68 ± 0.47 52.03 S7 Misty 86.64 ± 0.06 224.4 ± 8.5 80.41 S8 Stanley 133.9 ± 4.8 131.9 ± 16.4 170.0 S9 Nanda 47.16 ± 0.05 34.77 ± 0.42 25.07 S10 Sharpblue 96.23 ± 2.72 150.7 ± 18.2 71.78 S11 Cooper 122.6 ± 8.0 269.8 ± 1.5 99.74 S12 O'Neal 32.18 ± 0.01 35.99 ± 0.77 19.77 S13 Biloxi 123.6 ± 0.0 154.8 ± 1.6 62.33 S14 Meilan 1 115.0 ± 5.5 227.9 ± 7.1 113.7 S16 Ozarkblue 106.5 ± 3.4 205.2 ± 2.7 192.1 S17 A167 106.3 ± 3.6 165.6 ± 2.3 97.36 S19 Meilan 7 100.3 ± 16.3 122.2 ± 2.3 85.48 S20 Weierkang 73.45 ± 2.95 85.40 ± 0.57 87.74 S23 Nanjin 185.2 ± 3.8 304.8 ± 19.5 189.7 S24 Nanhao 78.58 ± 1.37 108.8 ± 7.1 42.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.53 9.3 7.3 7.53 6.7 9.36 5.49 6.5 3.10 8.65 4.71 1.75 6.56 2.0 9.7 4.03 4.04 5.24 8.9 2.43

214.4 274.1 303.4 155.8 229.2 269.2 314.2 315.7 184.2 279.0 300.3 143.6 263.0 304.6 342.6 246.0 373.2 251.1 586.6 267.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

14.4 24.5 26.8 22.8 33.8 0.8 16.8 6.4 30.2 26.9 3.1 16.5 4.8 2.5 8.1 5.3 11.2 34.6 33.8 8.4

239.0 465.4 279.1 431.7 311.0 322.5 314.5 299.5 64.94 452.3 274.3 150.4 296.5 297.1 382.9 339.5 400.5 227.7 862.4 517.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 0.9 79.2 7.1 65.5 63.5 73.6 25.9 1.55 5.5 40.5 1.9 73.9 74.0 5.6 69.4 43.6 40.8 12.9 1.6

636.8 ± 7.0 1614 ± 85 1541 ± 85 1582 ± 75 989.6 ± 51.8 827.0 ± 51.8 1296 ± 67 1230 ± 85 416.1 ± 19.7 1440 ± 73 998.5 ± 21.6 505.1 ± 27.2 1180 ± 41 1201 ± 52 1176 ± 36 1102 ± 72 1392 ± 45 903.3 ± 42.9 2675 ± 86 785.7 ± 4.6

459.1 ± 9.3 1094 ± 58 823.0 ± 30.9 607.5 ± 65.3 602.7 ± 41.4 918.6 ± 113.7 565.3 ± 9.3 725.0 ± 47.6 239.0 ± 43.8 646.6 ± 24.1 633.0 ± 11.5 175.8 ± 16.2 624.6 ± 44.2 928.7 ± 147.3 575.9 ± 30.8 914.2 ± 134.6 821.6 ± 60.9 876.1 ± 69.5 1003 ± 102 816.5 ± 38.7

Northern highbush blueberry Ericaceae, V. corymbosum L. N1 Bluecrop 91.80 ± 0.03 96.60 N2 Legacy 158.3 ± 10.3 143.6 N3 Duke 78.71 ± 0.04 56.28 N4 Nui 114.6 ± 0.1 130.3 N6 Coville 117.3 ± 1.0 160.7 N7 Berkeley 132.4 ± 2.7 191.6 N8 Brigitta 128.8 ± 2.9 173.6 N9 Reka 112.9 ± 5.4 167.4 N11 Bluejay 94.49 ± 1.38 78.82 N12 Jersey 85.67 ± 8.62 110.3 N13 Elliott 78.73 ± 1.33 132.8 N14 Spartan 104.2 ± 0.0 81.76 N15 Darrow 148.1 ± 0.1 215.0 N16 Earlyblue 77.40 ± 9.12 103.8 N17 Meader 123.2 ± 3.9 182.6 N19 Nelson 73.73 ± 0.01 105.1 N20 Sunrise 99.59 ± 4.81 90.88 N21 Rubel 101.4 ± 0.1 144.1 N22 Bluegold 80.40 ± 1.15 68.45 N23 D-II 114.4 ± 7.11 174.5 N25 Croaton 146.6 ± 2.8 220.9 N26 Bonus 54.39 ± 0.03 58.84 N27 Toro 75.07 ± 1.48 133.9 N28 Bluechip 107.4 ± 1.71 159.0 N29 Elizabeth 81.74 ± 0.03 49.34 N30 Chandler 123.1 ± 2.6 162.8 N32 Bluetta 101.9 ± 9.7 165.6 N33 Bluegarden 127.6 ± 0.6 232.1 N34 Puru 110.0 ± 0.0 73.46 N35 Chippewa 82.29 ± 0.04 66.24 N36 Roseblue 171.3 ± 5.0 307.4 N40 Blackpearl 224.1 ± 3.4 438.2 N42 Amatsububoshi 131.5 ± 7.0 242.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10.38 4.0 1.56 10.9 4.3 1.5 10.46 2.04 2.73 4.3 6.0 6.17 9.54 4.78 2.25 3.34 6.6 1.6 6.55 6.5 6.38 4.85 1.78 6.7 6.09 6.8 9.55 3.2 2.25 1.48 4.3 9.4 3.7

257.8 333.5 192.4 307.7 304.2 263.1 248.5 318.6 238.1 279.1 312.6 256.3 249.1 333.0 293.1 294.3 254.8 259.2 278.6 252.9 261.9 226.5 305.0 323.0 200.0 309.5 260.4 280.8 281.9 182.3 357.9 275.7 290.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.7 12.1 36.7 3.7 7.8 28.1 29.2 7.9 4.25 27.1 18.3 4.3 9.9 5.6 5.7 10.2 10.1 2.2 19.5 10.3 2.6 4.8 7.0 4.4 7.1 9.8 10.1 28.6 24.5 6.7 30.8 11.5 2.5

