Food Chemistry 145 (2014) 674680

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata Blanco) fruits
Yuanmei Zhang a,b, Yujing Sun b, Wanpeng Xi a,c, Yan Shen b, Liping Qiao b, Liezhou Zhong b, Xingqian Ye b,, Zhiqin Zhou a,c,
a b c

College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China Department of Food Science and Nutrition, School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310029, Zhejiang Province, China Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400715, China

a r t i c l e

i n f o

a b s t r a c t
As one of the most important centres of origin for the genus Citrus L., China is rich in wild mandarin germplasm. In this study, phenolic compounds in the peels of 14 wild mandarin genotypes native to China were determined and their antioxidant capacities were evaluated using DPPH, FRAP, ABTS and ORAC methods. We found that Nieduyeju had the highest total phenol content (51.14 mg/g DW), and Wulongsuanju had the highest total avonoid content (20.66 mg/g DW). Hesperidin, the dominant avonoid, was observed to be highest in Guangxihongpisuanju (55.98 mg/g DW). Ferulic acid was the most abundant phenolic acid analyzed, and Nieduyeju (7780.17 lg/g DW) and Guangxihongpisuanju (13,607.19 lg/g DW) had the highest contents of extractable and bound phenolic acid, respectively. Antioxidant potency composite (APC) index showed obvious variations ranging from 58.84 to 98.89 in the studied wild mandarins, among them, Nieduyeju had the highest APC index. Overall, Guangxihongpisuanju, Nieduyeju, Cupigoushigan and Daoxianyeju contained more phenolics and exhibited higher antioxidant capacities than the mandarin cultivars Satsuma and Ponkan. 2013 Published by Elsevier Ltd.

Article history: Received 31 January 2013 Received in revised form 20 July 2013 Accepted 2 August 2013 Available online 11 August 2013 Keywords: Wild mandarins Total phenolics Flavonoid Phenolic acid Antioxidant capacities

1. Introduction Plant polyphenols are a large group of secondary metabolites that have important functions in combating chronic diseases, such as type 2 diabetes, heart disease and various cancers, because of their high antioxidant activities (Chavez-Santoscoy, Gutierrez-Uribe, & Serna-Saldivar, 2009; Jiang & Dusting, 2003; Oboh & Ademosun, 2012). Citrus is one of the most important horticultural crops worldwide and their fruits are abundant in phenolic compounds. Methanol extracts of citrus peels are rich in avones and glycosylated avanones, while hydrolyzed extracts of citrus peels are rich in avonols and phenolic acids (Bocco, Cuvelier, Richard, & Berset, 1998). Citrus fruit juices contain avanones and phenolic acids (Rapisarda et al., 1999). Many studies have focused on the quantication of phenolic compounds and antioxidant capacity of citrus fruits such as lime, grapefruits, sweet orange, lemon, and tangerine (Abad-Garcia, Garmon-Lobato, Berrueta, Gallo, & Vicente, 2012; Goulas & Manganaris, 2012; Kelebek, Canbas, & Selli, 2008).

Corresponding authors. Tel.: +86 571 88982155; fax: +86 571 88982550 (X. Ye). Address: College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China. Tel.: +86 23 68250229; fax: +86 23 68251274 (Z. Zhou). E-mail addresses: pus@zju.edu.cn (X. Ye), zqzhou2013@swu.edu.cn (Z. Zhou).
0308-8146/$ - see front matter 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2013.08.012

However, only a few studies have sought to determine the phenolic compounds and antioxidant activity of wild citrus fruits, especially wild mandarin fruits. China is the most important centre of origin for the genus Citrus L., and some important wild citrus species or variety originated from the country. Over the past few decades, many wild mandarin genotypes native to China have been described, such as Citrusmangshanensis S.W. He & G.F. Liu and C. daoxianensis S.W. He & G.F. Liu (Liu & Deng, 2007). Thus far, several researchers have investigated the phylogeny of these wild mandarins using nuclear and chloroplast simple sequence repeat markers, nuclear LEAFY second intron and plastid trnLtrnF sequences, and random amplied polymorphic DNA (RAPD) (Leng et al., 2012; Li, Cheng, Guo, Xu, & Deng, 2006; Li, Cheng, Tao, & Deng, 2007). However, the phenolic compounds and antioxidant activity of wild mandarins have rarely been studied. The objective of the present study is to determine the content and composition of phenolic compounds in 14 Chinese wild mandarin genotypes and evaluate their antioxidant capacity. The results obtained were compared with those of the mandarin cultivars Satsuma and Ponkan, the most commonly cultivated mandarin fruits types, to provide useful information for future utilization of Citrus germplasm.

