Metabolic Profiling in Chinese Cabbage (Brassica rapa L. subsp

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Metabolic Profiling in Chinese Cabbage (Brassica rapa L. subsp. pekinensis) Cultivars Reveals that Glucosinolate Content Is Correlated with Carotenoid Content Seung-A Baek, Young-Ho Jung, Sun-Hyung Lim, Sang Un Park, and Jae Kwang Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01323 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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Journal of Agricultural and Food Chemistry

Metabolic Profiling in Chinese Cabbage (Brassica rapa L. subsp. pekinensis) Cultivars Reveals that Glucosinolate Content Is Correlated with Carotenoid Content

Seung-A Baek,† Young-Ho Jung,§ Sun-Hyung Lim,‡ Sang Un Park,*,# and Jae Kwang Kim*,†

†

Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National

University, Incheon 22012, Republic of Korea §

Biotechnology Institute, Nongwoo Bio Co., Ltd., Yeoju, Gyeonggi 12655, Republic of Korea

‡

National Academy of Agricultural Science, Rural Development Administration, Jeonju

54874, Republic of Korea #

Department of Crop Science, Chungnam National University, 99 Daehak-Ro, Yuseong-gu,

Daejeon 34134, Republic of Korea

*These authors contributed equally to this work.

*Corresponding Authors (S.U.P.) Tel: +82-42-821-5730; Fax: +82-42-822-2631; E-mail: [email protected] (J.K.K.) Tel: +82-32-835-8241; Fax: +82-32-835-0763; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Thirty-eight bioactive compounds, including glucosinolates, carotenoids, tocopherols, sterols,

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and policosanols, were characterized from nine varieties of Chinese cabbage (Brassica rapa

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L. subsp. pekinensis) to determine their phytochemical diversity and to analyze their

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abundance relationships. The metabolite profiles were evaluated with a principal component

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analysis (PCA), Pearson correlation analysis, and hierarchical clustering analysis (HCA).

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PCA and HCA identified two distinct varieties of Chinese cabbage (Cheonsangcheonha and

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Waldongcheonha) with higher levels of glucosinolates and carotenoids. Pairwise comparisons

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of the 38 metabolites were calculated using Pearson correlation coefficients. The HCA, which

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used the correlation coefficients, clustered metabolites that are derived from closely related

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biochemical pathways. Significant correlations were discovered between chlorophyll and

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carotenoids. Additionally, aliphatic glucosinolate and carotenoid levels were positively

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correlated. The Cheonsangcheonha and Waldongcheonha varieties appear to be good

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candidates for breeding because they have high glucosinolate and carotenoid levels.

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KEYWORDS: carotenoids, Chinese cabbage, glucosinolates, phytosterol, policosanol

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INTRODUCTION

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Chinese cabbage (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt) is a common vegetable

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in Asia, particularly in China, Japan, and Korea. In Korea, it has particular importance as the

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main ingredient of kimchi, a traditional fermented food. Chinese cabbage contains

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carotenoids and tocopherols and is a natural source of antioxidants. Yellow inner leaves of

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Chinese cabbage reflect lutein and carotene contents, and Asian consumers prefer yellower

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cultivars because they have high nutritional value.1 Watanabe et al. (2011)2 reported that

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orange Chinese cabbage contained higher amounts of carotenoids and phenolic compounds

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with radical-scavenging activity than normal Chinese cabbage. Antioxidants such as

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carotenoids and tocopherols have been associated with risk reductions for cardiovascular

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disease and several types of cancer.3 Carotenoids are essential accessory light-harvesting

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pigments with photoprotective functions and influence production of primary metabolites.4

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Secondary metabolites, such as terpenes, steroids, glucosinolates, and waxes, are synthesized

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from primary photosynthesis metabolites, and their production may be influenced by

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environmental conditions that can also affect photosynthesis.5 Chinese cabbage is also an

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important source of glucosinolates, which may reduce the risk of colon, bladder, lung, breast,

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and prostate cancers.6-7 Significant variations in carotenoids, tocopherols, and glucosinolates

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among cruciferous genotypes suggest differences in the beneficial properties of these

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vegetables.8-10 In Arabidopsis, biosynthesis of glucosinolates and sulfur assimilation were

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regulated by light period,11 with higher levels of glucosinolates during the day. Glucosinolate

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abundance in broccoli was affected by carbon dioxide and salinity.12 The total glucosinolate

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amount in broccoli increased under elevated carbon dioxide concentration. Conversely, a

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selenium treatment decreased glucosinolates and carotenoid biosynthesis in Arabidopsis.13

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However, no previous reports have examined correlations between phytochemicals such as

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carotenoids and glucosinolates.

