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

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LWT - Food Science and Technology

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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
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

a r t i c l e i n f o
Article history: Received 6 October 2013 Received in revised form 27 January 2014 Accepted 1 March 2014 Keywords: Brassica rapa L. ssp. pekinensis Chinese cabbage Glucosinolates LCeMS Antioxidant activity

a b s t r a c t
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 identied. 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 signicant antioxidant activities when compare with positive control. However, three antioxidant assays were not signicantly correlated with total GSLs. The presence of signicant quantities of glucobrassicin in some varieties should be studied more extensively, since GSL is the precursor of indole-3-carbinol, a potent anticancer isothiocyanate. 2014 Elsevier Ltd. All rights reserved.

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 classied 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.: 82 42 821 6738; fax: 82 42 822 7142. E-mail addresses: mvalanarasu@gmail.com (M.V. Arasu), kimsunju@cnu.ac.kr (S.-J. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.lwt.2014.03.001 0023-6438/ 2014 Elsevier Ltd. All rights reserved.

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

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. Accessions Field trial names 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 FV1 FV2 FV3 FV4 FV5 FV6 FV7 FC1 FC2 FC3 FC4 FC5 FC6 FC7 FC8 FC9 FV8 FV9 FV10 FC10 FC11 FC12 FC13 FC14 FC15 FC16 FI1 FI2 FI3 FI4 Varieties Bulam No.3 Line names Seminis Head formation Outer leaf color Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Green Viridian Viridian Gray egreen Viridian Green Viridian Viridian Viridian Viridian Viridian Viridian Viridian Green Viridian Viridian Green Inner leaf color Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Orange Yellow Yellow Brightyellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Head height Head width Harvest (cm) (cm) maturity 28.0 28.5 30.0 30.0 31.0 30.0 30.0 31.0 32.0 32.0 32.0 32.0 30.0 32.0 30.0 32.0 24.0 26.0 25.0 26.0 27.0 27.0 27.0 26.5 27.0 27.0 25.0 27.0 26.5 27.0 18.0 18.0 19.0 18.5 19.0 20.0 19.0 20.0 20.0 20.0 22.0 21.0 21.0 22.0 20.0 17.5 16.0 17.0 16.0 16.5 17.0 18.0 17.0 17.0 17.0 17.0 16.5 16.0 15.5 15.5 Medium-late type Medium-late type Medium-late type Medium-late type Late type Downy mildew Cold Clubroot Hot resistance tolerance

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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 ower 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

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

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

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

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

FI5 FI6 FI7 FI8 FI9 FI10 FI11 FI12 FI13 FI14 FI15 FI16 FI17 FI18 FI19 FI20 FI21 FI22 FI23 FI24 FI25 FI26 FI27 FI28 FI29 FI30 FI31 FI32 FI33

Bulam1 Shintam BulCheong PungDong Tamyoung Shintam PyeongDae75 Tamyoung ChuChoung CR DaemyoungHwanggo BulCheong PyeongDae75 WolHwang Whanggo65 No CR Myoung CR Daeno CR CR Dae CR Daeno CR Tamyoung CR DaemyoungTamyoung CR Dae CR DaemyoungTamyoung CR Daeno CR CR Dae Tamyoung CR Daeno CR Tamyoung CR Daeno CR Tamyoung CR DaemyoungTamyoung CR DaemyoungTamyoung CR DaemyoungHwanggo CR DaemyoungHwanggo Hano CR No KweonjiKweondan CR DaemyoungHwanggo

BA1 YM245-G7 73317C-G7 W23-G6 YM232-G6 OH25952-G7 YM232HN 1442-G6 YM23754 73317C58-G7 OH25951-G8 KN1 S241 955S S9415-G8 32S1553-G7 32S955 32S551 95S955 415 S955-G7 S415 32S553 32S155-G6 YM23751 32S13778 1042S510 H1896-G6 32S1-G6

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

Viridian Viridian Viridian Viridian Viridian Viridian Viridian Green Green Viridian Green Viridian Green Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Viridian Green Green Viridian

Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Orange Yellow

26.0 26.0 28.0 30.0 30.0 25.5 27.0 27.0 26.0 25.5 27.0 25.0 30.0 28.0 28.0 24.0 30.0 28.0 29.0 26.0 26.0 26.0 27.0 30.0 30.0 28.0 28.0 24.0 26.0

16.0 16.5 17.0 17.5 17.0 17.5 17.0 17.0 17.0 16.0 17.5 16.0 17.5 18.0 18.0 15.0 17.5 15.5 17.0 16.0 16.5 16.0 15.5 17.0 17.5 16.0 16.0 16.0 16.5

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

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

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

Medium type Late 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

Medium type Earlu medium type Early type Medium type

(continued on next page)

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

FV1 (F, eld trial; V, serial number; 1, accession number). V: control Var. C: F1 combination. I: inbred line. , Strongestrong; , strong; , strong-middle; , middle; , middle-weak; , weak.

