Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity

LWT - Food Science and Technology xxx (2014) 1e9

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Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity Min-Ki Lee a, Jin-Hyuk Chun a, Dong Hae Byeon b, Sun-Ok Chung c, Sang Un Park d, Suhyoung Park e, Mariadhas Valan Arasu f, *,1, Naif Abdullah Al-Dhabi f, Yong-Pyo Lim g, Sun-Ju Kim a, *,1 a

Department of Bio-Environmental Chemistry, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea Chinese Cabbage & Breeding, Suha-Ri 563, Sindun-Myon, Ichon-Si, Kyongki-Do 467-842, Republic of Korea Department of Biosystem Machinery Engineering, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea d Department of Crop Science, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea e Department of Horticultural Crop Research, National Institute of Horticultural and Herbal Science (NIHHS), Rural Development Administration (RDA), 475 Imok-dong, Jangan-gu, Suwon 440-706, Republic of Korea f Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia g Department of Horticultural Science, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 October 2013 Received in revised form 27 January 2014 Accepted 1 March 2014

Glucosinolate (GSL) and antioxidant activity in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) were determined by HPLC and DPPH, HRSA, and FRAP assays. Five aliphatic GSLs: progoitrin, sinigrin, glucoalyssin, gluconapin, and glucobrassicanapin; four indolyl GSLs: 4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin; one aromatic GSL: gluconasturtiin were identified. Glucobrassicanapin and gluconapin documented the most abundant (average 4.52 and 3.72 mmol/g DW, respectively). The contents of total GSLs varied extensively among 62 varieties (range from 2.83 to 48.53 mmol/g DW). Comprehensive differences in total and individual GSL contents have also been observed among different varieties. Indolyl and aromatic GSL together accounted 26% of the total GSLs; but there are few differences among varieties. FC7 and FI17 could be good candidates for future breeding programs since they had a high quantity of glucobrassicin (2.10 and 1.66 mmol/g DW, respectively). Most of the Chinese cabbage varieties showed significant antioxidant activities when compare with positive control. However, three antioxidant assays were not significantly correlated with total GSLs. The presence of significant quantities of glucobrassicin in some varieties should be studied more extensively, since GSL is the precursor of indole-3-carbinol, a potent anticancer isothiocyanate. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Brassica rapa L. ssp. pekinensis Chinese cabbage Glucosinolates LCeMS Antioxidant activity

1. Introduction Glucosinolates (GSLs) are negatively charged natural products mainly observed in the order Capparales with a various group of side chain and rich in sulfur containing amino acid methionine (Bennett, Mellon, & Kroon, 2004). They are classified into three major groups such as aliphatic, aromatic and indolyl GSLs based on their functional group of amino acids (Fahey, Zalcmann, & Talalay, 2001; Halkier & Gershenzon, 2006). GSLs are converted to the * Corresponding authors. Tel.: þ82 42 821 6738; fax: þ82 42 822 7142. E-mail addresses: [email protected] (M.V. Arasu), [email protected] (S.-J. Kim). 1 These authors contributed equally to this work.

corresponding aglycone by enzymatic hydrolysis with myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), which then decomposes to isothiocyanates, thiocyanates, or nitriles, depending on the substrate, pH, and availability of ferrous ions. At physiological pH, isothiocyanates are the major product. GSLs and myrosinase are segregated in intact plants (Kelly, Bones, & Rossiter, 1998). They are chemically very stable, unless they come in contact with catalytic enzyme myrosinases which are accumulated in different parts of cellular compartments to separate them from GSLs. GSLs and their breakdown products are known for its biological function such as fungicidal, bactericidal and in the treatment of cancer (Pedras, Chumala, & Suchy, 2003; Zasada & Ferris, 2004). Epidemiological studies have reported that GSLs breakdown products isothiocyanates have positive effects against bladder, colon and lung

http://dx.doi.org/10.1016/j.lwt.2014.03.001 0023-6438/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001

2

Accessions Field trial names

Varieties

Line names

1

FV1

Bulam No.3

Seminis

2

FV2

3

FV3

4

FV4

5

FV5

6

FV6

7

FV7

8

FC1

9

FC2

10

FC3

11

FC4

12

FC5

13

FC6

14

FC7

15

FC8

16

FC9

17

FV8

18 19

FV9 FV10

20

FC10

21

FC11

22

FC12

23

FC13

24 25 26 27

FC14 FC15 FC16 FI1

28

FI2

29

FI3

30

FI4

Head formation

Wrapped-over type ChuKwang Sakata Semi-wrappedover type ChuWeol Nongwoobio Wrapped-over type HwiParam Sakata Semi-wrappedover type NamdoJangkun Sakata Wrapped-over type BulamPlus Seminis Semi-wrappedover type HwiparamGold Sakata Semi-wrappedover type Tamyoung  CR DaemyoungHwanggo YM232HN  32S13778 Semi-wrappedover type Tamyoung  CR DaemyoungHwanggo 415  YM23754 Semi-wrappedover type CR DaemyoungHwanggo  Bulam1 YM23751  BA1 Wrapped-over type PyeongDae75  CR OH259  YM23754 Semi-wrappedDaemyoungHwanggo over type CR DaemyoungHwanggo  Bulam1 YM23752  BA1 Wrapped-over type CR DaemyoungHwanggo  WolHwang YM23752  KN1 Wrapped-over type CR DaemyoungHwanggo  Hwanggo65 YM23754  S241 Wrapped-over No CR Myoung type DaepyeongHano  ShinwolOjeon 1109JH  111533 Semi-wrappedover type SeoulOjeon  Hano S872C  9230252 Semi-wrappedover type ChungOk Woori flower seed & Semi-wrappedseedling over type CR NongShim Syngneta Joined-up type Sanjiwang No.2 Jung-ang tree Semi-wrappedover type IlChun  CR Daeno CR Tamyoung IL1  S415 Semi-wrappedover type Chunkwang  Hano CR No 040031  1042S510 Wrapped-over type CR Daemyoungnou  YoungjinJibu 0230027  866W53 Wrapped-over type IlChun  CR Daeno CR CR Dae IL1  955S Semi-wrappedover type CR Dae  CR Tamyoung 32S955  32S553 Joined-up type CR Dae  CR Tamyoung 32S955  32S551 Joined-up type CR Daeno CR CR Dae  Tamyoung 955S  415 Joined-up type PyeongDae75 OH259 Semi-wrappedover type CR DaemyoungHwanggo YM23752 Semi-wrappedover type ChunYoung HBR12 Wrapped-over type ChunYoung HBR21