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Other phytochemicals, including sterols and policosanols, may affect metabolism. Plant

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sterols can lower serum cholesterol.14 Some reports have suggested that policosanols can

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reduce total cholesterol and LDL and increase HDL.15-16 However, Ng et al. (2005)17 reported

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that policosanols have no antioxidant activity in human LDL. The policosanols have not been

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well-characterized in Chinese cabbage varieties. Therefore, we examined the diversity of

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carotenoids, tocopherols, glucosinolates, phytosterols, and policosanols within Chinese

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cabbage cultivars. Phytochemical characterization will aid breeding programs in developing

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and selecting new germplasm with improved nutritional value. Chinese cabbage has been

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biofortified to improve its nutritional quality via a transgenic engineering approach. Zang et

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al. (2008)18 metabolically engineered the indole glucosinolate pathway in Chinese cabbage,

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using three Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1. In addition, Zang et

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al. (2008)19 reported on the biofortification of alipathatic glucosinolate levels in Chinese

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cabbage by overexpression of Arabidopsis MAM1, CYP79F1, and CYP83A1. However,

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Chinese

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Phytochemicals must be characterized from multiple Chinese cabbage varieties if nutritional

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values are to be enriched through conventional breeding.

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Our objective was to profile and quantify the glucosinolates, carotenoids, tocopherols, sterols,

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and policosanols in nine Chinese cabbage varieties and to analyze the relationships among

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their abundance levels. Our results will provide Chinese cabbage breeders with critical data

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for developing new varieties with enhanced levels of bioactive compounds.

cabbage

has

seldom

been

biofortified

through

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conventional

breeding.

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MATERIALS AND METHODS

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Plant Materials and Culture Conditions.

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Seeds of seven cultivars of Chinese cabbage were provided by Nongwoo Bio Co., Ltd. (Yeoju,

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Gyeonggi, Republic of Korea): Cheongomabi, Cheonsangcheonha, Chuno, CRmat,

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Hwangkeummulgeul, Kangsimjang, and Waldongcheonha. Seeds of two additional varieties,

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Bularm3ho and Chukwang, were purchased from a local market. Seeds were germinated in

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soil in a 105-pot tray and grown under controlled conditions, at 20–25 °C and 50–60%

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relative humidity, in a greenhouse. Three-week-old seedlings were planted in an agricultural

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field and grown for 11 weeks. Three individuals from each cultivar were harvested after 11

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weeks. The edible portions were vertically cut, freeze-dried, powdered with a grinder

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(HR2860, Philips, Amsterdam, Netherlands), and stored at −70 °C prior to analysis.

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Desulfoglucosinolate Analysis Using HPLC and LC-QTOFMS.

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Crude glucosinolates were analyzed according to the procedure published by Kim et al.

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(2010)20. Desulfated glucosinolates were analyzed by a HPLC system (1200 series; Agilent

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Technologies, Waldbronn, Germany) at 227 nm. Desulfoglucosinolate extracts were separated

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on an Inertsil ODS-3 (C18) column (250 × 4.6 mm, 5 µm, GL Sciences, Tokyo, Japan). The

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mobile system was buffers A (water) and B (acetonitrile). The elution profile was: 0 min, 99%

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A; 0–18 min, 1–30% B in A (linear gradient); 18–30 min 30% B in A (isocratic). Quantitative

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analysis of glucosinolate was performed by the response factor of glucosinolate relative to

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sinigrin.21 For qualitative analysis of glucosinolates, extracts were separated on a C18 column

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(120 × 2.1 mm, 2.2 µm, Acclaim RSLC 120 C18) using a Dionex U3000 UHPLC-

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Quadrupole-time-of-flight mass spectrometer (QTOFMS) system (Bruker Daltonics, Bremen,

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Germany). The same elution buffers and elution program described previously were used.

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The flow rate was 0.4 mL/min, and the column temperature was 30 °C. The mass

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spectrometer was operated with full-scan acquisition in positive electrospray ionization

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source to acquire the mass range m/z 50–700. The spray voltage was set to 4.5 kV.

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Carotenoids and Chlorophyll Analysis.

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Carotenoids were analyzed using HPLC, as described previously.22 Freeze-dried samples

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were mixed at 500 rpm in 100% methanol at 70 °C for 30 min to extract total chlorophyll

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with a Thermomixer Comfort (model 5355; Eppendorf AG, Hamburg, Germany). The

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mixture was centrifuged at 800 × g and 4 °C for 10 min. Supernatant absorbance was

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measured with a spectrophotometer (Optizen POP; MECASYS CO., Daejeon, Republic of

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Korea) at 666 nm and 653 nm. Total chlorophyll content was calculated according to

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equations from Wellburn (1994).23

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Tocopherol, Phytosterol, Policosanol, and α-Amyrin Analysis Using GC-MS.