314.3 474.3 303.6 452.6 367.1 451.1 191.3 457.7 148.5 257.3 571.5 447.6 455.8 349.9 586.7 201.6 260.1 476.7 242.1 332.1 302.8 161.1 338.1 360.0 274.0 331.4 290.2 343.3 249.9 379.8 301.2 586.5 371.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

37.4 1.8 2.2 17.7 44.8 4.5 36.3 4.5 6.8 29.2 19.0 0.9 5.6 18.0 23.7 25.6 43.1 2.7 58.9 2.7 8.5 15.5 69.1 68.6 41.5 22.2 40.0 30.2 52.5 51.3 39.7 87.0 39.3

937.6 ± 84.9 415.3 ± 39.5 547.9 ± 36.6 683.3 ± 65.7 989.3 ± 28.9 557.1 ± 8.0 933.5 ± 17.6 587.7 ± 40.1 550.7 ± 6.3 1055 ± 80 492.6 ± 33.5 358.2 ± 26.2 614.5 ± 16.1 257.3 ± 15.4 539.0 ± 3.9 920.8 ± 47.3 408.4 ± 15.1 1098 ± 92 710.5 ± 53.2 823.3 ± 44.3 1026 ± 60 692.5 ± 16.2 1386 ± 97 1056 ± 48 665.0 ± 27.8 565.5 ± 22.8 1230 ± 153 1435 ± 152 945.7 ± 74.5 607.1 ± 98.6 1374 ± 56 1601 ± 69 981.2 ± 80.1

464.2 ± 81.7 680.1 ± 24.2 720.6 ± 38.1 1140 ± 87 1018 ± 176 859.6 ± 105.3 538.1 ± 37.4 686.9 ± 36.0 419.6 ± 87.2 481.1 ± 28.6 816.8 ± 35.1 695.1 ± 30.3 933.9 ± 35.0 585.3 ± 109.2 992.1 ± 17.2 862.3 ± 42.5 1183 ± 91 665.1 ± 37.7 509.9 ± 44.3 594.1 ± 27.8 755.0 ± 73.3 276.9 ± 10.9 273.1 ± 71.0 654.7 ± 25.8 341.8 ± 20.1 1036 ± 123 821.7 ± 53.8 1072 ± 21 738.3 ± 66.2 737.4 ± 76.4 1188 ± 27 1422 ± 30 1111 ± 31

Rabbiteye blueberry Ericaceae, Vaccinium ashei R1 Premier 103.1 ± 2.2 R2 Bluebelle 200.5 ± 9.6 R3 Choice 146.3 ± 7.1 R4 Beckyblue 130.4 ± 11.0 R5 Briteblue 153.6 ± 1.7 R6 Austin 112.8 ± 2.7 R7 Garden Blue 211.6 ± 3.5 R8 Brightwell 132.6 ± 0.7 R9 Climax 90.57 ± 1.81 R10 Powderblue 105.7 ± 8.8 R11 Baldwin 141.4 ± 1.2 R12 Tifblue 124.7 ± 5.6 R13 Woodard 135.7 ± 1.4

± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.4 12.1 2.1 7.5 17.8 13.0 4.3 2.5 2.6 5.4 2.7 4.2 3.1

0.64 19.6 0.18 0.7 3.5 3.4 7.6 0.1 2.67 0.5 4.1 0.31 0.6 6.9 6.3 2.1 2.58 0.8 13.89 3.2 14.2 0.53 2.8 17.6 0.21 1.8 0.6 5.5 0.23 0.15 1.1 11.9 0.4

56.10 101.3 44.11 154.4 132.9 205.0 59.65 96.40 30.89 122.4 105.6 52.27 34.49 53.97 88.49 62.58 104.7 145.7 81.96 120.2 85.34 45.53 96.15 106.6 53.26 148.9 85.50 106.5 80.13 12.74 105.4 240.7 260.7

(continued on next page)

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Table 1 (continued) Sample code

blueberry cultivar

TPC

TFC

PAC

DPPH

ABTS

FRAP

ORAC

(mg GAE/g DW)

(mg RTE/g DW)

(mg CTE/g DW)

(μmol TEAC /g DW)

(μmol TEAC /g DW)

(μmol FEAC/g DW)

(μmol TEAC/g DW)

Half-high blueberry Ericaceae, V. corymbosum L. × V. H1 Northblue 73.53 ± 0.02 H2 Polaris 104.0 ± 1.1 H3 Northland 160.9 ± 9.2 H4 Northcountry 103.6 ± 1.7 H5 Tophat 105.3 ± 1.2

angustifolium Ait. 123.6 ± 2.8 151.0 ± 6.3 179.9 ± 12.9 187.5 ± 6.2 179.6 ± 14.9

93.53 156.2 110.3 280.8 39.89

281.4 294.7 223.7 351.5 253.0

375.0 408.0 454.3 395.5 413.4

8.6 10.2 5.6 22.6 12.5

533.7 ± 23.9 544.4 ± 8.14 560.1 ± 17.2 556.3 ± 27.8 1030 ± 40

539.0 ± 23.1 939.9 ± 35.6 1002 ± 90 1002 ± 30 1267 ± 115

Lowbush blueberry Ericaceae, V. angustifolium Ait. L1 Brunswick 170.0 ± 11.1 L3 Chignecto 88.38 ± 0.03

351.7 ± 18.6 54.71 ± 0.21

229.7 ± 5.6 54.80 ± 1.38

552.8 ± 20.2 352.8 ± 24.1

1553 ± 124 483.0 ± 59.4

1235 ± 59 639.3 ± 34.9

± ± ± ± ±

14.26 6.4 8.4 8.7 0.81

± ± ± ± ±

25.1 1.9 25.2 7.7 18.1

253.1 ± 11.1 212.8 ± 25.7

± ± ± ± ±

TPC, TFC, and PAC are the abbreviations of total phenolic content, total flavonoid content, and total proanthocyanidin content, respectively. DPPH, ABTS, FRAP, and ORAC are the abbreviations of the DPPH radical scavenging activity, ABTS radical cation scavenging activity, ferric reducing antioxidant power, and oxygen radical absorbance capacity, respectively. GAE, RTE, CTE, TEAC, and FEAC represent the gallic acid equivalent, rutin equivalent, catechin equivalent, trolox equivalent antioxidant capacity, and Fe (II) equivalent antioxidant capacity, respectively.

were added into a tube wrapped with foil. A 1.500 mL of concentrated hydrochloric acid was added, and the reaction solution was thoroughly mixed at RT for 15.00 min. The absorbance was measured at 500.0 nm. The PACs were calculated using the standard curve of catechin and were expressed in catechin equivalent (CTE), i.e., mg CTE/g DW.

analytical column was a Zorbax SB-C18 column (250.0 mm × 4.600 mm, 5.000 μm). The HPLC conditions were the same as those described above. The ESI capillary voltage was 3.000 kV in negative ion (NI) mode with a capillary temperature of 350.0 °C. A nebulizing gas of 1.500 L/min and a drying gas of 10.00 L/min were applied for the ionization using nitrogen (N2). ESI was performed with a scan range of m/z 200.0–2000.