Outer leaf color

Inner leaf color

Head height Head width Harvest (cm) (cm) maturity

Downy mildew

Cold Clubroot Hot resistance tolerance

Viridian

Yellow

28.0

18.0

þþþþþ

þþþþþ

þ

þ

Viridian

Yellow

28.5

18.0

þþþþþ

þþþþþ

þ

þ

Viridian

Yellow

30.0

19.0

þþþþþ

þþþþþ

þ

þ

Viridian

Yellow

30.0

18.5

þþþþþ

þþþþþ

þ

þ

Viridian

Yellow

31.0

19.0

Medium-late type Medium-late type Medium-late type Medium-late type Late type

þþþþþ

þþþþþ

þ

þ

Viridian

Yellow

30.0

20.0

Medium type þþþþþ

þþþþþ

þþþþþ þ

Viridian

Yellow

30.0

19.0

Medium type þþþþþ

þþþþþ

þþþþþ þþþ

Viridian

Yellow

31.0

20.0

Medium type þþþþþ

þþþþþ

þþþþþ þþþþþ

Viridian

Yellow

32.0

20.0

Medium type þþþþþ

þþþþþ

þþþþþ þþ

Viridian

Yellow

32.0

20.0

þþþþþ

þþþþþ þþ

Viridian

Yellow

32.0

22.0

Medium-late þþþþþ type Medium type þþþþþ

þþþþþ

þþþþþ þþþ

Viridian

Yellow

32.0

21.0

þþþþþ

þþþþþ

þþþþþ þþ

Viridian

Yellow

30.0

21.0

þþþþþ

þþþþþ

þþþþþ þþ

Green

Yellow

32.0

22.0

þþþþ

þþþþþ þþ

Viridian

Yellow

30.0

20.0

Medium-late þþþþ type Medium type þþþþ

þþþþ

þ

þþ

Viridian

Orange

32.0

17.5

Early type

þþþþþ

þþþþ

þ

þþþ

Gray egreen Viridian Green

Yellow

24.0

16.0

Early type

þþþþþ

þþþþ

þþþþþ þþþþþ

26.0 25.0

17.0 16.0

Early type Early type

þþþþþ þþþ

þþþþ þþþþ

þþþþþ þþþþþ þþþþþ þþþþ

Viridian

Yellow Brightyellow Yellow

26.0

16.5

Early type

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian

Yellow

27.0

17.0

Early type

þþþþþ

þþþþ

þþþþþ þþþþ

Viridian

Yellow

27.0

18.0

Early type

þþþþþ

þþþþ

þþþþþ þþþþ

Viridian

Yellow

27.0

17.0

Early type

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian Viridian Viridian Green

Yellow Yellow Yellow Yellow

26.5 27.0 27.0 25.0

17.0 17.0 17.0 16.5

þþþþþ þþþþþ þþþþþ þþþþ

þþþþ þþþþ þþþþ þþþþ

þþþþþ þþþþþ þþþþþ þ

Viridian

Yellow

27.0

16.0

Early type Early type Early type Medium-late type Medium type

þþþþþ

þþþþþ

þþþþþ þþþ

Viridian

Yellow

26.5

15.5

Medium-late type

þþþþþ

þþþþ

þ

þ

Green

Yellow

27.0

15.5

þþþþþ

þþþþþ

þ

þ

Medium-late type Late type

þþþþþ þþþþþ þþþþþ þþ

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001

Table 1 Agronomic characteristics of 62 varieties of Chinese cabbage.