Y. Zhang et al. / Food Chemistry 145 (2014) 674680

675

2. Materials and methods 2.1. Chemicals Eriocitrin, taxifolin, narirutin, neohesperidin, rhoifolin, quercitrin, eridictyol, didymin, poncirin, naringenin, luteolin, kaempferol, diosmetin, sinensetin, nobiletin, tangeretin, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, chlorogenic acid, FolinCiocalteu phenol reagent, 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH), 2,20 -azino-bis(3-ethylbenzthiozoline-6)-sulphonic acid (ABTS), 2,4,6-tris (2-pyridyl)-S-triazine (TPTZ), 2,20 -azobis (2-methylpropionamidine) dihydrochloride (AAPH), trolox (6-hydrox-2,5,7,8-tetramethylchromane-2-carboxylic acid) and uorescein were purchased from Sigma (St. Louis, MO, USA). Naringin and hesperidin were obtained from the National Institutes for Food and Drug Control (Beijing, China). The methanol of high-performance liquid chromatography (HPLC) grade was purchased from Merck KgaA (Darmstadt, Germany). All the other reagents of analytical grade were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.6. Analysis of avonoids by HPLC About 0.5 g of freeze-dried sample powder was extracted with methanol (80%, 12 mL) and dimethyl sulphoxide (1:1, v/v) following the procedures described in Section 2.3. The avonoids contents were determined by HPLC method as described in our previous report (Zhang et al., 2012). After ltration on Millipore membrane (0.22 lm), 10 lL of the solution was injected into the HPLC system. Chromatographic separation was performed using a reverse phase column (ZORABX SB-C18, 250 4.6 mm internal diameter). The mobile phase was composed of (A) 0.1% formic acid (aqueous) and (B) methanol. Gradient elution was performed as follows: from 0 to 20 min, 37% to 50% B; from 20 to 35 min, 50% to 80% B; from 35 to 40 min, 80% to 100% B; from 40 to 50 min, 100% B; from 50 to 60 min, 37% to 50% B. The column temperature was maintained at 25 C and the ow rate was 0.7 mL/min. Flavanones, avones and avonols were detected at wavelengths of 283, 330 and 367 nm, respectively. 2.7. Statistical analysis All data are expressed as the means standard deviation of three replicates. Statistical analysis was performed using SPSS v19.0 software (SPSS for Windows, Release 19.0, SPSS Inc.). Significant differences among the samples were calculated using oneway ANOVA, followed by Duncans multiple-range test at 5% level (p 6 0.05). 3. Results and discussion 3.1. Flavonoid contents In the present study, a total of 18 avonoids were identied, including 10 avanones, seven avones and one avonol compounds (Table 1). We found that the variation patterns of avonoid components and contents were largely the same for the different genotypes studied (Fig. 1). This result provides direct evidence that phenolic compounds are genetically controlled. Flavanones were the major avonoids of the wild mandarins tested, and hesperidin was the major avanone, followed by narirutin and eriocitrin. The contents of these avanones varied from not detected (ND) to 55.98 mg/g dry weight (DW), ND to 6.89 mg/ g DW, and 0.798.51 mg/g DW, respectively. Guangxihongpisuanju had the highest content of hesperidin, and Cupigoushigan the highest narirutin and eriocitrin contents. Guangxihongpisuanju had a higher hesperidin content than the cultivars Satsuma and Ponkan. The eriocitrin contents of Cupigoushigan, Daoxianyeju and Nieduyeju were 4.210.91 times higher than those of the cultivars Satsuma and Ponkan. In general, mandarin fruits have a distinct avanone prole that is dominated by hesperidin and narirutin (Peterson et al., 2006), and this is largely conrmed by our results. Instead of hesperidin and narirutin, however, naringin and neohesperidin were found to be dominant in Xichuanzhoupigan and Wangcangzhoupigan. The peels of them contained approximately 4 times more naringin and 10 times more neohesperidin than the rest of materials studied. These results suggested that Xichuanzhoupigan and Wangcangzhoupigan may be excellent sources of naringin and neohesperidin. Among the seven avones identied in this study, nobiletin was the most abundant avone composition, followed by tangeretin and sinensetin (Table 1). The contents of these avones varied from 2.25 to 6.83 mg/g DW, 0.87 to 2.92 mg/g DW, and 0.12 to 4.71 mg/g DW in the wild materials, respectively. Wulongsuanju and Nieduyeju had the highest levels of nobiletin and sinensetin, respectively,

2.2. Fruit materials All citrus fruits were collected from the National Citrus Germplasm Repository, Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing, China (Table 3). Fruits were harvested at commercial maturity stage based on external colour and size uniformity. After harvest, pooled peel samples of each genotype were freeze-dried, ground, and sieved through a 40-mesh sieve. The powders were stored at 80 C until analysis.

2.3. Sample extraction The extraction procedure was based on the method of Ramful, Tarnus, Aruoma, Bourdon, and Bahorun (2011) with some modications. Methanol (80%, 24 mL) was added to 1 g of lyophilized powder samples. The mixture was shaken for 12 h and centrifuged at 3000g for 10 min at 4 C. Methanol (80%, 24 mL) was added to the residue and the same procedure was repeated. Supernatants from both extractions were collected and diluted to 50 mL with methanol. The solutions were then stored at 20 C for determination of total phenol, total avonoid, extractable phenolic acids, and antioxidant activity. The residue was further used to determine bound phenolic acids using the method of Ye et al. (2011).

2.4. Determination of total phenolic content, total avonoid content, and antioxidant capacity Total phenolic content was determined using the FolinCiocalteu method (Xu et al., 2008). The total avonoid content was determined using the method of Wang, Chuang, and Hsu (2008). DPPH, ABTS, ferric reducing ability of plasma (FRAP) and oxygen radical antioxidant capacity (ORAC) assays were used to evaluate the antioxidant capacity (Almeida et al., 2011; Rapisarda, Fabroni, Peterek, Russo, & Mock, 2009; Xu et al., 2008).

2.5. Analysis of phenolic acids by HPLC Phenolic acid contents, including both extractable and bound phenolic acids, were determined using the method of Ye et al. (2011). The HPLC condition was followed as described by Xu et al. (2008).

676

Table 1 The avonoid contents in the peels of the 14 wild mandarin genotypes and 2 cultivars analyzed in this study (mg/g DWA).B
Flavone Hesperidin 20.39 0.23g 28.41 0.89d ND ND 0.07 0.00ef 0.05 0.01f 0.37 0.05 0.15 0.05d 0.05 0.01f ND ND 0.08 0.01fg 0.10 0.01 0.12 0.01def 0.45 0.02a 0.09 0.02efg 0.27 0.01b ND 0.07 0.02f 0.10 0.01 0.16 0.01c 0.02 0.00g 1.52
e efg b

Genotypes Neohesperidin 0.21 0.04c 0.87 0.01b 0.22 0.01c 0.15 0.01c 0.89 0.04 ND ND ND ND 0.63 0.01bc 0.74 0.00bc ND ND ND ND ND ND ND 0.86 2.01 0.03e 23.41 ND 2.1 0.92 0.01e 12.41 0.12 0.01de 2.47 0.55 0.03gh 17.13 0.13 0.00cde 2.78 ND 2.82 0.05b 2.02 0.19 0.06 0.00j
b b

Flavanone Eridictyol 0.07 0.00f 0.12 0.01c 0.13 0.00b ND 0.46 0.01 0.09 0.01e ND ND ND 3.79 0.00a 0.51 0.03k ND ND ND 0.34 0.01b ND ND 0.30 0.05 0.05 0.01g
b a