2.5. High-performance liquid chromatographic (HPLC) analysis The main phenolic compounds in blueberry leaves were analyzed based on our previous report with some modifications (Huang et al., 2012). The samples were filtered using a 0.220 μm polyvinylidene fluoride (PVDF) membrane, and then analyzed using an Agilent 1100 HPLC system (Agilent Technologies, USA) that was equipped with a binary pump and a diode-array detector (DAD). Chromatographic analysis was conducted using a 250.0 mm × 4.600 mm, 5.000 μm particle size, and end-capped reverse-phase Zorbax SB-C18 column (Agilent Technologies, USA). The running temperature was 20.00 °C, and the injection volume was 10.00 μL. The detection was conducted at 280.0, 320.0, and 360.0 nm at a flow rate of 0.600 mL/min. Mobile phase A was 1.000% formic acid (TFA), whereas mobile phase B was 100.0% methanol. The elution gradients were as follows: 10.00% to 60.00% B (from 0.000 to 25.00 min), 60.00% to 80.00% B (from 25.00 to 40.00 min), and 80.00% to 10.00% B (from 40.00 to 45.00 min). The phenolics were identified by comparing both the retention times (tR) and UV spectra with the standards and/or the literature data (Wang et al., 2015). Eight major phenolics were quantified using their relative standard curve. The results were expressed as milligrams of each phenolic compound per gram of the dried blueberry leaves, i.e., mg/g DW. The standard curves at 280 nm of the quantified individuals were y = 40.68x + 26.82 (R2 = 0.999), y = 80.41x − 86.85 (R2 = 0.999), y = 42.45x + 60.61 (R2 = 1.000), y = 51.00x − 6.233 (R2 = 1.000), y = 48.17x − 94.50 (R2 = 1.000), y = 26.32x + 69.63 (R2 = 0.999), y = 36.77x + 39.44 (R2 = 1.000), and y = 36.37x − 35.32 2 (R = 0.998), where y was the peak area, and x was 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, quercetin-3-O-galactoside, quercetin-3-O-glucoside, and kaempferol-3-O-glucoside, respectively, in μg/mL.

2.7. Assays of different antioxidant capacities 2.7.1. DPPH radical (DPPHˑ) scavenging activity The assay was examined according to the method of Xiao et al. (2014). Briefly, 2.000 mL of sample was added to 2.000 mL of 0.200 mmol/L DPPH solution. The mixture was shaken immediately and allowed to stand in the dark for 30.00 min. The absorbance was then recorded at 517.0 nm. Different concentrations of Trolox in 85.00% methanol containing 0.500% formic acid were prepared and assayed as a standard. The extraction solvent without the sample was used as a blank control. The results were expressed in terms of the Trolox equivalent antioxidant capacity (TEAC), i.e., μmol TEAC/g DW. 2.7.2. ABTS radical cation (ABTSˑ+) scavenging activity As reported by Cai, Luo, Sun, and Corke (2004), ABTSˑ+ was generated by oxidizing ABTS (7.000 mmol/L) with potassium persulfate (2.450 mmol/L) after incubation in the dark for 16.00 h. The freshly prepared ABTSˑ+ solution was diluted with ethanol in order to obtain the absorbance at 734.0 nm of 0.700 ± 0.020. A 1.000 mL of sample was added to 4.000 mL of ABTSˑ+ solution and mixed thoroughly. The reaction mixture was then allowed to stand in the dark for 6.000 min. The extraction solvent and Trolox were also assayed as a control and a standard, respectively. The absorbance at 734.0 nm was recorded. The results were also expressed in terms of their TEAC, i.e., μmol TEAC/g DW. 2.7.3. Ferric reducing antioxidant power (FRAP) The FRAP evaluation was determined according to Razak, Rashid, Jamaluddin, Sharifudin, and Long (2015)'s report with slight modifications. A 100.0 mL of 0.300 mol/L acetate buffer (pH 3.600), 10.00 mL of 10.00 mmol/L TPTZ solution in 40.00 mmol/L HCl, and 10.00 mL of 20.00 mmol/L ferric chloride were mixed and warmed to 37.00 °C in order to prepare the FRAP solution. A 1.000 mL of sample was added to 5.000 mL of fresh FRAP solution. Then the mixture was placed in the dark at 37.00 °C for 20.00 min. The absorbance was measured at 593.0 nm. Different concentrations of ferrous sulfate were prepared and assayed to obtain a standard curve. The FRAP results were

2.6. HPLC-ESI-MS analysis HPLC-MS analysis was carried out using an Agilent-1100 HPLC system equipped with a UV detector and a LC-MSD Trap VL ion-trap mass spectrometer (MS) via an electrospray ionization (ESI) interface (Agilent Technologies, USA) (Li, Huang, Wang, & Liu, 2013). The 551

