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Acta Chromatographica 20(2008)3, 489–496 DOI: 10.1556/AChrom.20.2008.3.14

Isolation of Glucosinolates from Broccoli Seeds by Use of High-Speed Countercurrent Chromatography

Q. DU*, L. ZHANG, H. CUI, K. WANG, L. XIU AND L. SHENG

Institute of Food and Biological Engineering, Zhejiang Gongshang University, Hangzhou, 310035, China E-mail: qizhendu@163.com

Summary. Cleavage of glucosinolates with myrosinase yields thioglycosidic compounds which have cancer chemoprevention activity. In this paper, glucosinolates in an extract (2.0 g) of broccoli seeds (Brassica oleracea var. italica) were separated by high-speed coun- tercurrent chromatography (HSCCC) with the solvent system n-butanol–acetonitrile– 10% ammonium sulfate solution 1:0.5:2.2 (v/v) to yield five glucosinolate compounds af- ter desalting and decolorizing by MCI column chromatography. The five compounds, 7-methylsulfinylheptyl glucosinolate (22.4 mg), 4-pentenyl glucosinolate (33.6 mg), 3-butenyl glucosinolate (24.0 mg), 4-methylsulfinylbutyl glucosinolate (161.4 mg), and 3-methylsulfinylpropyl glucosinolate (29.6 mg), were identified by ESI-MS, 1 H NMR and

13 C NMR. The purity of the products was >98%, and 7-methylsulfinylheptyl glucosi- nolate was obtained from broccoli seeds for the first time.

Key Words: broccoli seeds, glucosinolates, isolation, high-speed countercurrent chroma- tography

Introduction

Accurate analysis of the levels of glucosinolates in different vegetables is important because of the growing awareness of scientists and consumers of glucosinolates, which play a role in cancer prevention. Lack of pure glu- cosinolates currently limits quantitative analysis worldwide. Broccoli is a typical Brassica plant rich in glucosinolates [1–4] and is an excellent material for preparing several glucosinolate monomers. Flash chromatography and preparative HPLC have been used to separate the glucosinolates in an ex- tract of broccoli seeds [5, 6]. This yielded mainly pure 4-methylsulfinylbutyl

0231–2522 © 2008 Akadémiai Kiadó, Budapest

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glucosinolate (glucoraphanin) and was unsuitable for simultaneous isola- tion of other glucosinolate monomers. High-speed countercurrent chromatography (HSCCC) is an all-liquid chromatographic system, working without solid support, in which separa- tion is based on partitioning of the analytes between two immiscible liquid phases [7]. Irreversible adsorption and artifact formation is thus minimized. Application of HSCCC in natural product chemistry is steadily increasing, because of this advantage and the excellent recoveries achieved [8]. This paper reports a rapid method for isolation and purification of the five glu- cosinolate monomers 7-methylsulfinylheptyl glucosinolate, 4-methylsul- finylbutyl glucosinolate, 3-methylsulfinylpropyl glucosinolate, 4-pentenyl glucosinolate, and 3-butenyl glucosinolate (Fig. 1) from broccoli seeds by use of HSCCC.

( Fig. 1 ) from broccoli seeds by use of HSCCC. Fig. 1. Structures of the

Fig. 1. Structures of the glucosinolate compounds isolated from broccoli seeds:

1, 3-butenyl glucosinolate; 2, 4-pentenyl glucosinolate; 3, 7-methylsulfinylheptyl glucosinolate; 4, 4-methylsulfinylbutyl glucosinolate; 5, 3-methylsulfinylpropyl glucosinolate

HSCCC Isolation of Glucosinolates from Broccoli Seeds

Experimental

Materials

491

Broccoli seeds (Brassica oleracea var. italica; 1 kg) were provided by Hang- zhou Seeds Company (Hangzhou, China). All solvents for extraction and separation (analytical grade) were purchased from Hangzhou Huadong Chemicals, China. Tetramethyl ammonium bromide (TMAB) was obtained from Sigma Chemical Company, Shanghai, China. MCI gel (Mitsubishi Chemical, Japan) was purchased from Greenherbs Science and Technology, Beijing, China.