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Tocopherols, phytosterols, policosanols, and α-amyrin were extracted according to previously

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described methods.24 Lipophilic metabolites were released from the freeze-dried samples (50

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mg) by the addition of 3 mL of ethanol containing 0.1% ascorbic acid (w/v), and 0.05 mL of

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5α-cholestane (10 µg/mL) was added as an internal standard. The sample was vortexed for 20

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s, then incubated in a water bath at 85 °C for 5 min. After incubation, 120 µL of potassium

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hydroxide (80%, w/v) was added and the samples were vortexed before being returned to the

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water bath for 10 min for saponification. Samples were immediately put on ice, and 1.5 mL

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of deionized water was added. Each sample received 1.5 mL of hexane, was vortexed briefly,

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and was then centrifuged at 1200 × g for 5 min. The upper layer was pipetted into a new tube,

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and the pellet was re-extracted using 1.5 mL of hexane. The hexane fraction was dried in a

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centrifugal concentrator (CC-105, TOMY, Tokyo, Japan). For derivatization, 30 µL of N-

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methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and 30 µL of pyridine were added and the

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mixture was incubated at 60 °C for 30 min at 1200 rpm. The same GC-MS procedure

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described previously was used.24 Each derivative sample was analyzed with the GCMS-

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QP2010 Ultra system (Shimadzu, Japan), using an Rtx-5MS column (30 m, 0.25 mm i.d.,

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film 0.25 µm).

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Statistical Methods.

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All analyses were performed in triplicate at a minimum. The glucosinolate, carotenoid,

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policosanol, phytosterol, chlorophyll, and α-amyrin levels in Chinese cabbage varieties were

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analyzed using principal component analysis (PCA) (SIMCA-P version 12.0; Umetrics,

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Umeå, Sweden). Additionally, samples were clustered based on their metabolite profiles

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through hierarchical clustering analysis (HCA), with Pearson correlation and average linkage.

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We performed Pearson correlation analysis and HCA using SAS 9.2 software (SAS Institute,

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Cary, NC, USA). Data were scaled to unit variance. MULTIEXPERIMENT VIEWER version

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4.4.0 software (http://www.tm4.org/mev/) was used for the analysis of heat map and HCA

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visualization of the correlation coefficients.

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RESULTS AND DISCUSSION

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Chinese Cabbage Metabolite Profiling.

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For the first time, glucosinolates, carotenoids, policosanols, phytosterols, chlorophyll, and α-

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amyrin from Chinese cabbage grown in Korea were identified by HPLC, LC-MS, and GC-

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MS. Carotenoids and tocopherols are lipophilic antioxidants with important functions in

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plants and humans.3 Phytosterols are bioactive lipophilic compounds that have an ability to

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reduce serum cholesterol.14-16 Glucosinolates and their breakdown products are known for

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their bactericidal biological functions, and they are used in cancer treatments.6-7 In this study,

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individual glucosinolates were separated by HPLC and confirmed by LC-QTOFMS in

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positive mode (Table 1 and Supplementary figure 1). Eleven kinds of glucosinolates were

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detected in all varieties. Glucocochlearin was only present in Waldongcheonha. Tocopherols,

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phytosterols, policosanols, and α-amyrin were identified by GC-MS analysis (Table 2 and

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Supplementary figure 2). Twenty-two types of lipophilic compounds were detected in most

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varieties. Quantification was performed using selected ions (Table 2). The mass spectra of

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policosanol trimethylsilyl derivatives showed that the molecular ion [M-15]+ (through the

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loss of a methyl group) was generally dominant. The carotenoid composition in Chinese

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cabbage leaves is shown in Supplementary figure 3.

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PCA and HCA.

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Metabolite profiling has been combined with chemometrics to direct breeding strategies, with

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the goal of enhancing specific components that are desirable. Similarities in metabolite

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abundance in nine Chinese cabbage varieties were first examined with PCA (Figure 1). The

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components revealed no clear separation among nine Chinese cabbage varieties. However,

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the first component separated Cheonsangcheonha and Waldongcheonha from the other

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varieties. An HCA with metabolite profiles also clearly distinguished these two cultivars.

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Factor loadings in component 1 were compared to identify the compounds with the highest

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contributions, which were carotenoids and glucosinolates. The variation was mainly

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attributable to α-carotene, lutein, glucobrassicanapin, and β-carotene, for which the

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eigenvectors were 0.2592, 0.2539, 0.2491, and 0.2479, respectively, indicating that the

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compounds were present at higher levels in the two varieties (Cheonsangcheonha and

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Waldongcheonha) than in others. However, the corresponding loading was mainly negative

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for phytosterols such as brassicasterol and stigmasterol, indicating a negative correlation

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between the levels of carotenoids and phytosterols.