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expressed in Fe(II) equivalent antioxidant capacity (FEAC), i.e., μmol FEAC/g DW. 2.7.4. Oxygen radical antioxidant capacity (ORAC) In this assay, fluorescein was used as the fluorescent probe (Li et al., 2013). In brief, the reaction was carried out at 37.00 °C in 75.00 mmol/ L phosphate buffer (pH 7.400). One hundred microliters of sample at different concentrations and 50.00 μL of 0.200 μmol/L fluorescein were mixed in a black 96-well microplate. After pre-incubation at 37.00 °C for 15.00 min, a 50.00 μL of 80.00 mmol/L AAPH solution was added immediately using a multichannel pipette. The fluorescence was recorded by a LB 941 TriStar Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) with 485-P excitation and 535-P emission filters and was controlled by Mikro Win Microplate Data Reduction 2000 (Mikrotek Laborsystem GmbH, Overath, Germany) every minute during the 100.0 min process. The phosphate buffer and the Trolox were used as the blank and standard, respectively. The regression equations between the net AUC (the area under the fluorescence decay curve) and the test sample concentrations were calculated. The ORAC value was calculated from the slope of the sample equation divided by the slope of the Trolox curve that was obtained in the same assay. The results were also expressed in their TEAC, i.e., μmol TEAC/g DW. 2.8. Statistical analysis All the data were expressed as the means ± standard deviation (SD) from triplicate experiments. The areas under the fluorescence decay curve were calculated using GraphPad Prism Version 5.01 (GraphPad Software, Inc., San Diego, CA, USA), and the slopes of the regression equations were obtained by Microsoft Excel 2007. Analysis of variance (ANOVA) and Duncan's multiple comparison tests were used to determine the significant differences (P < 0.050) using IBM SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). Cluster analysis (CA) was performed and visualized using the Multi Experiment Viewer software (http://www.tm4.org/#/welcome). Principal component analysis (PCA) was conducted using JMP Pro 10 software (SAS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. TPC, TFC and PAC in phenolic extracts Phenolics are one major class of phytochemicals with discrete bioactivities towards biochemistry and metabolism (Dillard & German, 2000). The consumption of fruits and vegetables rich in phenolics can prevent degenerative and chronic diseases (Qin, Xing, Zhou, & Yao, 2015). As shown in Table 1, the TPC values of rabbiteye blueberries ranged from 90.57 ± 1.81 (R9, Climax) to 211.6 ± 3.5 (R7, Garden Blue) mg GAE/g DW, whose mean value (137.6 ± 35.4 mg GAE/g DW) was much higher than that of the other blueberries (P < 0.010 and P < 0.050 vs. S and N categories, respectively) (Fig. 1A). For half-high blueberries, the highest and the lowest TPC were 160.9 ± 9.2 (H3, Northland) and 73.53 ± 0.02 (H1, Northblue) mg GAE/g DW, respectively. Similarly, a significant difference was found between the two lowbush cultivars. Brunswick (L1) reached 170.0 ± 11.1 mg GAE/ g DW, while Chignecto (L3) only contained 88.38 ± 0.03 mg GAE/g DW. Southern and northern highbush blueberries exhibited more diverse TPCs. The highest TPCs were 185.2 ± 3.8 (S23, Nanjin) and 224.1 ± 3.4 (N40, Blackpearl) mg GAE/g DW, and the lowest were 32.18 ± 0.01 (S12, O'Neal) and 54.39 ± 0.03 (N26, Bonus) mg GAE/ g DW, respectively. There were three cultivars (N40, R7, and R2) with the values over 200.0 mg GAE/g DW, while only two (S12 and S9) had values < 50.00 mg GAE/g DW. In this study, the blueberry leaves from different cultivars contained a wide range of TPCs, which was consistent with the previous study on the leaf extracts of V. formosum (Deng et al., 2014). According to the database URL: http://www.phenol-

Fig. 1. Total phenolic content (A), total flavonoid content (B) and proanthocyanidin content (C) of blueberry leaf extracts from 73 different cultivars. ( ), ( ), ( ), ( ) and ( ) indicate the rabbiteye (R), southern highbush (S), northern highbush (N), half-high (H) and lowbush (L) blueberry cultivars.

explorer.eu, the mean TPCs of rabbiteye and highbush blueberry fruits were 5.500 and 2.234 mg GAE/g FW (19.93 and 8.09 mg GAE/g DW, fruits' moisture was approximately 72.40%), respectively. Compared with the fruits, the blueberry leaves was indicated to possess much higher contents of the total phenolics. Interestingly, the leaves that Elliot and Nelson harvested in October contained higher TPCs than these two cultivars (N13 and N19) that were collected in July in this study (Routray & Orsat, 2014). Environmental factors, such as climatic factors, soil composition (organic and mineral nutrients), weather availability, irrigation intensity, and sulfur fertilization, might contribute to the different phenolic contents (Alcaraz-Mármol, NuncioJauregui, Garcia-Sanchez, Martinez-Nicolas, & Hernandez, 2017). The TFC values of the blueberry leaves of different cultivars also 552

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varied from each other (Table 1). The southern/northern highbush and lowbush cultivars had wider TFC ranges than the rabbiteye and halfhigh ones. Consistently, S23 (304.8 ± 19.5 mg rutin/g DW) and N40 (438.2 ± 11.9 mg rutin/g DW) also had the highest TFC values, while H1 (123.6 ± 2.8 mg rutin/g DW) had the lowest value among each category. L1 (351.7 ± 18.6 mg rutin/g DW) possessed much more flavonoids than L3 (54.71 ± 0.21 mg rutin/g DW). Most rabbiteye blueberries contained high levels of flavonoids with a mean value of 249.9 ± 80.6 mg rutin/g DW, which was significantly higher than that of others (P < 0.010, P < 0.001, and P < 0.050 vs. the S, N, and H categories, respectively) (Fig. 1B). However, N40 reached the highest among all the 73 cultivars. The maximum PAC values of the corresponding categories were obtained from R7, S2 (Magnolia), N42 (Amatsububoshi), H4 (Northcountry), and L1 (Table 1), which were approximately three-, ten-, twenty-, seven-, and four-fold of the minimum values, respectively. Moreover, Fig. 1C shows the PAC distribution in different samples. Obviously, the rabbiteye blueberries had much higher PACs (mean value at 311.0 ± 104.2 mg catechin/g DW) than the other categories (P < 0.001, P < 0.001, and P < 0.010 vs. S, N, and H categories, respectively). The samples with PAC values > 400.0 mg catechin/g DW were all distributed in rabbiteye blueberries (R3, R7, and R13), whereas the values under 40.00 mg catechin/g DW were evenly spread in highbush or half-highbush blueberries, including the S (S9 and S12), N (N11, N15, and N35), and H (H5) categories. The 73 different cultivars among the five categories that were tested in our study were all collected from the same place in one season. The rabbiteye blueberry leaves contained relatively more phenolics than the other four categories probably for two other reasons. One reason is due to the genotypical difference among the cultivars. Various cultivars accumulate different quantities of phenolics, which result in major organoleptic characteristics of vegetables and fruits, especially, the properties of color and taste (Alcaraz-Mármol et al., 2017). The other reason is due to the differences in gene expression. The various blueberry species exhibit different effects with respect to the expression of phenolic genes, enzymes and metabolites. For example, the induction of phenolics may be mediated by the increase in reactive oxygen species (ROS). Since the phenylalanine ammonia-Liaza (PAL) enzyme functions in the main biosynthetic pathway of phenolic compounds, the activation of the PAL enzyme may occur as a consequence of an increase in ROS. These chain reactions lead to the increased accumulation of phenolic metabolites in cultivars (Kupper et al., 2009).