FI5

Bulam1

BA1

32

FI6

Shintam

YM245-G7

33

FI7

BulCheong

73317C-G7

34

FI8

PungDong

W23-G6

35

FI9

Tamyoung Shintam

YM232-G6

36

FI10

PyeongDae75

OH25952-G7

37

FI11

Tamyoung

YM232HN

Viridian

Yellow

26.0

16.0

Viridian

Yellow

26.0

16.5

Earlumedium type Medium-late þþþþþ type Medium type þþþþþ

Viridian

Yellow

28.0

17.0

Medium type þþþþþ

þþþþþ

þ

Viridian

Yellow

30.0

17.5

Late type

þþþþþ

þþþþþ

þþþþþ þ

Viridian

Yellow

30.0

17.0

þþþþþ

þ

þ

Viridian

Yellow

25.5

17.5

Medium-late þþþþþ type Medium type þþþþ

þþþþ

þ

þ

Viridian

Yellow

27.0

17.0

þþþþþ

þþþþ

þ

þ

þþþþþ

þþþþ

þþþþþ þ

þþþþþ

þþþþþ þþþ

þþþþþ

þ

þþ

þþþþ

þ

þ þ

þþþþþ

þ

þ

þþþþþ

þ

þ þþ

Green

Yellow

26.0

17.0

Viridian

Yellow

25.5

16.0

Earlumedium Earlumedium Earlumedium Medium

Green

Yellow

27.0

17.5

Medium type þþþþþ

Viridian

Yellow

25.0

16.0

Late type

þþþþþ

þþþþþþ þ

Green

Yellow

30.0

17.5

þþþþ

þþþ

þþþþþ þ

Viridian

Yellow

28.0

18.0

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian

Yellow

28.0

18.0

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian

Yellow

24.0

15.0

þþþþþ

þþþ

þþþþþ þþþþþ

Viridian

Yellow

30.0

17.5

þþþþþ

þþþ

þþþþþ þþþþ

Viridian

Yellow

28.0

15.5

þþþþþ

þþþ

þþþþþ þþþþþ

Viridian

Yellow

29.0

17.0

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian

Yellow

26.0

16.0

þþþþþ

þþþ

þ

Viridian

Yellow

26.0

16.5

þþþþþ

þþþþ

þþþþþ þþþþþ

Viridian

Yellow

26.0

16.0

þþþþþ

þþþþ

þþþþþ þþþþþ

32S553

Semi-wrappedover type Semi-wrappedover type Joined-up type

Viridian

Yellow

27.0

15.5

þþþþþ

þþþ

þþþþþ þþþþþ

CR DaemyoungTamyoung

32S155-G6

Joined-up type

Viridian

Yellow

30.0

17.0

þþþþþ

þþþ

þþþþþ þþþþþ

FI29

CR DaemyoungHwanggo

YM23751

Viridian

Yellow

30.0

17.5

þþþþþ

þþþþþ

þþþþþ þþþ

56

FI30

CR DaemyoungHwanggo

32S13778

Viridian

Yellow

28.0

16.0

Medium type þþþþþ

þþþþþ

þþþþþ þþþ

57

FI31

Hano CR No

1042S510

Green

Yellow

28.0

16.0

þþþþþ þþ

FI32

KweonjiKweondan

H1896-G6

Green

Orange

24.0

16.0

Earluþþþþþ medium type Early type þþ

þþþþ

58

Semi-wrappedover type Semi-wrappedover type Wrapped-over type Semi-wrappedover type

Medium-late type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Earlumedium type Medium type

þþ

þ

59

FI33

CR DaemyoungHwanggo

32S1-G6

Viridian

Yellow

26.0

16.5

Medium type þþþþþ

þþþþþ

38

FI12

ChuChoung

1442-G6

39

FI13

CR DaemyoungHwanggo

YM23754

40

FI14

BulCheong

73317C58-G7

41

FI15

PyeongDae75

OH25951-G8

42

FI16

WolHwang

KN1

43

FI17

Whanggo65 No CR Myoung

S241

44

FI18

CR Daeno CR CR Dae

955S

45

FI19

CR Daeno CR Tamyoung

S9415-G8

46

FI20

CR DaemyoungTamyoung

32S1553-G7

47

FI21

CR Dae

32S955

48

FI22

CR DaemyoungTamyoung

32S551

49

FI23

CR Daeno CR CR Dae

95S955

50

FI24

Tamyoung

415

51

FI25

CR Daeno CR Tamyoung

S955-G7

52

FI26

CR Daeno CR Tamyoung

S415

53

FI27

CR DaemyoungTamyoung

54

FI28

55

Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Joined-up type

Green

Yellow

27.0

17.0

type type þþþþþ type type þþþþþ

þþþþþ

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001

31

Semi-wrappedover type Wrapped-over type Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Wrapped-over type Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Semi-wrappedover type Wrapped-over type Wrapped-over type Semi-wrappedover type Semi-wrappedover type Joined-up type

þþþ

þþþþþ þþþþþ 3

(continued on next page)

þþþ þþ þ þ þþþþþ þþþ S872C 1109JH SeoulOjeon DaepyeongHano FI35 FI36 61 62

“FV1” (F, field trial; V, serial number; 1, accession number). V: control Var. C: F1 combination. I: inbred line. þþþþþþ, Strongestrong; þþþþþ, strong; þþþþ, strong-middle; þþþ, middle; þþ, middle-weak; þ, weak.

Viridian Green

Orange Orange

31.0 29.0

14.5 19.0

Medium type þþþþþ Earluþþþþ medium type

þ þþþþ Medium type þþþþ 17.0 25.0 Orange Green FI34 60

ShinwolOjeon

111533

Semi-wrappedover type Semi-wrappedover type Joined-up type Semi-wrappedover type

Downy mildew Head height Head width Harvest (cm) (cm) maturity Inner leaf color Outer leaf color Head formation Line names Varieties Accessions Field trial names

Table 1 (continued )