Line names

111533

S872C 1109JH

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 signicant 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 countrys 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 signicantly 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 identied 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 ve species of Chinese Brassica campestris vegetables and conrmed glucobrassicin as a predominant individual GSL. Therefore, the objectives of this study were to determine GSL contents and proles 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

Cold Clubroot Hot resistance tolerance

Medium type 17.0 25.0 Orange Green ShinwolOjeon FI34 60

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

Inner leaf color

Outer leaf color

Head formation

Table 1 (continued )

Accessions Field trial names

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61 62

FI35 FI36

SeoulOjeon DaepyeongHano

Varieties

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

Viridian Green

Orange Orange

31.0 29.0

14.5 19.0

Medium type Earlu medium type

Downy mildew

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

the greenhouse in the eld 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 eld 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). Briey, 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 ltered through 0.45 mm Teon PTFE syringe lter and analyzed immediately by HPLC or stored at 20  C until further chemical analysis. 2.4. Separation and identication 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 ow 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 quantied with the external standard sinigrin with their HPLC area and response factor (ISO 9167-1, 1992). For the identication 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).

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 modied 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 (%) [(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 modied 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 Pearsons 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 identication 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 conrmed by LCeESIeMS analysis. Identication of GSLs was done at both negative and positive ion mode. The DSeGSLs were conrmed according to their [M H] and [M Na] quasimolecular ions. At positive ion scan mode, the DSeGSLs share a common glucosyl structure. HPLC prole 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 signicant 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 signicant 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 deciency 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
Table 2 Glucosinolates identied in Chinese cabbage. No.a 1 2 3 4 5 6 7 8 9 10
a b c d

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 benecial 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 identied 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.

RTb (min) 5.97 7.09 8.26 13.19 15.90 19.23 22.10 24.59 25.45 30.18

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

Semisystematic names of R-groups (2R)-2-Hydroxy-3-butenyl 2-Propenyl 5-Methylsunylpentyl 3-Butenyl 4-Hydroxy-3-indolylmethyl Pent-4-enyl 3-Indolymethyl 4-Methoxy-3-indolylmethyl 2-Phenethyl N-Methoxy-3-indolylmethyl

Compound groups Aliphatic Aliphatic Aliphatic Aliphatic Indolyl Aliphatic Indolyl Indolyl Aromatic Indolyl

[M H] (m/z)c 310 280 372 294 385 308 369 399 344 399

Response factord 1.09 1.00 1.07 1.11 0.28 1.15 0.29 0.25 0.95 0.20

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 Table 3 Glucosinolate contents (mmol/g dry wt.) in 62 varieties of Chinese cabbage (n 3). Accessions/eld 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 Identied glucosinolates based on HPLC retention time (no.a) 1 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 2 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 3 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 4 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 5 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 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 7 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 8 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 9 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 10 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 Total

Mean values (n 3) indicated by the same letters in a line do not signicantly different at 5% level using Tukeys 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 signicant 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

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

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

importance of avor 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 avor. Therefore, breeding and identication of suitable Chinese cabbage for health benets metabolites should be associated with enhanced levels of the major anti-carcinogenic GSLs, which do not have signicant effects on avor, 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 signicant 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 signicantly 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

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

biological or food systems, making the radicals stable and unreactive. Because of the complex mechanism of antioxidant activity, the above tests are normally not enough to evaluate precisely the antioxidant activity of the potential antioxidant. Consequently various oxidative stress models for different varieties of Chinese cabbage should deserve more in-depth research in the future. 4. Conclusions Ten GSLs were separated and identied in Chinese cabbage using by HPLC and LCeMS method. The composition and contents of GSLs among 62 different varieties were analyzed. Remarkable differences in the total and individual GSL contents were observed among different varieties. The antioxidant activities of the Chinese cabbage samples were concentration-dependent manner. Chinese cabbage contained rich anti-carcinogenic GSLs, and is an excellent choice for human daily consumption. Some varieties with higher contents of health promoting GSL glucobrassicin in edible part of Chinese cabbage, such as FC7 and FI17 are promising varieties for future breeding initiatives. Acknowledgements This research was supported by Bio-industry Technology Development Program (311022-05-3-SB020), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS). References
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