acid (peak 10) < 4,5-dicaffeoylquinic acid (peak 13). The peaks with mass spectra patterns [M-H]− ion at m/z 463.3 were quercetin (m/z 301.1) glycosylated by the sugar moieties of hexose (glucose or galactose, m/z 162.2). According to the polarities of sugar moieties, the RTs of quercetin derivates were in the order: quercetin-3-O-galactoside (peak 14) < quercetin-3-O-glucoside (peak 15). In addition, peak 19 with mass spectra patterns [M-H]− ion at m/z 447.2 were kaempferol (m/z 287.2) glycosylated by the hexose (m/z 162.0), which was confirmed to kaempferol-3-O-glucoside by referencing the standard. Caffeoylquinic acids and quercetin and kaempferol glycosides were the major phenolic constituents in blueberry leaves. Table 2 also listed the numbers of samples in each category containing the individual phenolic. Most phenolics were widely distributed in rabbiteye, southern highbush, northern highbush, half-high, and lowbush blueberry cultivars. Meanwhile, some phenolics were unequally distributed. Quinic acid (12/13, 92.31%) and myricetin glucuronide (9/13, 69.23%) were mainly distributed in rabbiteye cultivars. Kaempferol-3-O-gluconide was mainly distributed in southern (16/20, 80.00%) and northern (30/ 33, 90.91%) highbush, and half-high (5/5, 100.0%) blueberries. However, quercetin-3-O-rutinoside, myricetin glucuronide, and 5-galloylquinic acid were not detected in the two lowbush cultivars, while 5galloylquinic acid was not found in all the rabbiteye blueberries. There were 23 phenolics that were detected in the 73 cultivars, eight of which were furthermore quantified using the relative standards (Table 2, Fig. 2A-E). Similarly, the contents of the phenolics varied greatly in the different cultivars. According to Fig. 2F, 3-O-caffeoylquinic acid, quercetin-3-O-galactoside, and 5-O-caffeoylquinic acid were the three most dominant phenolics, accounting for 33.79 to 56.67%, 14.54 to 27.96%, and 5.762 to 14.90% in the five categories, respectively. The H contained the highest level of 3-O-caffeoylquinic acid (20.90 ± 6.49 mg/g DW, 56.67%), and the L contained the highest level of quercetin-3-O-galactoside (5.082 ± 5.030 mg/g DW, 27.96%). Eight phenolics exhibited a relative average distribution (from 4.473% to 33.79%) in the R except for 4-O-caffeoylquinic acid (1.245%). However, the R exhibited the lowest mean value (29.56 mg/ g DW) of these eight phenolics among five categories. Some other phenolics listed in Table 2 probably contributed to the R's highest TPC. The limits of detection (LOD) and the limits of quantification (LOQ) for standards were in the range of 0.320–0.873 μg/mL and 0.971–2.645 μg/mL, respectively (Chanda, Biswas, Kar, & Mukherjee, 2019). Since these LODs and LOQs were very low, it was realistic to conclude that the HPLC method was precise and accurate. The linearity ranges were similar for all standards and ranged between 0.010 and 0.500 mg/mL. The blueberry leaves had similar phenolic compositions, but different contents of the main constituents. The change in the amounts of individual phenolics in different cultivars can be explained in terms of the biosynthetic pathways of compounds (Kalinowska, Bielawska, Lewandowska-Siwkiewicz, Priebe, & Lewandowski, 2014). In this study, quercetin-3-O-galactoside, 3-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid were the dominant constituents. Among the 23 phenolics that were identified, there were 13 flavonoids, more than half of which were quercetin derivatives. This result was consistent with previous reports on berries, such as bilberries, blueberries, and black currants, which were good sources of flavonoids, especially quercetin and myricetin (Maatta-Riihinen, Kamal-Eldin, & Torronen, 2004). Compared with quercetin, the kaempferol contents in most cultivars were lower, and myricetin could not be detected in some cultivars. Paunovic, Maskovic, Nikolic, and Miletic (2017) also found that quercetin was the dominant flavonoid in the leaves from black currants (Ribes nigrum L.); Tabart, Kevers, Pincemail, Defraigne, and Dommes (2006) reported that myricetin varied widely among cultivars, and the kaempferol content was at a relatively low level. Here, 3-O-caffeoylquinic acid was the most abundant of the nine phenolic acids that were detected, followed by 5-O-caffeoylquinic acid. This finding was in accordance with the previous report on bilberry (Vaccinium myrtillus)

3.2. Identification and quantification of phenolic compounds By comparing the chromatographic behavior, UV–vis spectral characteristics, and mass spectra with the standards and literature data that were previously reported (Gavrilova, Kajdzanoska, Gjamovski, & Stefova, 2011; Jiao, Kilmartin, Fan, & Quek, 2018; Tian et al., 2017; Wang et al., 2015; Wang et al., 2019), 23 phenolics were tentatively identified, including nine phenolic acids, thirteen flavonoids (seven quercetin derivatives, four kaempferol derivatives, and two myricetin derivatives), and one proanthocyanidin (B-type procyanidin dimer) (Table 2). The representative HPLC chromatograms of the leave samples from each category were provided as supplemented data. There were three peaks that had an [M-H]− ion at m/z 353.0, an [M-H-162]− ion at m/z 191.0 (quinic acid), an [2 M-H]− ion at m/z 707.0, and a λmax approximately at 320.0 nm were identified as 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid. According to the different polarities of the derivates, the retention times (RT) on the C18 column were with the order: 5-O-caffeoylquinic acid (peak 2) < 3O-caffeoylquinic acid (peak 4) < 4-O-caffeoylquinic acid (peak 5). Moreover, the two peaks which had an [M-H]− ion at m/z 515.2 and an [M-H-162]− ion at m/z 353.2 (caffeoylquinic acid) and a λmax approximately at 330.0 nm, were identified as dicaffeoylquinic acids. And the RTs of them increased in the following order: 3,5-dicaffeoylquinic 553

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Table 2 Phenolic compounds identified from blueberry leaves of 73 different cultivars. Peak No.