Flavonol Quercitrin 0.86 0.21a ND 0.07 0.00c ND 0.46 0.00 ND 0.07 0.01c ND ND ND ND 0.12 0.01d 0.38 0.01a 0.74 0.01fg 0.13 0.01ij 0.12 0.00 0.07 0.01j
ij b

Eriocitrin 0.84 0.02j 1.58 0.04g 2.28 0.01d 1.10 0.06hi 2.52 0.01 1.84 0.02f 0.98 0.03ij 0.66 0.09k 1.09 0.23 1.37 0.01d 0.48 0.02fg 0.92 0.01e 2.26 0.12a 0.25 0.03 ND
c hi c

Taxifolin 0.14 0.04ij 0.17 0.02ij 0.50 0.02fg 0.37 0.01gh 1.58 0.06 0.83 0.02e 0.28 0.01hi ND 0.54 0.01 0.49 0.01a 0.47 0.03a 0.10 0.00def 0.25 0.02c ND 0.19 0.02ij 0.34 0.00hi 0.07 0.01j 0.93 0.02ef 3.94 0.01a 4.20 0.18b 1.40 0.00e
f c

Narirutin 0.82 0.13f 1.87 0.02bc 0.74 0.01fg 2.09 0.37b 1.80 0.03 1.09 0.01e 0.73 0.16fg 0.58 0.16g 1.41 0.01
d c

Naringin 0.09 0.01efg 0.44 0.06a 0.16 0.01cd 0.18 0.01c 0.15 0.03 0.15 0.01cd 0.49 0.00h 2.15 0.27d 0.33 0.00
hi cd

Didymin 0.06 0.02f 0.35 0.01b 0.11 0.00de ND 0.06 0.01 0.03 0.01g 4.51 0.08d 6.83 0.43a 4.09 0.14
ef f

Poncirin 0.15 0.06ij 3.54 0.06c 4.71 0.29a 0.94 0.10f 0.56 0.01 0.23 0.01ij
gh

Naringenin 4.10 0.18ef 3.02 0.09g 4.29 0.23de 5.46 0.29c 5.43 0.02 2.74 0.07g
c

Rhoifolin 2.00 0.08f 1.55 0.03gh 1.71 0.11g 2.79 0.02bc 2.92 0.02 2.73 0.08cd 2.49 0.02e 2.19 0.14f 2.55 0.09
de b

Luteolin

Diosmetin

Sinensetin

Nobiletin

Tangeretin

Kaempferol ND 0.08 0.00 0.10 0.00 ND ND ND ND ND ND

DK DX 20.38 0.46g 26.42 0.90e 29.52 0.68 55.98 1.23a 18.23 0.51h 29.39 0.54d 33.30 0.57 24.93 0.63f 17.53 1.01h 29.37 0.33d 0.75 0.01i ND 36.18 0.81b 33.29 0.39c 404.05 ND 26.9 11.69 0.46 ND
a c d

0.79 0.20j 7.75 0.35b

0.51 0.10cd 0.34 0.02def

1.28 0.26g 6.60 0.91b

ND 1.35 0.11c

ND BY No. 1
b

7.72 0.27c 1.35 0.01ij

ND 0.49 0.01cd

5.83 0.18c 1.49 0.04g

ND 0.12 0.01d

BY No. 2 GX

1.12 0.11 3.43 0.05d

1.35 0.06 ND

4.01 0.15 5.09 0.06d

2.78 0.15 0.56 0.01d

TG WL 0.12 0.01
de

1.03 0.08j 2.90 0.47def 0.30 0.01

b

0.16 0.01fg 0.81 0.23b 3.85 0.10f 4.51 0.26d 6.23 0.07b 2.22 0.04h 2.25 0.03 0.51 0.03i
h

1.16 0.01g 3.44 0.59e

0.04 0.00d 0.48 0.11d

JJ

2.77 0.14

ef

0.50 0.08

cd

3.44 0.16

ND

CP XP 0.24 0.00c 11.27 0.55a 1.17 0.02h 0.25 0.04l

8.51 0.13a 2.66 0.07efg

0.37 0.01de ND

6.89 0.13b 3.54 0.08e

ND ND

1.45 0.07h 2.67 0.30cde 2.02 0.02f 0.87 0.01i 0.88 0.01 0.19 0.01j 6.49 0.25b 66.54 5.56 0.19a 34.57
i

0.08 0.00 ND 0.03 0.00 ND ND ND ND 0.29

JZ XC
a

2.13 0.08gh 2.59 0.20fg

0.66 0.01bc 0.25 0.06ef

2.66 0.02f 1.52 0.02g

ND 4.54 0.05a

WC ST

3.21.59 0.78 0.00j

de

ND 0.06 0.01g

ND 13.61 0.32a

4.28 0.92 ND

PK Total

1.84 0.09hi 50.04

0.27 0.01ef 5.46

2.34 0.15f 62.85

ND 14.13

0.89 0.02f 20.63

Y. Zhang et al. / Food Chemistry 145 (2014) 674680

ND, not detectable. A Data are expressed as means standard deviation of triplicate samples. B Different superscripts between rows represent signicant differences between samples (p < 0.05).