þ

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

Cold Clubroot Hot resistance tolerance

4

cancers (Cartea & Velasco, 2008). Other breakdown products of aliphatic GSLs, such as glucoraphanin and glucoiberin are also considered to reduce the risk of cancers. Similar mechanism and activation of cancer preventing enzymes were observed in breakdown products of indolyl GSLs, glucobrassicin (Kim & Milner, 2005). Moreover, consumption of vegetables rich in glucobrassicin is thought to yield significant amounts of indole-3-carbinol or related substituted compounds. The anticancer activity of phenylethyl isothiocyanate, a hydrolyzed product obtained from gluconasturtiin, is excellent as it induces cyto-protective genes mediated by Nrf2 and AhR transcription factors, represses NF-kB, and inhibits both cytochrome P450 and histone deacetylase (Hayes, Kelleher, & Eggleston, 2008). Furthermore, hydrolysis products of other GSLs such as glucoiberin and gluconasturtiin have been used as protective agents against different carcinogenesis (Smith, Lund, & Johnson, 1998). Therefore the presence of naturally occurring GSLs should be monitored in vegetables under Brassica because of their immense role in the diet and medical values. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an original Chinese vegetable belonging to the Brassicaceae family and a major ingredient in pickle kimchi a traditional fermented food in Republic of Korea. Chinese cabbage and other vegetables that have been trimmed, cut, salted, and seasoned before fermentation (CAC, 2001). In addition, kimchi is the country’s most important processed food product and an essential staple side dish in Republic of Korea. Ninety percent of domestic Chinese cabbage production is earmarked for kimchi processing. Chinese cabbage is produced on a total area of over 30,000 ha in Republic of Korea and every citizen consume as much as the annual amount of 120e150 kg per year, nearly half a kg per person per day. Though, recently a price of kimchi significantly increased due to its higher cost for manufacturing. However, its popularity attracts other countries for the cultivation thereby increase in the production of Chinese cabbage. In the previous study, Kim et al. (2010) reported the composition and content of GSLs in 24 varieties of Korean B. rapa L. ssp. pekinensis identified glucobrassicanapin, 4methoxyglucobrassicin, gluconapin and glucobrassicin as the main GSL compounds, whereas Chen, Zhu, Gerendas, and Zimmermann (2008) compared the composition and content of GSLs in five species of Chinese Brassica campestris vegetables and confirmed glucobrassicin as a predominant individual GSL. Therefore, the objectives of this study were to determine GSL contents and profiles in 62 varieties of Chinese cabbage collected from Republic of Korea. 2. Materials and methods 2.1. Chemicals HPLC grade acetonitrile (CH3CN) and methanol (MeOH) were obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). DEAESephadex A-25, sinigrin (2-propenyl GSL) and aryl sulfatase (type H-1, EC 3.1.6.1) were obtained from SigmaeAldrich (St. Louis, MO, USA). 2,2-diphenyl-1-picrylhydrazyl (DPPH) and other chemicals were purchased from SigmaeAldrich (St. Louis, MO, USA). 2.2. Plant materials Sixty-two varieties of Chinese cabbage have been collected in Republic of Korea, and the lines of them were developed with selfpollination method by the co-operated breeding company called ‘Chinese Cabbage & Breeding’. Individual names, source details (e.g., Company & line), and agronomic characteristics are described in Table 1. The seeds were sown in 72-holled plastic tray on August 16 and intensively grown in a nursery house until September 3, 2011 at

Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

the greenhouse in the field of the breeding company, Chinese Cabbage & Breeding (Ichon, Republic of Korea). After three weeks, the seedlings having 5 or 6 leaves were transplanted in the experimental field containing 3000 kg/10a compost, 60 kg/10a urea [(NH2)2CO], 45 kg/10a potassium chloride (KCl), 100 kg/10a fused phosphate, 100 kg/10a hydrated lime [Ca(OH)2], and boron trihydroxide (B2O3) together with based fertilizer and supplied fertilizer twice according to the Standard Farming Manual. The matured Chinese cabbages (90 days grown) were harvested and collected in sampling bags with three replicates and stored at cooling refrigerator at 0e2  C. Following day, the 3e4 outside leaves of cabbages were removed because of dust particles, and sliced into four parts with a kitchen knife. The one eighth was immediately stored at 70  C and then lyophilized for the analysis of GSLs. 2.3. Extraction of crude glucosinolates (GSLs) and their desulfation Desulfo (DS) e GSLs were extracted according to the procedure of Kim et al. (2007) and ISO 9167-1 (1992). Briefly, the fresh leaves of Chinese cabbage was freeze-dried at 70  C in a freeze-dried for three days, and powdered using mortar and pestle and stored at desiccator for until for further extraction. Crude GSLs from freezedried materials (100 mg) were extracted using 1.5 ml of boiling 70% (v/v) methanol in water bath at 70  C for 5 min. After centrifugation (12,000 g, 4  C, 10 min), the supernatant was immediately transferred to a clean test tube, and the residue was further reextracted twice to complete extraction of GSLs. The combined supernatant was considered as the crude of GSLs. Separately 0.5 mg of sinigrin was dissolved in 5 ml distilled water which used as an external standard (0.001792 mmol). Desulfation of the crude extracts were performed on DEAE anion exchange column which was prepared by adding a slurry of Sephadex A-25 previously activated with 0.5 M sodium acetate (ca. 40 mg as dry matrix), whereas desulfation of sinigrin (external standard) was carried out separately in a DEAE anion exchange column. The crude GSL extracts were loaded onto a pre-equilibrated column. After that the column was washed with ultra-pure water 1 ml (2 times) to remove neutral and positive ions. Aryl sulfatase (E.C.3.1.6.1) (75 ml) was then loaded onto each column. After desulfation reaction through overnight (16 h) at room temperature, the desulfated GSLs were eluted with 0.5 ml (3 times) of distilled water. The eluates were filtered through 0.45 mm Teflon PTFE syringe filter and analyzed immediately by HPLC or stored at 20  C until further chemical analysis. 2.4. Separation and identification of glucosinolates DSeGSLs were analyzed by 1200 series HPLC system (Agilent Technologies, CA, USA) equipped with an Inertsil ODS-3 (C18) column 150  3.0 mm i.d., particle size 3 mm (GL Science, Tokyo, Japan). The HPLC analysis was carried out with a flow rate of 1.0 ml/ min at a column oven temperature of 40  C and a wavelength of 227 nm. The solvent system employed was (A) ultra-pure water (PURELAB Option-Q, ELGA) and (B) 100% acetonitrile. The solvent program was used as follows: 0 min solvent B 7%, 18 min solvent B 24%, then kept constant at solvent B 24% by 32 min, further down to solvent B 7% at 32.1 min, and then kept constant at solvent B 7% for 10 min (total 40 min). The individual GSLs were quantified with the external standard sinigrin with their HPLC area and response factor (ISO 9167-1, 1992). For the identification of the individual GSLs, the MS analysis was carried out with an ESI interface operated in the positive ion mode. The MS operating conditions were as follows: ion spray voltage, 5.5 kV; curtain gas (20 Pa), nebulizing gas (50 Pa) and heating gas (50 Pa), high purity nitrogen (N2); heating gas temperature, 550  C; spectra range, m/z 100e800 (scan time 4.8 s).