Compounds

tR (min)a

λmax (nm)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Number of samples in each category Quinic acid 5-O-Caffeoylquinic acid B-type Procyanidin dimer 3-O-Caffeoylquinic acid 4-O-Caffeoylquinic acid O-p-Coumaroylquinic acid Quercetin-3-O-arabinoside Malonyl-caffeoylquinic acid Kaempferol-3-O-arabinoside 3,5-Dicaffeoylquinic acid Quercetin-3-O-glucuronide Quercetin-3-O-rutinoside 4,5-Dicaffeoylquinic acid Quercetin-3-O-galactoside Quercetin-3-O-glucoside Quercetin-3-O-acetylhexoside Quercetin-3-O-xyloside Kaempferol-3-O-rutinoside Kaempferol-3-O-glucoside Myricetin glucuronide Kaempferol-3-O-glucuronide 5-Galloylquinic acid Myricetin-3-O-hexose

of blueberry cultivars 4.390 210.0, 13.60 234.0, 16.24 216.5, 17.11 234.0, 17.83 234.0, 19.85 240.0, 21.15 256.0, 22.79 246.0, 24.24 254.0, 24.57 210.0, 25.57 254.0, 25.82 257.3, 26.20 210.0, 26.43 257.0, 26.73 256.0, 27.37 257.2, 27.77 257.2, 28.49 254.0, 29.06 263.7, 30.94 267.0, 31.73 253.0, 32.19 210.0, 32.85 260.0,

280.0 325.0 282.1 325.0 325.0 314.0 356.0 326.0 355.0 329.0 351.0 352.8 329.0 354.6 356.0 355.1 353.8 355.0 347.9 352.0 357.0 280.0 356.0

[M-H]− (m/z)

Rc

S

N

H

L

Total

191.5 191.5, 289.1, 191.3, 191.5, 191.3, 433.2 439.1 417.1 353.2, 477.2 463.3, 353.2, 301.1, 301.1, 505.2 433.2 593.5 287.2, 493.1 461.1 343.2 501.2

13 12d 13 8 13 13 10 8 11 8 13 9 1 13 13 13 9 9 10 13 9 5 0 5

20 15 20 19 20 20 20 14 20 12 20 17 18 20 20 20 19 19 12 20 17 16 4 16

33 20 33 30 33 33 29 19 23 15 33 18 8 33 33 33 31 30 27 33 5 30 7 18

5 1 5 5 5 5 5 2 5 4 5 4 2 5 5 5 4 4 3 5 3 5 3 2

2 1 2 2 2 2 2 1 2 1 2 1 0 2 2 2 2 2 2 2 0 1 0 1

73 49 73 64 73 73 66 44 61 40 73 49 29 73 73 73 65 64 54 73 34 57 14 37

353.2, 707.2 577.3 353.5, 707.3 353.2, 707.2 337.1

515.2 609.3 515.2 463.3 463.3

447.2

a

tR is the retention time of LC-MS. λmax is the maximum absorption wavelength. c R, S, N, H and L represent rabbieye blueberry, southern highbush blueberry, northern highbush blueberry, half-high blueberry and lowbush blueberry, respectively. d numbers indicate the number of samples which contain the corresponding phenolic compound in the same row of Table 2. b

leaves that hydroxycinnamic acid derivatives accounted for 82% of the total phenolics, mostly as 3-O-caffeoylquinic acid (Tian et al., 2018). According to a report on crabapples, their flowers and leaves were rich in flavonoids, while phenolic acids were abundant in fruits (Liu, Wang, & Wang, 2018). However, the blueberry leaves examined here were detected to have both a large amount of chlorogenic acid and a high level of flavonoids. For proanthocyanidin, the primary B-type procyanidin dimer was detected in 87.67% of blueberry cultivars. Similarly, Tian et al. (2018) also found that procyanidin dimers and trimers were rich in the leaf extracts of other fruits, such as lingonberry (Vaccinium vitis-idaea), hawthorn (Crataegus spp.), and bilberry (Vaccinium myrtillus).

S7, S8, S11, S14, S16, S19, and S23) and 30.30% of the N (N2, N4, N6, N9, N13, N16, N27, N28, N30, and N36) had DPPH values over 300.0 μmol TEAC/g DW. For either the rabbiteye or the half-high blueberries, only one cultivar (R6 and H4) could reach this level. Fig. 3A also showed the changes in the DPPH values in different categories. The mean DPPH values of the S (280.9 ± 92.7 μmol TEAC/g DW), the N (275.2 ± 40.6 μmol TEAC/g DW), and the H (280.9 ± 40.0 μmol TEAC/g DW) were relatively higher than that of the R (254.9 ± 25.3 μmol TEAC/g DW) and the L (232.9 ± 28.6 μmol TEAC/g DW), respectively (P > 0.050). However, the variations among the different cultivars of the S, N, and H were also greater than those of the R and L. The highest ABTS value of each category was 450.8 ± 0.9 (R6), 862.4 ± 12.9 (S23), 586.7 ± 23.7 (N17, Meader), 454.3 ± 5.6 (H3), and 552.8 ± 20.2 (L1) μmol TEAC/g DW, which were 3.071, 13.280, 3.951, 1.212, and 1.567 folds greater than the corresponding lowest ABTS value (R12, S9, N11, H1, and L3), respectively (Table 1). There were six cultivars (S23, S24, N13, N17, N40, and L1) with ABTS values over 500.0 μmol TEAC/g DW, while six (R12, S9, S12, N8, N11, and N26) were under 200.0 μmol TEAC/g DW. The H cultivars showed moderate and concentrated ABTS radical scavenging activities with values from 375.0 ± 8.6 to 454.3 ± 5.6 μmol TEAC/g DW. No significant difference in the ABTS means existed among the five categories; however, substantial changes existed among the different cultivars (Fig. 3B). Most cultivars had high FRAP values (Table 1). The highest value among the 73 cultivars reached 2674 ± 86 μmol FEAC/g DW (N23, DII). In each category, the percentages of the samples with FRAP values higher than 1000 μmol FEAC/g DW were 60.00% (S), 53.85% (R), 50.00% (L), 27.27% (N), and 20.00% (H). However, one S cultivar (S9), four N cultivars (N2, N13, N14, and N16), and one L cultivar (L3) had low FRAP values under 500.0 μmol FEAC/g DW. As shown in Fig. 3C, the FRAP values of blueberry leaves were mainly located between 200.0 and 2000 μmol FEAC/g DW, thus reflecting the dispersed distribution of their ferric reducing antioxidant power. The mean FRAP value of