Fig. 1. Variations pattern of avonoid components and contents in the peel extracts of the 14 wild mandarin genotypes and two cultivars analyzed in this study.

while Banyeshengjuzi No. 2 had the highest tangeretin content. The nobiletin content of Wulongsuanju was 1.0513.40-fold higher than those of the cultivars Satsuma and Ponkan. The sinensetin contents of Nieduyeju, Cupigoushigan and Daoxianyeju were 3.9867.29-fold higher than those of the cultivars Satsuma and Ponkan. Kaempferol was only found in four genotypes, namely, Daoxianyeju, Nieduyeju, Cupigoushigan and Jizigan. Lu, Zhang, Bucheli, and Wei (2006) found that Zaoju (C. subcompressa Tanaka) has the highest nobiletin content (5.9 mg/g DW) among 13 citrus species, while Gongju (C. kinokuni hort. ex. Tanaka), Yongchunlugan (C. reticulata Blanco) and Hamlin (C. sinensis Osbeck) has the highest sinensetin contents (0.5 mg/g DW) in the citrus species analyzed. Bermejo, Llosa, and Cano (2011) found that the nobiletin, tangeretin and sinensetin contents in the peels of mandarin fruits ranged from 0.45 to 0.61 mg/g DW, 0.27 to 0.86 mg/g DW, and 0.12 to 0.25 mg/g DW, respectively. In this study, we found that the nobiletin contents of Wulongsuanju and Jizigan were higher than those reported by Lu et al. (2006). We also noticed that the nobiletin and tangeretin contents of our wild mandarin fruits were higher than those reported by Bermejo et al. (2011). Moreover, the sinensetin contents in most of the wild mandarin peels, especially in those of Nieduyeju, Cupigoushigan, Daoxianyeju and Wulongsuanju, were much higher than those reported in current literatures (Bermejo et al., 2011; Lu et al., 2006).

3.2. Phenolic acid content

The extractable and bound form phenolic acids, including hydroxybenzoic acid, hydroxycinnamic acid and chlorogenic acid of the 14 wild mandarin genotypes were determined and the results were shown in Table 2 and Fig. 2. Ferulic acid was the most dominant extractable phenolic acid, followed by caffeic acid, p-coumaric acid and sinapic acid. The contents of these extractable phenolic acids varied from 1730.93 to 7780.17 lg/g DW, 96.53 to 1256.20 lg/g DW, 198.66 to 834.77 lg/g DW, and ND to 342.84 lg/g DW in the tested materials, respectively. Nieduyeju had the highest ferulic acid content, while Guangxihongpisuanju had the highest caffeic acid and p-coumaric acid contents. Dakengyeju had the highest sinapic acid content. Overall, Nieduyeju, Dakengyeju, Guangxihongpisuanju, Jinju, Xipigoushigan and

Table 2 The extractable and bound phenolic acid contents in the peels of the 14 wild mandarin genotypes and 2 cultivars analyzed in this study (lg/g DWA).B Genotypes PA typesC Benzoic acids Protocatechuic DK DX ND BY No. 1 BY No. 2 GX TG WL JJ CP XP JZ XC WC ST PK Total DK DX ND BY No. 1 BY No. 2 GX TG WL JJ CP XP JZ XC WC ST PK Total Extractable 3.66 0.50h 11.49 0.47ef 19.87 0.38cd 18.30 3.01d 13.26 0.30e 12.62 1.20ef 18.41 0.55d 12.71 0.55ef 12.99 0.53ef 6.26 0.59gh 23.22 0.37c 6.08 0.32gh 44.77 3.16a 39.02 6.13b 8.85 1.24fg 10.68 0.58ef 262.17 19.98 1.64f 19.53 0.94fg 19.57 0.03fg 18.63 1.04fg 20.98 0.95ef 24.79 2.32cde 15.68 1.99g 24.44 2.47cde 18.82 0.21fg 22.18 1.23def 25.98 2.06cd 19.69 2.21fg 48.06 2.57a 28.29 2.50bc 31.74 0.26b 21.37 0.55ef 379.74 p-Hydroxybenzoic 52.57 0.38c 49.34 1.42c 50.64 0.98c 31.42 1.80e 30.46 2.59e 40.16 1.04d 21.72 1.58f 38.10 1.75d 31.50 1.12e 16.55 3.58g 31.19 1.70e 33.14 2.56e 84.26 3.16a 79.72 1.92b 29.52 2.66e 30.54 0.73e 650.85 80.83 1.77b 68.58 1.11c 83.00 5.33b 27.04 2.04def 32.66 2.30def 65.65 5.00c 26.15 3.96ef 35.45 4.71d 33.77 0.93de 95.91 7.79a 24.12 1.53f 63.62 4.84c 84.36 0.29b 63.30 3.56c 78.39 0.04b 26.97 2.45def 889.78 Vanillic 60.57 1.79ef 60.76 3.78ef 63.67 2.30e 73.28 5.73d 76.24 1.76d 88.28 1.59c 57.25 2.50fg 52.70 4.31g 95.09 1.74b 23.99 4.57i 63.91 4.29e 53.75 0.83g 117.62 2.86a 119.28 4.77a 45.26 3.49h 63.98 0.35e 1115.64 74.09 1.51de 81.44 5.96d 83.77 6.11d 40.71 2.20h 79.33 3.42de 101.66 9.19c 54.54 7.61fg 66.91 4.92ef 78.64 9.38de 150.22 11.43b 83.24 6.02d 103.44 6.98c 182.81 5.58a 144.48 8.27b 43.91 1.76gh 59.00 5.31f 1428.16 Cinnamic acids Caffeic 504.87 5.67bc 337.24 16.22de 458.53 36.74c 565.22 24.70b 460.31 17.92c 1256.20 80.61a 331.57 30.12de 255.36 11.37fg 376.53 7.40d 96.53 15.04h 300.29 5.44efg 320.24 4.97def 249.48 0.57g 251.71 24.61g 46.24 2.89h 1273.47 77.61a 7083.79 472.59 51.49ef 433.40 25.54efg 486.10 20.23ef 477.95 4.15ef 664.77 43.93d 2055.79 137.74a 462.10 38.06efg 537.03 39.49e 358.03 40.29gh 382.10 3.80fgh 499.18 54.76e 1017.21 27.15b 306.14 1.15hi 251.56 15.10ij 193.19 8.93j 908.84 67.08c 9505.98 p-Coumaric 248.92 3.45i 450.97 21.23de 642.44 5.28b 323.73 9.51gh 212.56 5.03j 834.77 29.81a 260.54 14.73i 350.85 10.19g 426.05 12.49ef 198.66 30.58j 487.35 18.81c 299.94 20.75h 464.29 4.41cd 400.25 31.34f 154.55 9.79k 416.44 20.96f 6172.30 636.61 51.34hi 921.28 34.51fg 1265.87 125.73e 586.96 4.31i 801.45 33.06gh 3642.63 242.46a 841.94 53.45fg 1307.48 101.63de 571.93 56.82i 1472.80 43.46cd 1724.16 80.22b 1625.66 76.65bc 1278.15 11.85e 853.60 84.23fg 461.33 32.89i 1016.23 71.20f 19007.06 Ferulic 3467.14 66.08e 5839.99 259.01b 7780.17 563.30a 3711.07 101.08e 2772.10 58.93fg 5438.18 310.18b 2846.02 203.00f 2447.13 57.97fgh 5022.94 119.41c 2155.94 323.15hi 4270.57 275.70d 2363.94 162.75gh 1917.57 32.20ij 1730.93 156.47j 1613.34 99.30j 3322.97 194.57e 56700.01 5941.67 517.84cde 6551.71 264.31c 8305.33 675.73b 4111.24 18.62g 6102.41 325.37cde 13607.19 837.02a 5249.85 503.07ef 5578.61 386.36de 3910.52 256.09gh 8095.63 139.51b 7563.51 375.37b 6433.09 357.66cd 4328.05 82.28g 3124.48 236.33h 4419.71 278.68fg 4548.02 321.60fg 97871.00 Sinapic 342.84 1.59a 173.86 2.69de 203.14 13.57c 180.68 8.37d 132.88 1.98gh ND 150.33 12.32fg 118.68 4.15h 157.24 5.22ef 75.80 11.53ij 230.11 25.12b 87.43 7.53i 152.39 5.63fg 87.24 14.37i 60.79 3.52j 83.13 6.06i 2236.54 100.19 4.06bc 110.07 1.94b 103.76 7.35bc 66.97 3.54e 84.52 7.61d ND 66.32 5.59e 54.14 3.51f 55.72 3.81ef 167.23 10.16a 86.30 3.08d 56.37 2.91ef 97.59 5.73c 63.06 5.11ef 34.99 3.81g 43.52 3.42g 1190.74 20.51 0.35f 28.81 0.52d 32.58 0.74c 25.45 0.76e 19.31 0.90fg 21.97 1.32f 8.85 0.19h 16.81 1.69g 29.19 0.44d 9.75 0.74h 26.75 1.49e 7.72 0.42h 46.68 0.78b 61.50 4.96a 16.79 1.04g 9.29 0.18h 381.93 34.02 4.49gh 45.18 4.91g 37.38 3.44gh 14.51 0.82j 107.24 12.86de 128.44 4.28c 28.31 5.13hi 19.03 0.05ij 24.72 1.33hij 74.20 0.99f 114.21 13.58d 25.67 1.51hij 264.93 4.83a 175.96 3.84b ND 98.04 0.24e 1191.79 Chlorogenic