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Differences between 38 Metabolites in Chinese Cabbage Samples.

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Glucosinolate and carotenoid accumulation in plants are heavily influenced by genetic and

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environmental factors. Kang et al. (2006)25 reported that significant phenotypic variation in

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glucosinolate levels existed among commercial Chinese cabbage germplasm, so levels could

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be increased through genetic selection. In our study, the total glucosinolate content of

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Chinese cabbage ranged from 5.70 to 32.79 µmol/g of dry weight (DW) (Table 3). Previous

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work has shown that total glucosinolate content of 24 Chinese cabbage varieties ranged from

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4.48 to 31.58 µmol/g DW.20 Our results showed that Cheonsangcheonha and

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Waldongcheonha had the highest total glucosinolate levels of the nine varieties examined.

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The predominant aliphatic glucosinolates were gluconapin and glucobrassicanapin. The

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hydrolysis products of gluconapin and glucobrassicanapin are isothiocyanates, nitriles, and

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epithionitriles.20

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In this study, total carotenoids ranged from 36.22 to 141.96 µmol/kg of DW (Table 4). The

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predominant carotenoids were lutein (20.18–66.59 µmol/kg DW) and β-carotene (8.99–64.08

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µmol/kg DW). These results were consistent with those of previous studies.26 Lutein and β-

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carotene are the dominant carotenoids in other cruciferous vegetables.22 Lutein is considered

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to increase the density of the macular pigment in the eye. β-Carotene is a precursor to vitamin

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A via the action of beta-carotene 15,15'-monooxygenase. Varietal differences in carotenoid

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accumulation have been noted for kale.27 The total pigment levels were much higher in

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Cheonsangcheonha and Waldongcheonha than in other varieties, echoing the pattern for

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glucosinolate levels. Therefore, Cheonsangcheonha and Waldongcheonha are the most

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promising varieties for future breeding efforts because they have the highest total contents of

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glucosinolates and carotenoids.

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Our research included the first measurements of policosanols in Chinese cabbage varieties

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(Table 5). C26 (hexacosanol, 147.12–337.27 µmol/kg DW) was the predominant policosanol

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in all varieties. Kim et al. (2015)24 also reported that C26 was the most abundant policosanol

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in Brassicaceae species, including kale, mustard, and tatsoi. In wheat, policosanol

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composition varied considerably among 34 varieties.28 Although a recent study failed to

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observe a cholesterol-lowering effect from a policosanol supplement,29 these compounds,

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which are a mixture of long chain alcohols extracted from plant waxes, are important for

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agronomic reasons because of their significance in water loss and mechanical damage. As

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shown in this study, Cheonsangcheonha and Waldongcheonha had high policosanol contents.

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Phytosterol production may be influenced by environmental conditions as well as genotype.

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In this study, the predominant phytosterols were campesterol (1.33–2.10 µmol/g DW) and β-

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sitosterol (2.30–3.35 µmol/g DW), and α-tocopherol (22.27–48.16 µmol/kg DW) was the

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predominant tocopherol in all varieties; these results agree with those of Kim et al. (2015).25

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Amaral et al. (2006)30 reported that in hazelnuts, genotype does not significantly influence

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tocopherol and tocotrienol composition. No significant differences in total tocols were

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observed among Chinese cabbage varieties analyzed in this paper. However, environmental

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growth factors such as water loss, air temperature, and photoperiod can influence carotenoid,

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glucosinolate, policosanol, and phytosterol accumulation within Chinese cabbage.

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Correlations between 38 Metabolites in Chinese Cabbage Leaves.

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A correlation analysis can establish relationships between metabolite signals in a biological

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system.31 To determine detailed relationships between the 38 studied compounds in Chinese

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cabbage, we performed Pearson’s correlation analyses on the accessions. The resulting matrix

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is called the metabolite-to-metabolite correlation matrix (Figure 2). An HCA with the

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correlation coefficients provided two major metabolite clusters (boxed within dotted lines).

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One group contained all pigments, glucosinolates except for glucoerucin, and policosanols

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except for heneicosanol. The other group contained most of the plant sterols. The Pearson

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correlation analysis provided correlations between metabolites that participate in common or

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closely related pathways. For example, chlorophyll is associated with isoprenoid biosynthesis.

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Chlorophyll concentrations were positively correlated with β-carotene (r = 0.7277, P

Metabolic Profiling in Chinese Cabbage (Brassica rapa L. subsp

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