3.3. Antioxidant activities of phenolic extracts In this study, several assays were used to determine the antioxidant effects of blueberry leaf extracts. As a stable lipophilic free radical, the DPPH radical is commonly employed in order to evaluate the free radical scavenging potential of plant extracts (Huang et al., 2012). FRAP is evaluated by the Fe3+ to Fe2+ transformations and acts as an important indicator of antioxidant activity (Lee, Seo, Lim, & Cho, 2011). ORAC may be one of the most suitable methods to assess the in vitro antioxidant capacity because it utilizes a biological relevant radical source (Prior & Gao, 1999). According to the present evidence, the antioxidant activities of blueberry leaves were varied due to the inherent variability in the phenolic contents among different cultivars. This observation was similar to Ehlenfeldt and Prior (2001)'s, which revealed that the genetic expression in highbush blueberries was deduced to be a dominant determinant of the antioxidant properties. Table 1 provides the results of four different antioxidant assays. The DPPH assay showed that southern highbush cultivars had the highest value of 586.6 ± 33.8 μmol TEAC/g DW (S23) and the lowest of 143.6 ± 16.5 μmol TEAC/g DW (S12). Moreover, both southern and northern highbush cultivars possessed relatively better DPPH radical scavenging activity than the others. Among them, 40.00% of the S (S3, 554

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Fig. 2. Contents of eight individual phenolics in rabbiteye blueberry (A), southern highbush blueberry (B), northern highbush blueberry (C), half highbush blueberry (D) and lowbush blueberry (E), and distribution of the corresponding total contents of the eight phenolics in each category (F).

southern highbush cultivars was 1175 ± 493 μmol FEAC/g DW, which was comparatively higher than the others. The half-high cultivars had a relatively low FRAP with a mean value of 645.0 ± 216.0 μmol FEAC/g

DW. The variation in the ORAC values among the different categories and cultivars were also exhibited in Table 1 and Fig. 3D. The highest 555

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Fig. 2. (continued)

and 30.00% (6/20) of the S cultivars were distributed in Clusters I, II, III, and IV, respectively. In addition, the N cultivars were separated in Cluster I (27.27%, 9/33), II (39.39%, 13/33), III (12.12%, 4/33), and IV (21.21%, 4/7). This finding indicated that most of the S gathered in Clusters III and IV (65.00%), while the N were mainly in Clusters I and II (66.66%). Thus, the S and N were the two highbush blueberry categories with the greatest differences. In addition, among the five categories, the R had the best correlation between the 15 parameters and the cultivars since 69.23% (9/13) of the R were located in Cluster III. On the other side, all the parameters that were considered were separated into four groups. The TPC, TFC, PAC, and two dicaffeoylquinic acids were in Group III, and the four antioxidant indices and three caffeoylquinic acids were in Group IV. In this condition, the TPC, TFC, PAC, and the dicaffeoylquinic acids contributed equally to the classification of the cultivars, and among the eight quantified compounds, the caffeoylquinic acids and the antioxidant capacities were closely associated. The FRAP was slightly separated from the DPPH, ABTS, and ORAC, which was probably due to the different standard substances that were used in the detection methods. The PCA provided valuable information on the relationships among the phenolic compositions and antioxidant capacities. There were four PCs (PC1, PC2, PC3 and PC4) explaining up to 66.23% of total variance. PC1 and PC2 were the first two principal components extracted from the statistical analysis, which accounted for 30.39% and 14.28%, totaling 44.67% of the cumulative variance in the data set. The score plot (Fig. 4B) indicated that the samples were arranged from left to right according to the category on the dimension plot. Obviously, the rabbiteye blueberry leaves converged in the bottom-right area (PC1 > 0.000% and PC2 < 0.000%), and most of the northern highbush leaves gathered together in the bottom-left area (PC1 < 0.000% and PC2 < 0.000%) of the plot. In addition, Fig. 4C provides the PCA loadings of the parameters. The TPC, TFC, PAC, DPPH, ABTS, FRAP, ORAC and three (5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-Ocaffeoylquinic acid) of the eight phenolics were well correlated with their high loadings on PC1, which reflected the potential correlations between the phenolic contents and the antioxidant activity. Similarly, this result also indicated that the caffeoylquinic acids in blueberry leaves perhaps contributed some to their antioxidant activities. Consistent with the PCA, R2 analysis (4-O-caffeoylquinic acid vs. FRAP = 0.786, 4-O-caffeoylquinic acid vs. ABTS = 0.800, etc.) showed the obvious positive correlations between some of the phenolics and the antioxidant activities. As shown by the PCA, the activities that were measured in DPPH,