Y. Zhang et al. / Food Chemistry 145 (2014) 674680

Bound

ND, not detectable. A Data are expressed as means standard deviation of triplicate samples. B Different superscripts between rows represent signicant differences between samples (p < 0.05). C PA type are expressed as phenolic acid type.

677

678

Y. Zhang et al. / Food Chemistry 145 (2014) 674680

3.3. Total phenolic content, total avonoid content and antioxidant capacities The total phenolic contents of methanol extracts from the peels of 14 wild mandarin genotypes and two cultivars were measured. Their antioxidant capacities were evaluated by DPPH, FRAP, ABTS, and ORAC methods. The total phenolic contents showed obvious variations among the different genotypes ranging from 29.38 to 51.14 mg/g DW (gallic acid equivalents) (Table 3). This variation range is higher than that (15.59518.950 mg gallic acid equivalents/g DW) of ethanol extracts of C. reticulata Blanco cv. Ougan fruits (Chen, Yuan, & Liu, 2010), but lower than that (104.2 172.1 mg gallic acid equivalents/g DW) of extracts of C. reticulata Blanco fruits (Ghasemi, Ghasemi, & Ebrahimzadeh, 2009). Nieduyeju had the highest total phenolic content, followed by Dakengyeju, Cupigoushigan and Daoxianyeju. Wulongsuanju had the lowest total phenolic content. In addition, we found that the total phenolic contents of Nieduyeju, Dakengyeju, Cupigoushigan, Daoxianyeju, Xipigoushigan and Banyeshengjuzi No. 2 were all higher than those of the cultivars Satsuma and Ponkan. The total avonoid contents differed signicantly (p < 0.05) among the wild mandarin genotypes tested. The total avonoid contents varied from 7.95 to 20.66 mg/g DW (rutin equivalents) (Table 3). These data are partially in accordance with the results (0.331.1 mg quercetin equivalents/g DW) reported by Ghasemi et al. (2009), but much higher than those (4.6715.796 mg rutin equivalents/g DW) obtained by Chen et al. (2010). The highest total avonoid content (20.66 mg/g DW) was found in Wulongsuanju, and it is signicantly different (p < 0.05) from other wild genotypes tested. No signicant differences (p > 0.05) were observed between the total avonoid contents of Daoxianyeju and Nieduyeju, and they ranked second in term of the total avonoid content among all the genotypes studied. The total avonoid contents of Guangxihongpisuanju and Jizigan were more or less the same, and they ranked third among all the genotypes studied. It is noteworthy that the total avonoid contents of Wulongsuanju, Nieduyeju, Daoxianyeju, Jizigan, Guangxihongpisuanju and Cupigoushigan were all higher than those of the cultivars Satsuma and Ponkan. The DPPH assay has been widely used for the determination of primary antioxidant capacity. DPPH radical could be decreased by reactions with antioxidant compositions that can donate hydrogen (Kumaran & Joel Karunakaran, 2007). The DPPH values of the wild mandarins varied from 29.04 to 50.46 lmol trolox equivalents (TE)/g DW. Cupigoushigan and Nieduyeju had the highest DPPH values and no signicant difference was observed between them (p > 0.05). On the other hand, Tuju had the lowest DPPH value. Six out of 14 wild mandarins tested, including Cupigoushigan, Nieduyeju, Dakengyeju, Jinju, Guangxihongpisuanju and Daoxianyeju, had higher DPPH values than those of the cultivars Satsuma and Ponkan. The FRAP method is commonly applied to determine the antioxidant activity of plant materials, and it measures the capacity of the sample to reduce ferric complex to the ferrous form (ContrerasCaldern, Caldern-Jaimes, Guerra-Hernndez, & Garca-Villanova, 2011). The FRAP values of the 14 wild mandarins analyzed ranged from 26.50 to 46.98 lmol TE/g DW. The highest FRAP value was found in Nieduyeju, whereas the lowest FRAP value was found in Wulongsuanju. Six out of 14 wild mandarins tested, including Nieduyeju, Cupigoushigan, Daoxianyeju, Dakengyeju, Jinju and Xipigoushigan, had higher FRAP values than those of the cultivars Satsuma and Ponkan. The ABTS method is also commonly used to study the antioxidant capacity of plants based on the capacity to scavenge the radical cation ABTS+ generated in the system (Kim, Lee, Lee, & Lee, 2002). The ABTS values of the 14 wild mandarins analyzed ranged from 65.62 to 108.60 lmol TE/g DW. The highest ABTS value was