5

In this study, all the samples were designated as GSLs even though DSeGSLs were determined. 2.5. Preparation of plant materials 500 mg of lyophilized powder was mixed with 5 ml of methanol by vortexing for 5 min and then kept in an orbital shaker at 150 g for 24 h at room temperature for through extraction. After incubation the samples were centrifuged at 13,000 rpm for 15 min at 4  C. The resulting supernatant was used for the analysis of antioxidant assay. 2.6. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay The DPPH quenching ability of extracts was measured by the modified method of Hanato, Kagawa, Yasuhara, and Okuda (1988). All solutions were prepared freshly. A methanol DPPH solution (0.15%) (w/v) was mixed with different concentrations of plant extracts and after 10 min, the absorbance was read at 515 nm using a micro plate reader. Vitamin C was used as standard (10, 20, 30 mg/ ml). The ability to scavenge the DPPH radical was calculated using the following equation, DPPH scavenging effect (%) ¼ [(A0  A1)/ A0  100]. Where, A0 is the absorbance of the control at 10 min, and A1 is the absorbance of the sample at 10 min. All the samples were analyzed in triplicates. 2.7. Hydroxyl radical-scavenging (HRSA) assay The assay was performed as described by the method of Elizabeth and Rao (1990) with minor changes. All solutions were prepared freshly. One milliliter of the reaction mixture contained 100 ml of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), various concentrations of extract, 200 ml of 200 mM FeCl3 and 1.04 mM EDTA (1:1, v/v), 100 ml H2O2 (1 mM) and 100 ml ascorbic acid (1 mM). After an incubation period of 1 h at 37  C the extent of deoxyribose degradation was measured by the TBA reaction. The absorbance was read at 532 nm against the blank solution using micro plate reader. Vitamin C was used as a positive control. The scavenging activity was calculated using equation mentioned in Section 2.6. 2.8. Ferric reducing antioxidant power (FRAP) assay The reducing power of the extracts was evaluated by the modified method of Oyaizu (1986). All solutions were prepared freshly. Different amounts of the extracts were suspended in distilled water and mixed with 750 ml of 1% K3Fe (CN) 6. The mixture was incubated at 50  C for 20 min; after that 750 ml of 10% TCA was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of solution (100 ml) was mixed with distilled water (100 ml) and FeCl3 (20 ml, 0.1%), and the absorbance was measured at 700 nm using a micro plate reader. Increase in absorbance of the reaction mixture indicated reducing power. Vitamin C was used as standard. 2.9. Statistical analysis Data were analyzed by the application of the Pearson’s correlation test at p  0.05, using SPSS statistical software (version 21 for Windows, SPPS Inc., Chicago, IL, USA). The data shown in all the tables are the means of three replicates. 3. Results and discussion Separation and identification of different DSeGSLs of Chinese cabbage were performed by using HPLC according to their retention

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M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

Fig. 1. HPLC chromatogram of glucosinolates (GSLs) isolated from Chinese cabbage. Peak numbers refer to the GSLs listed in Table 2. Peak No. 1, progoitrin; 2, sinigrin; 3, glucoalyssin; 4, gluconapin; 5, 4-hydroxyglucobrassicin; 6, glucobrassicanapin; 7, glucobrassicin; 8, 4-methoxyglucobrassicin; 9, gluconasturtiin; 10, neoglucobrasscin.

times and confirmed by LCeESIeMS analysis. Identification of GSLs was done at both negative and positive ion mode. The DSeGSLs were confirmed according to their [M þ H]þ and [M þ Na]þ quasimolecular ions. At positive ion scan mode, the DSeGSLs share a common glucosyl structure. HPLC profile of DSeGSLs in Chinese cabbage was shown in Fig. 1. Ten GSLs, belonging to the three chemical classes, were detected in the whole collection of the Chinese cabbage varieties studied (Table 2). Ten GSLs were detected as follows progoitrin, sinigrin, glucoalyssin, gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin, 4methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin. The total GSL content ranged from 2.83 to 48.53 mmol/g DW (mean 14.27) (Table 3). The results revealed that significant differences in the GSL contents occurred among the varieties. The aliphatic GSLs were predominant, representing over 68% of the total GSL content, followed by indolyl GSL (26) and aromatic GSL (6). All the varieties showed comparatively significant differences in the total aliphatic GSL contents and individual aliphatic GSL, notably sinigrin, gluconapin, and progoitrin. Aliphatic GSL derived from amino acid methionine, its side-chain elongation and alteration for synthesis of different GSLs are regulated by a limited number of genes that triggers to create variation in different aliphatic GSLs (Giamoustaris & Mithen, 1996). Sulfur is the main element for the backbone of amino acid methionine, its deficiency diminishes the concentration of aliphatic GSL. The highest content of progoitrin was observed in FC6 (5.72) and FC9 (4.33 mmol/g DW), while the highest contents of total GSLs and