ORAC value of rabbiteye cultivars was obtained from Baldwin (1348 ± 50 μmol TEAC/g DW, R11), while the lowest was obtained from Austin (391.9 ± 33.8 μmol TEAC/g DW, R6). Moreover, the highest values were 1094 ± 58 (S2), 1422 ± 30 (N40), 1267 ± 115 (H5) and 1235 ± 59 (L1) μmol TEAC/g DW, and the lowest values were 175.8 ± 16.2 (S12), 273.1 ± 71.0 (N27, Toro), 539.0 ± 23.1 (H1) and 639.3 ± 34.9 (L3) μmol TEAC/g DW for the S, N, H, and L cultivars, respectively. In addition, most of the ORAC values over 1000 μmol TEAC/g DW belonged to the H (60.00%; H3, H4, and H5) and the N (21.21%; N4, N6, N20, N30, N36, N40, and N42), and the values lower than 500.0 TEAC/g DW were mainly in the N category (18.18%, N1, N11, N12, N26, N27, and N29) but were not detected in the H and L. Similarly, the ORAC values of different cultivars changed greatly, but the difference among the five categories was not significant. According to the database URL: http://oracdatabase.com, the average ORAC value of blueberry fruits was 234.7 μmol TEAC/g FW (850.4 μmol TEAC/g DW). In comparison with the data above, the majority of leaves in our study performed better antioxidant activities and their extracts had the potential to be developed as nutraceuticals. The different phenolic components displayed various bioactivities due to their structure-activity relationships. The bioactive properties of phenolics were mainly due to the neighboring hydroxyl groups that were present, allowing them to act as reducing agents, hydrogen donors, and singlet oxygen (Kim, Cho, & Han, 2013). With respect to the flavonoids, which are the most common components in blueberry leaves, there are three essential structural features impacting their antioxidant properties: an ortho-dihydroxyl group in the B ring, a C2]C3 double bond conjugated with the 4-oxo group, and the hydroxyl groups in the C3 and/or C5 of C ring (Balasundram, Sundram, & Samman, 2006). The antioxidant activity of flavan-3-ols is attributed to the catechol group in the B ring and the C3-OH in C ring (Heim, Tagliaferro, & Bobiya, 2002). In the present study, phenolic acids and their derivatives were also investigated. Through the functional groups, such as hydroxyl (OH) and carboxylic acid (-COOH), phenolic acids acted as the antioxidant agents and complemented the activity of antioxidant enzymes, especially superoxide dismutase (SOD), mainly by the ROS-scavenging mechanism (Zouari et al., 2016). 3.4. CA and PCA models According to the dendrogram from the CA, a heat map (Fig. 4A) was generated demonstrating that the 73 cultivars were clustered into four big clusters. Obviously, 25.00% (5/20), 10.00% (2/20), 35.00% (7/20), 556

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Fig. 3. DPPH radical scavenging activity (A), ABTS radical cation scavenging activity (B), ferric reducing antioxidant power (C) and oxygen radical absorbance capacity (D) of blueberry leaf extracts from 73 different cultivars. ( ), ( ), ( ), ( ) and ( ) indicate the rabbiteye (R), southern highbush (S), northern highbush (N), half-high (H) and lowbush (L) blueberry cultivars.

ABTS, FRAP, and ORAC were highly correlated with caffeoylquinic acids and quercetin glycosides. It has been acknowledged that the sugar moieties in flavonoids may reduce the radical scavenging activity by diminishing the coplanarity of the B ring and by occupying more hydroxyl groups. The type and position of the sugar substituents in the molecule may have a significant influence on the antioxidant activities (Heim et al., 2002). Tian et al. (2018) also found a positive correlation between the DPPH radical scavenging activity and the flavone di- and tri-glycosides. Based on the ORAC assay of Wang et al. (2017), blueberry fruits had a significantly higher value (4826 μmol TEAC/100 g DW) than other kinds of fruits, including pomegranates (4479 μmol TEAC/100 g DW), oranges (2887 μmol TEAC/100 g DW), etc. In this study, the average ORAC value of blueberry leaf extracts was almost 10 times higher than that of blueberry fruits, thus showing that the leaves are a superior resource of antioxidants. Unexpectedly, the rabbiteye blueberry leaves had higher TPC, TFC, and PAC than the other four categories but had weaker antioxidant activities than the highbush leaves. These findings could be explained by the lower mean values of the eight quantified phenolics in the R compared with the S and N. It was speculated that the eight phenolics, including the caffeoylquinic acids and glycosides of quercetin and kaempferol, might be primary in the blueberry leaves and mainly contribute to the antioxidant activity. Consistent with this hypothesis, the PCA also revealed that the three caffeoylquinic acids were the most antioxidant related compounds for blueberry leaves. Otherwise, some phenolics that were responsible for the bioactivities in the leaves were perhaps present in other forms. 4. Conclusions The leaves that were tested from 73 blueberry cultivars were rich in phenolics and had strong in vitro antioxidant capacities. The total phenolic, total flavonoid, and proanthocyanidin contents varied considerably, which led to the various antioxidant activities of different cultivars. The rabbiteye blueberry cultivars had significantly higher TPC, TFC, and PAC than the other four categories. In addition, the caffeoylquinic acids and the quercetin and kaempferol glycosides were the dominant phenolics in blueberry leaves, which contributed to the stronger antioxidant activity in southern and northern highbush cultivars. Several sources of variability in these assays that were described have been taken into account, including the origin of the cultivars, storage conditions, analytical errors, etc. Thus, the leaf extracts from the different blueberry cultivars could be compared by neglecting the effecting factors besides the cultivar variety at the same level. The individual phenolic contents and four antioxidant indices were well-separated by the PCA, and the different cultivars were grouped into four prime clusters. This survey should be important for the breeding programs and overall utilization of blueberries. However, future studies are needed to clarify the mode-of-action of the bioactive phenolics in blueberries, as well as the interactions between the phenolics and the other compounds existing in the matrix. Due to the climatic limitations in southern China, the sample size of lowbush blueberry cultivars in this study was not large enough. It would be interesting to further study the characteristics of the blueberries from different regions and/or at different times. The blueberry cultivars with significantly better phenolic performance in their leaves would also be selected for follow-up intensive research.

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Fig. 4. The dendrogram from cluster analysis (CA) and the plots of principal component analysis (PCA) involving the 15 variables on blueberry leaves of 73 different cultivars. All values in the heat map of fig. (A) are transformed into Z-scores and then clustered using Euclidean distances. Figs. (B) and (C) indicate PCA scores of the samples and PCA loadings of the parameters, respectively. , , , , represent the rabbiteye, southern highbush, northern highbush, half-high and lowbush blueberry cultivars, respectively.

Acknowledgements

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