Fig. 2. Variations pattern of phenolic acid components and contents in the peel extracts of the 14 wild mandarin genotypes and two cultivars analyzed in this study. (A) Extractable phenolic acids and (B) bound phenolic acids.

Banyeshengjuzi No. 1 had higher ferulic acid contents than those of the cultivars Satsuma and Ponkan. The ferulic acid was also the most dominant bound phenolic acid, followed by p-coumaric acid and caffeic acid. Guangxihongpisuanju had the highest contents of ferulic acid, p-coumaric acid and caffeic acid, while Wangcangzhoupigan had the lowest ferulic acid and caffeic acid contents. Most of the wild mandarin genotypes tested had a higher ferulic acid content than the cultivars Satsuma and Ponkan. In particular, the ferulic acid content of Guangxihongpisuanju was 3-fold higher than those of the cultivars Satsuma and Ponkan. In addition, Guangxihongpisuanju had higher p-coumaric acid and caffeic acid contents than the cultivars Satsuma and Ponkan. Bocco et al. (1998) found that ferulic acid is the main phenolic acid in citrus fruits, and the abundance of hydroxycinnamic acids in citrus varied in the following order: ferulic acid (0.036 1.580 mg/g DW) > sinapic acid (0.0300.954 mg/g DW) > p-coumaric acid (0.0710.193 mg/g DW) > caffeic acid (0.0060.229 mg/ g DW). Wang et al. (2008), however, reported that chlorogenic acid rather than ferulic acid is the main phenolic acid in the peels of citrus fruits, and the contents of phenolic acids varied as the following order: chlorogenic acid (145339 lg/g DW) > p-coumaric acid (41.7346 lg/g DW) > ferulic acid (30.3150 lg/g DW) > sinapic acid (10.1178 lg/g DW) > caffeic acid (3.0680.0 lg/g DW). Our results showed that ferulic acid is the most abundant phenolic acid in the peels of the wild mandarins tested, similar to the results obtained by Bocco et al. (1998). However, the variation order of the hydroxycinnamic acid contents observed in our study was different as follows: ferulic acid > p-coumaric acid and caffeic acid > sinapic acid. It is noteworthy that the contents of ferulic acid, pcoumaric acid, caffeic acid and sinapic acid of the wild mandarins tested in this study were all higher than those reported by previous studies. The chlorogenic acid content, however, was lower than that obtained by Wang et al. (2008). These differences may be attributed to the genetic backgrounds of the citrus species and/or environmental factors.

Y. Zhang et al. / Food Chemistry 145 (2014) 674680

679

Table 3 The fruit materials, the total phenolic, total avonoids content (mg/g DWA),B and antioxidant capacities (lM TE/g DW) of the fruits analyzed in this study.

Total avonoids

45.38 0.82b 43.46 0.35bcd 51.14 0.26a 39.86 0.55ef 41.43 1.36de 38.37 1.18fg 36.02 0.62gh 29.38 2.63j 35.27 0.69hi 44.64 2.02bc 42.32 3.28cd 31.09 1.17j 33.55 1.67i 34.48 0.13hi 39.71 1.21ef 36.54 0.63gh

9.70 0.29ef 16.28 0.83b 16.80 0.34b 8.93 0.24fg 7.95 0.09g 12.56 0.31c 10.34 0.85e 20.66 1.53a 8.87 0.32fg 11.55 0.58d 8.81 0.84fg 13.53 0.94c 8.33 0.26g 8.12 0.26g 6.28 0.24h 10.59 0.12de