major GSLs (progoitrin, sinigrin, glucoalyssin, gluconapin, and glucobrassicanapin) were found in the varieties (FI13 and FI29). The amount of gluconapin (7.03) was marginally higher than progoitrin in FC6 and slightly lower (2.76 mmol/g DW) than FC9. Remarkably high levels of glucobrassicanapin (24.78) and intermediate levels of gluconapin (16.87 mmol/g DW) were detected in FI29. The contents of degradation products and its bitter tastes of GSLs have been reported by several researchers (Cartea & Velasco, 2008). In B. rapa GSLs such as gluconapin and glucobrassicanapin were involved for bitterness (Padilla, Cartea, Velasco, De Haro, & Ordas, 2007). Enzymatic breakdown of glucobrassicanapin and gluconapin leads to the production of isothiocyanates, nitriles, and epithio nitriles. Some GSLs have been studied for its nutritional and beneficial effects in order to decrease the risk of certain types of tumor in humans. Indole-3-carbinol is a derived product of glucobrassicin and sulphoraphane together are the most potent anticancer compounds found in the GSLs in cruciferous vegetables (Zhang & Talalay, 1994). Glucoiberin, progoitrin, and gluconasturtiin hydrolysis products have also been identified as inhibiting agents in protecting human and animal cells carcinogens (Staack, Kingston, Wallig, & Jeffery, 1998; Zhang & Talalay, 1994). Although the hydrolysis products of glucoraphanin have a remarkable anticancer effect, its alkenyl product gluconapin, the primary GSL in Chinese cabbage has not been reported to be anti-carcinogenic. From this comparative study, it is clearly displayed that Chinese cabbage not only contains high amounts of GSLs, but is also resident for rich anticancer GSLs.

Table 2 Glucosinolates identified in Chinese cabbage. No.a

RTb (min)

Trivial names

Semisystematic names of R-groups

Compound groups

[M þ H]þ (m/z)c

Response factord

1 2 3 4 5 6 7 8 9 10

5.97 7.09 8.26 13.19 15.90 19.23 22.10 24.59 25.45 30.18

Progoitrin Sinigrin Glucoalyssin Gluconapin 4-Hydroxyglucobrassicin Glucobrassicanapin Glucobrassicin 4-Methoxyglucobrassicin Gluconasturtiin Neoglucobrasscin

(2R)-2-Hydroxy-3-butenyl 2-Propenyl 5-Methylsufinylpentyl 3-Butenyl 4-Hydroxy-3-indolylmethyl Pent-4-enyl 3-Indolymethyl 4-Methoxy-3-indolylmethyl 2-Phenethyl N-Methoxy-3-indolylmethyl

Aliphatic Aliphatic Aliphatic Aliphatic Indolyl Aliphatic Indolyl Indolyl Aromatic Indolyl

310 280 372 294 385 308 369 399 344 399

1.09 1.00 1.07 1.11 0.28 1.15 0.29 0.25 0.95 0.20

a b c d

No., the elution order of glucosinolates from HPLC chromatograms in Fig. 1. RT, retention time. Bennett et al. (2004). International Organization for Standardization (ISO 9167-1, 1992).

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M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

7

Table 3 Glucosinolate contents (mmol/g dry wt.) in 62 varieties of Chinese cabbage (n ¼ 3). Accessions/field trial names 1/FV1 2/FV2 3/FV3 4/FV4 5/FV5 6/FV6 7/FV7 8/FC1 9/FC2 10/FC3 11/FC4 12/FC5 13/FC6 14/FC7 15/FC8 16/FC9 17/FV8 18/FV9 19/FV10 20/FC10 21/FC11 22/FC12 23/FC13 24/FC14 25/FC15 26/FC16 27/FI1 28/FI2 29/FI3 30/FI4 31/FI5 32/FI6 33/FI7 34/FI8 35/FI9 36/FI10 37/FI11 38/FI12 39/FI13 40/FI14 41/FI15 42/FI16 43/FI17 44/FI18 45/FI19 46/FI20 47/FI21 48/FI22 49/FI23 50/FI24 51/FI25 52/FI26 53/FI27 54/FI28 55/FI29 56/FI30 57/FI31 58/FI32 59/FI33 60/FI34 61/FI35 62/FI36

Identified glucosinolates based on HPLC retention time (no.a)

Total

1

2

3

4

5

6

7

8

9

10

0.98def 2.1b 1.19c 2.87b 0.88cd 1.32cd 0.94ce 1.26c 1.71c 1.09c 1.09e 1.33de 5.72cd 2.33cd 1.95cd 4.33c 1.06de 0.91cde 1.66c 0.88de 0.70c 1.24c 1.03c 1.17c 1.16b 1.04c 1.46bc 1.48cdf 2.35b 1.16b 0.94c 1.07c 0.99bc 2.45d 0.86c 0.73cd 1.25c 1.62b 1.64e 1.33c 0.55cd 1.06b 2.69cd 1.32cd 1.22d 1.39cd 2.04bc 1.03c 2.00d 1.46c 1.23de 1.53d 1.40c 1.23bc 1.19d 0.96d 0.85e 1.11cd 0.97d 1.57cd 2.54cd 2.21c

1.38cde 1.17cd 1.03c 1.03c 0.95cd 0.77de 1.07bce 0.62c 0.66c 0.43c 0.36f 0.47ef 0.53ef 0.46cd 0.77cd 0.72g 0.49e 0.65def 0.47ef 0.38de 0.09c 0.84c 0.20d 0.68cd 0.32c 0.45c 0.22c 0.27df 0.77cd 0.44c 0.23d 0.09c 1.20bc 1.11e 0.36cd 0.36d 0.43def 0.42c 0.81efgh 0.66cdef 0.13d 0.24bc 0.60g 0.17e 0.63efg 0.90de 0.37c 0.42de 1.32ef 0.45ef 0.50fgh 0.10f 0.98cde 0.39cd 0.26d 0.56de 0.31e 1.96cd 0.57d 1.07de 0.34e 0.58d