found in Nieduyeju, whereas the lowest ABTS value was found in Wulongsuanju. Seven out of 14 wild mandarins tested, including Nieduyeju, Banyeshengjuzi No. 1, Banyeshengjuzi No. 2, Dakengyeju, Xipigoushigan, Jinju, and Daoxianyeju, had higher ABTS values than those of the cultivars Satsuma and Ponkan. The ORAC assay has found broader application for determining the antioxidant capacity of botanical and biological samples (Prior et al., 2003; Rapisarda et al., 2009). This method is based on a hydrogen atom transfer reaction mechanism, which is most relevant to human biology (Prior et al., 2003). The ORAC values of the 14 wild mandarins tested ranged from 395.66 to 834.37 lmol TE/g DW. The highest ORAC value was found in Nieduyeju, whereas the lowest ORAC value was found in Jizigan. Among the 14 wild mandarins tested, only Nieduyeju had a higher ORAC value than those of the cultivars Satsuma and Ponkan. Given that the four methods used above resulted in different antioxidant capacities for the same genotypes, an overall antioxidant potency composite (APC) index was calculated for each genotype using the method described by Seeram et al. (2008). The resulting APC indices are shown in Table 3. The APC index of different mandarins showed obvious variation ranging from 58.84 to 98.89. Nieduyeju had the highest APC index, followed by Cupigoushigan, Dakengyeju and Daoxianyeju. Wulongsuanju had the lowest APC index. Phenolic compounds, including avonoids and phenolic acids, are known to be responsible for antioxidant activity in fruits. Fruits with higher total phenolic content generally showed stronger antioxidant capacity (Rice-Evans & Miller, 1996). The high antioxidant activity of Nieduyeju, Cupigoushigan, Dakengyeju, and Daoxianyeju may be attributed to their phenolic compositions and contents. 4. Conclusions The phenolic compositions and antioxidant capacities of the Chinese wild mandarins are reported for the rst time in the present study. Signicant variations in total phenol and total avonoid contents were observed in the 14 genotypes studied. We found that the wild mandarin fruits were rich in polyphenols. A total of 18 avonoids and eight phenolic acids were identied from the genotypes studied. Among the compounds identied, hesperidin, narirutin, eriocitrin, nobiletin and ferulic acid were found to be the major phenolic compounds. Among the 14 wild mandarins tested, Nieduyeju and Wulongsuanju had the highest total phenolic and total avonoid contents, respectively. Nieduyeju had the highest ferulic acid content of the extractable phenolic acid, while Guangxihongpisuanju had the highest ferulic acid content of the bound phenolic acids. The eriocitrin levels of Nieduyeju, Cupigoushigan, and Daoxianyeju were 410-fold higher than those of the cultivars Satsuma and Ponkan. Wulongsuanju had 113-fold higher nobiletin content than the cultivars Satsuma and Ponkan. Guangxihongpisuanju had the highest hesperidin content. The APC index also showed obvious variation ranging from 58.84 to 98.89 in the wild mandarins studied, among them, Nieduyeju had the highest APC index. In a word, our ndings provide useful information for future study and utilization of the wild mandarin germplasm in China and abroad. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 31171930 and 31071635), the Fundamental Research Funds for the Central Universities (XDJK2013A014), the Program for Chongqing Innovation Team of University (KJTD201333), and the 111 Project (B12006).

Rank APC indexC ORAC ABTS FRAP Total phenolics DPPH

39.95 1.41b 38.71 3.16bc 48.21 1.75a 36.11 0.11bcd 32.05 0.41de 38.75 0.18bc 32.59 0.35de 29.04 1.20ef 39.45 1.38bc 50.46 3.57a 33.59 1.87cde 33.84 1.64cde 33.82 0.53cde 35.15 0.58bcd 25.14 0.58f 36.35 1.27bcd

40.47 2.12bc 42.90 2.71ab 46.98 0.89a 34.81 1.50defg 32.83 0.57fgh 39.03 0.46bcd 30.27 0.50gh 26.50 0.44h 36.93 1.00cde 46.55 1.64a 36.41 2.82cde 31.15 0.13gh 30.08 2.96gh 32.66 2.92efg 35.06 1.15defg 35.58 1.05de

104.22 3.58a 91.01 2.87cd 108.60 0.24a 94.61 5.24bc 104.27 4.74a 85.00 0.24def 88.54 1.91cd 65.62 1.43g 84.27 1.18def 91.69 6.67cd 101.18 2.62ab 66.97 2.68g 77.59 0.72f 80.45 2.63ef 87.53 2.11cde 88.88 3.34cd

642.49 34.46c 625.40 20.21cd 834.37 16.98a 529.42 60.49efg 645.30 17.60c 610.76 8.43cd 557.64 7.69def 508.85 45.37efg 421.49 14.29hi 631.05 43.41cd 577.72 29.38cde 395.66 14.42i 487.85 14.50fgh 462.03 30.95ghi 748.21 21.07b 578.86 27.55cde

84.57 81.69 98.89 74.06 76.69 77.83 69.34 58.84 71.22 89.78 76.62 60.61 65.49 67.16 73.68 74.74

3 4 1 9 6 5 12 16 11 2 7 15 14 13 10 8

Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Data are expressed as means standard deviation of triplicate samples. Different superscripts between rows represent signicant differences between samples (p < 0.05). Antioxidant index score = [(sample score/best score) 100].

Abbreviation Chinese name Repository number Scientic name

Citrus daoxianensis S.W. He Citrus daoxianensis S.W. He Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus reticulata Blanco Citrus unshiu Marc. Citrus poonensis Hort. Ex Tanaka

GPGJ0757 GPGJ0144 GPGJ0790 GPGJ0785 GPGJ0782 GPGJ1243 GPGJ0042 GPGJ0402 GPGJ0089 GPGJ0041 GPGJ0036 GPGJ0339 GPGJ0986 GPGJ0030 GPGJ0667 GPGJ00948

Dakengyeju Daoxianyeju Nieduyeju Banyeshengjuzi No. 1 Banyeshengjuzi No. 2 Guangxihongpisuanju Tugan Wulongsuanju Jinju Cupigoushigan Xipigoushigan Jizigan Xichuanzhoupigan Wangcangzhoupigan Satsuma Ponkan