0.40ef 0.39def 0.41c 0.41cd 0.41cd 0.39def 0.42e 0.40c 0.37c 0.38c 0.41f 0.41ef 0.47f 0.50cd 0.36cd 0.37g 0.34e 0.35ef 0.27ef 0.25e 0.28c 0.45c 0.41d 0.5de 0.38c 0.41c 0.38c 0.35df 0.50d 0.38cd 0.29d 0.51c 0.19c 0.22f 0.27cd 0.28d 0.18 fg 0.29c 0.57fgh 0.46def 0.15d 0.14c 0.24g 0.20de 0.22fgh 0.28e 0.23c 0.16e 0.19g 0.17f 0.22gh 0.14ef 0.19e 0.15d 0.16d 0.18e 0.16e 0.17d 0.16d 0.18f 0.15e 0.18d

2.49c 0.88de 4.21b 0.73cd 0.26d 2.28bc 2.04bc 7.11b 8.69b 9.48b 6.14b 5.72c 7.03c 7.50b 0.75cd 2.76d 6.43c 1.12cd 0.37ef 3.64c 0.95c 0.25c 0.10d 0.17de 0.12c 0.14d 1.48bc 6.89b 0.26d 0.18cdf 0.09d 19.72b 0.61c 3.63c 0.17cd 0.99cd 0.37efg 0.26c 26.02b 0.86cde 5.75b 0.27bc 2.93c 0.95cde 4.19c 2.04c 1.70bc 1.01c 3.81c 0.88e 7.38c 3.83c 2.78b 1.08bcd 16.87c 4.98b 4.66c 5.19b 13.01c 0.44ef 2.92c 1.05cd

0.02f 0.04f 0.05c 0.04d 0.11d 0.06e 0.06e 0.17c 0.05c 0.04c 0.06f 0.11f 0.08f 0.04d 0.01d 0.05h 0.07e 0.07f 0.08f 0.04e 0.03c NDb 0.02d ND ND 0.02d 0.02c 0.05f 0.02d ND ND 0.02c 0.03c 0.04f 0.02d 0.03d 0.04g 0.05c 0.09h 0.10f 0.11d 0.11c 0.17g 0.07e 0.06h 0.09e 0.08c 0.11e 0.06g 0.13f 0.20h 0.11f 0.17e 0.14d 0.32d 0.22e 0.13e 0.11d 0.18d 0.08f 0.11e 0.07d

6.79b 2.78b 5.05b 2.17b 1.46c 2.92b 2.43bc 8.30b 8.62b 7.47b 5.37c 7.37b 11.71b 8.48b 4.33b 6.97b 12.71b 1.54bc 1.23d 5.93b 1.39bc 1.01c 0.20d 0.47de 0.16c 0.41c 1.70bc 3.80c 1.11c 0.44c 0.17d 0.30c 1.04bc 4.89b 0.27cd 2.49b 0.81d 0.53c 13.45c 1.23c 5.40b 0.49bc 5.95b 2.70b 6.48b 3.06b 4.35b 1.88b 5.22b 2.96b 8.90b 5.72b 3.50b 1.79b 24.78b 4.69b 5.63b 3.02bc 18.98b 2.76b 6.31b 5.95b

0.49def 0.37ef 0.32c 0.29cd 0.68cd 0.31de 0.38e 0.57c 0.35c 0.25c 0.48f 0.43ef 0.80ef 2.10cd 1.04cd 0.60gh 1.10de 0.37ef 0.25ef 0.20e 0.14c 0.49c 0.12d 0.4de 0.11c 0.13d 0.46c 0.58df 0.43d 0.13df 0.10d 0.48c 0.74c 0.67ef 0.14cd 0.63cd 0.31efg 0.66c 1.41ef 1.02cd 0.45cd 0.11c 1.66ef 0.56cde 0.36fgh 0.53de 0.78c 0.46de 1.18f 0.91de 0.83efg 0.15ef 0.38de 0.51cd 0.78d 0.95d 0.86e 1.14cd 0.60d 0.80ef 0.42e 0.90d

1.72cd 1.87c 1.51c 2.00b 2.86b 2.04bc 2.73b 2.18c 1.67c 1.87c 2.38d 2.32d 3.35de 2.79c 2.55bc 2.29e 2.80d 1.85b 2.32b 1.83d 2.73b 4.35b 3.95b 2.77b 1.28b 2.06b 2.31b 2.86cd 2.35b 1.26b 1.63b 2.79b 2.84b 3.20c 2.38b 1.97bc 2.66b 1.87b 3.24d 3.74b 0.77c 0.21c 1.88de 1.25cd 1.11de 1.05cde 1.03bc 0.99c 1.86de 1.37cd 1.76d 0.74e 1.11cd 0.83bcd 1.48d 1.49c 2.44d 2.28cd 1.27d 2.02bc 0.91e 1.11cd

0.87def 0.88de 0.76c 0.83cd 0.56cd 0.66de 0.90ce 0.81c 0.72c 0.79c 0.34f 0.93ef 1.98ef 1.18cd 0.51cd 1.41f 1.40de 0.82de 0.47e 0.46de 0.12c 0.60c ND 0.41de 0.14c 0.32c 0.17c 0.35df 0.36d 0.35cd 0.24d 0.28c 0.67c 2.18d ND 0.14d 0.68de 0.40c 1.16efg 1.10cd 0.59cd 0.10c 0.93 fg 0.92cde 0.71def 0.84de 0.81c 0.82cd 1.55def 0.61ef 1.08ef 0.47ef 0.93cde 0.78bcd 2.29d 0.71d 0.52e 2.61c 1.58d 0.96de 1.28de 0.73d

0.22ef 0.17ef 0.06c 0.09d 0.17d 0.06e 0.09e 0.06c 0.06c 0.03c 0.05f 0.06f 0.13f 0.37cd 0.13d 0.10h 0.25e 0.03f 0.02f 0.04e 0.04c 0.08c 0.01d 0.09de 0.01c 0.02d 0.07c 0.04f 0.07d 0.03f 0.01d 0.10c 0.05c 0.16f ND 0.13d 0.05 fg 0.16c 0.15gh 0.21ef 0.20cd 0.09c 0.47g 0.15e 0.15gh 0.22e 0.08c 0.11e 0.47g 0.14f 0.11h 0.08f 0.17ef 0.16d 0.13d 0.12e 0.24e 0.11d 0.09d 0.18f 0.14e 0.22d

15.35a 10.64a 14.59a 10.45a 8.33a 10.81a 11.05a 21.48a 22.89a 21.83a 16.68a 19.16a 31.81a 25.77a 12.38a 19.61a 26.65a 7.69a 7.14a 13.65a 6.48a 9.31a 6.04a 6.66a 3.66a 5.00a 8.26a 16.66a 8.22a 4.36a 3.69a 25.36a 8.37a 18.55a 4.46a 7.75a 6.77a 6.26a 48.53a 10.71a 14.11a 2.83a 17.51a 8.29a 15.11a 10.39a 11.46a 7.00a 17.67a 9.08a 22.20a 12.87a 11.61a 7.07a 48.26a 14.85a 15.81a 17.72a 37.40a 10.05a 15.12a 13.00a

Mean values (n ¼ 3) indicated by the same letters in a line do not significantly different at 5% level using Tukey’s multiple range test. a No., the elution order of glucosinolates from HPLC chromatograms in Fig. 1. b ND, not detected.

The differences in total and individual GSL contents found among varieties in Chinese cabbage are in coincidence with previous observations in Brassica vegetables and ornamental plants (Cartea & Velasco, 2008; Yang & Quiros, 2010). It is believed that the most important factor in identifying the GSL contents is genotype

and the genetic variation has a significant effect on the GSL synthesis pattern. Gluconapin (3-butenyl GSL) and glucobrassicanapin (4-pentenyl GSL) are the predominant (mean 3.72 and 4.52 mmol/g DW) GSLs in Chinese cabbage, as the breakdown products of these two GSLs, particularly the isothiocyanates, are considered the

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8

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

Fig. 2. Antioxidant activities in 60 varieties (except for accession 14 and 61 in Table 1) of Chinese cabbage (n ¼ 3) (:, 20; -, 40; A, 60 mg/ml). a) DPPH assay; b) HRSA assay; c) FRAP assay.

importance of flavor and anticancer properties, whereas the larger amounts of gluconapin have implications for sensory properties. Moreover, 2-propenyl isothiocyanate derived from sinigrin is involved in pungency, bitterness effects and special flavor. Therefore, breeding and identification of suitable Chinese cabbage for health benefits metabolites should be associated with enhanced levels of the major anti-carcinogenic GSLs, which do not have significant effects on flavor, as well as glucobrassicin as the major indolyl GSLs in Chinese cabbage. Numerous antioxidants are present in plant samples; therefore measuring each antioxidant can be tedious and time consuming. Hence, several methods have been developed to measure antioxidant activities as a whole and these methods are more useful and can be applied to the samples from different varieties of Chinese cabbage. Furthermore, these methods are reliable and easy to perform and can be used for determining antioxidant capabilities of plant samples (Thaipong, Boonprakob, Crosby, CisnerosZevallos, & Byrne, 2006). Protective effects of nutritional antioxidants in health come from their ability to scavenge free radicals by acting as a hydrogen/electron donor or direct reaction with them; chelate transition metal ions (thus preventing the formation of free radicals via the Fenton reactions); inhibit radical-producing enzymes such as cyclooxygenase and lipoxygenase or increase the expression of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In this study, the ability of antioxidants in different varieties of Chinese cabbage has been investigated.

The radical-scavenging activities of the samples from different varieties of Chinese cabbage were estimated by comparing the percentage inhibition of formation of DPPH radicals by the test samples. Fig. 2 depicts a steady increase in the percentage inhibition of the absorbance of the DPPH radicals by the test samples by concentration-dependent manner. The DPPH radical-scavenging capacity of samples from different varieties of Chinese cabbage is lower than the positive control (mean 91.15%). Much of the oxidative damage to biomolecules can also be induced by eOH, the more reactive one among ROS species (Yang, Liu, Han, & Sun, 2006). As illustrated in Fig. 2, the hydroxyl radical-scavenging activity initiated by samples from different varieties of Chinese cabbage is similar to positive control in a concentration-dependent manner. This result proved that test samples had a significant effect on scavenging hydroxyl radical. The ferric reducing assay gives fast and reliable results in the samples from different varieties of Chinese cabbage. In this assay, samples are used in a redox linked reaction whereby the antioxidants present in the plant sample act as the reductants while the reagent, containing excess ferric ions act as the oxidants. Both samples from different varieties of Chinese cabbage and positive control showed dose dependent ferric reducing activity. Antioxidant capacities of 60 varieties (except for accessions 14 and 61) of Chinese cabbage were not significantly correlated with their total GSLs (data were not shown). The reductive ability of the samples assessed in this study suggests that the extracts were able to donate electron, hence they should be able to donate electrons to free radicals in actual

Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001

M.-K. Lee et al. / LWT - Food Science and Technology xxx (2014) 1e9

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Please cite this article in press as: Lee, M.-K., et al., Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.03.001