DK DX ND BY No. 1 BY No. 2 GX TG WL JJ CP XP JZ XC WC ST PK

680

Y. Zhang et al. / Food Chemistry 145 (2014) 674680 Leng, X. P., Li, H. R., Zhong, G. Y., Song, C. N., Sun, X., & Fang, J. G. (2012). Employment of a new strategy for identication of loose-skin mandarin (Citrus reticulata Blanco) cultivars using RAPD markers. Romanian Biotechnological Letters, 17(2), 70737083. Li, Y. Z., Cheng, Y. J., Guo, W. W., Xu, Q., & Deng, X. X. (2006). Genetic diversity in mandarin landraces and wild mandarins from China based on nuclear and chloroplast simple sequence repeat markers. Journal of Horticultural Science and Biotechnology, 81, 371378. Li, Y. Z., Cheng, Y. J., Tao, N. G., & Deng, X. X. (2007). Phylogenetic analysis of mandarin landraces, wild mandarins, and related species in China using nuclear leafy second intron and plastid trnLtrnf sequence. Journal of the American Society for Horticultural Science, 132(6), 796806. Liu, Y. Z., & Deng, X. X. (2007). Citrus breeding and genetics in China. The Asian and Australasian Journal of Plant Science and Biotechnology, 1(1), 2328. Lu, Y., Zhang, C., Bucheli, P., & Wei, D. (2006). Citrus avonoids in fruit and traditional Chinese medicinal food ingredients in China. Plant Foods for Human Nutrition, 61(2), 5765. Oboh, G., & Ademosun, A. O. (2012). Characterization of the antioxidant properties of phenolic extracts from some citrus peels. Journal of Food Science and Technology, 49(6), 729736. Peterson, J. J., Dwyer, J. T., Beecher, G. R., Bhagwat, S. A., Gebhardt, S. E., Haytowitz, D. B., et al. (2006). Flavanones in oranges, tangerines (mandarins), tangors, and tangelos: A compilation and review of the data from the analytical literature. Journal of Food Composition and Analysis, 19, S66S73. Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., et al. (2003). Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry, 51(11), 32733279. Ramful, D., Tarnus, E., Aruoma, O. I., Bourdon, E., & Bahorun, T. (2011). Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps. Food Research International, 44(7), 20882099. Rapisarda, P., Fabroni, S., Peterek, S., Russo, G., & Mock, H. P. (2009). Juice of new citrus hybrids (Citrus clementina Hort. ex Tan. C. sinensis L. Osbeck) as a source of natural antioxidants. Food Chemistry, 117(2), 212218. Rapisarda, P., Tomaino, A., Lo Cascio, R., Bonina, F., De Pasquale, A., & Saija, A. (1999). Antioxidant effectiveness as inuenced by phenolic content of fresh orange juices. Journal of Agricultural and Food Chemistry, 47(11), 47184723. Rice-Evans, C. A., & Miller, N. J. (1996). Antioxidant activities of avonoids as bioactive components of food. Biochemical Society Transactions, 24(3), 790795. Seeram, N. P., Aviram, M., Zhang, Y., Henning, S. M., Feng, L., Dreher, M., et al. (2008). Comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the United States. Journal of Agricultural and Food Chemistry, 56(4), 14151422. Wang, Y. C., Chuang, Y. C., & Hsu, H. C. (2008). The avonoid, carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chemistry, 106(1), 277284. Xu, G. H., Liu, D. H., Chen, J. C., Ye, X. Q., Ma, Y. Q., & Shi, J. (2008). Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chemistry, 106(2), 545551. Ye, X. Q., Chen, J. C., Liu, D. H., Jiang, P., Shi, J., Xue, S., et al. (2011). Identication of bioactive composition and antioxidant activity in young mandarin fruits. Food Chemistry, 124(4), 15611566. Zhang, Y. M., Zhou, Z. Q., Sun, Y. J., Shen, Y., Zhong, L. Z., Qiao, L. P., et al. (2012). Simultaneous determination of 18 avonoids in citrus fruits by highperformance liquid chromatography. Scientia Agricultura Sinica, 45(17), 35583565 (in Chinese).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 08.012.

References
Abad-Garcia, B., Garmon-Lobato, S., Berrueta, L. A., Gallo, B., & Vicente, F. (2012). On line characterization of 58 phenolic compounds in Citrus fruit juices from Spanish cultivars by high-performance liquid chromatography with photodiode-array detection coupled to electrospray ionization triple quadrupole mass spectrometry. Talanta, 99, 213224. Almeida, M. M. B., de Sousa, P. H. M., Arriaga, . M. C., do Prado, G. M., Magalhes, C. E., Maia, G. A., & de Lemos, T. L. G. (2011). Bioactive compounds and antioxidant activity of fresh exotic fruits from northeastern Brazil. Food Research International, 44(7), 21552159. Bermejo, A., Llosa, M. J., & Cano, A. (2011). Analysis of bioactive compounds in seven citrus cultivars. Food Science and Technology International, 17(1), 5562. Bocco, A., Cuvelier, M. E., Richard, H., & Berset, C. (1998). Antioxidant activity and phenolic composition of citrus peel and seed extracts. Journal of Agricultural and Food Chemistry, 46, 21232129. Chavez-Santoscoy, R. A., Gutierrez-Uribe, J. A., & Serna-Saldivar, S. O. (2009). Phenolic composition, antioxidant capacity and in vitro cancer cell cytotoxicity of nine prickly pear (Opuntia spp.) juices. Plant Foods for Human Nutrition, 64(2), 146152. Chen, X. T., Yuan, K., & Liu, H. L. (2010). Phenolic contents and antioxidant activities in ethanol extracts of Citrus reticulata Blanco cv. Ougan fruit. Journal of Food, Agriculture and Environment, 8(2), 150155. Contreras-Caldern, J., Caldern-Jaimes, L., Guerra-Hernndez, E., & GarcaVillanova, B. (2011). Antioxidant capacity, phenolic content and vitamin C in pulp, peel and seed from 24 exotic fruits from Colombia. Food Research International, 44(7), 20472053. Ghasemi, K., Ghasemi, Y., & Ebrahimzadeh, M. A. (2009). Antioxidant activity, phenol and avonoid contents of 13 citrus species peels and tissues. Pakistan Journal of Pharmaceutical Sciences, 22(3), 277281. Goulas, V., & Manganaris, G. A. (2012). Exploring the phytochemical content and the antioxidant potential of Citrus fruits grown in Cyprus. Food Chemistry, 131(1), 3947. Jiang, F., & Dusting, G. J. (2003). Natural phenolic compounds as cardiovascular therapeutics: Potential role of their antiinammatory effects. Current Vascular Pharmacology, 1(2), 135156. Kelebek, H., Canbas, A., & Selli, S. (2008). Determination of phenolic composition and antioxidant capacity of blood orange juices obtained from cvs. Moro and Sanguinello (Citrus sinensis (L.) Osbeck) grown in Turkey. Food Chemistry, 107(4), 17101716. Kim, D. O., Lee, K. W., Lee, H. J., & Lee, C. Y. (2002). Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. Journal of Agricultural and Food Chemistry, 50(13), 37133717. Kumaran, A., & Joel Karunakaran, R. (2007). In vitro antioxidant activities of methanol extracts of ve Phyllanthus species from India. LWT Food Science and Technology, 40(2), 344352.