Extraction and Pre-Separation

Broccoli seeds (1 kg) were marinated in 3 L methanol for 2 h at 70°C to inac- tivate endogenous myrosinase and to prepare the sample for extraction of the glucosinolates. After filtration of the extract solution, the broccoli resi- dues were pulverized and extracted again with 5 L methanol for 2 h at 70°C. The two extracts were combined and evaporated to dryness, and the residue was defatted with 1 L of diethyl ether. The residue was then lyophi- lized to yield 137 g crude extract which was subjected to HSCCC isolation of the glucosinolates.

Apparatus

The high-speed countercurrent chromatograph used in this study was con- structed at the Institute of Food and Biological Engineering, Zhejiang Gong- shang University, Hangzhou, China. The apparatus was equipped with a 1200-mL column with six layers of coils made from 5.0 mm i.d. polytetra- fluoroethylene (PTFE) tubing. A K-1800 Wellchrom preparative HPLC pump (Knauer, Germany), a 100-mL sample loop made of 3 mm i.d. PTFE tubing, a 210 UV-detector (Elite, Dalian, China), and a B-684 collector (Büchi, Switzerland) with 15-mL tube racks were used to establish the isola- tion system.

High-Speed Countercurrent Chromatographic Isolation Procedure

HSCCC was performed with a two-phase solvent system comprising n- butanol–acetonitrile–10% ammonium sulfate solution 1:0.5:2.2 (v/v), which was selected after experimental partition of the crude extract in a series of

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solvent systems prepared from n-butanol, acetonitrile, 10% ammonium sul- fate solution in different volume ratios. After thorough equilibration of the solvents in a separatory funnel, the two resulting phases were separated shortly before use. The multilayer coil column was completely filled with the aqueous lower phase as the stationary phase. The apparatus was then rotated at 700 rpm. The sample solution was prepared by dissolving 2.0 g crude extract in 50 mL organic mobile phase. Injection into the HSCCC sys- tem was achieved by use of the PTFE sample loop, followed by of the pumping mobile phase at a flow rate of 3.0 mL min 1 . The HSCCC elution mode of mobile phase was head to tail. The effluent was monitored by UV detection at 233 nm and collected by use of the fraction collector, 15 mL for each fraction. When the separation was complete the solvents in the column were pushed out and the retention of the stationary phase was measured to be 55%.

TLC Analysis

To estimate their purity the compounds from HSCCC separation were ana- lyzed by TLC on Merck aluminum foil plates coated with silica gel GF 254 ; the mobile phase was chloroform–methanol–water 15:15:1. Spots were de- tected by treatment with a 10% solution of sulfuric acid in ethanol and heat- ing at 110°C.

Desalting and Decolorizing by MCI Column Chromatography

The compounds from HSCCC separation were dissolved in water and the solutions were subjected to MCI column chromatography, and eluted with pure water and 50% methanol. The solutions in 50% methanol were evapo- rated under vacuum to yield pure glucosinolate compounds.

HPLC Analysis

HPLC was performed with Waters (Milford, MA, USA) equipment – a 2695 pump, a 717 autosampler, and a 996 diode-array detector. Compounds were separated on a 300 mm × 3.9 mm i.d., 10-μm particle, C 18 Bondapak column (Waters) fitted with a C 18 guard column. The mobile phase was 0.005 M TMAB dissolved in 2% (v/v) methanol–water. The flow rate was 1.5 mL min 1 and the separations were monitored at 233 nm. Waters Mil- lennium HPLC 2010 software was used to process chromatographic data.

HSCCC Isolation of Glucosinolates from Broccoli Seeds

ESI-MS and NMR

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All ESI-MS experiments were performed with a Bruker (Bremen, Germany) Esquire LC–MS ion-trap multiple mass spectrometer in negative-ionization mode, with analysis of ions up to m/z 2200. 1 H and 13 C NMR spectra were recorded in CD 3 OD with a Bruker (Karlsruhe, Germany) Avance 500, at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. ESI-MS analysis of com- pounds 1, 2, 3, 4, and 5 from HSCCC separation gave pseudomolecular ions

m/z (neg.) a: 371, 385, 477, 435, and 421, respectively. 1 H and 13 C NMR data

are listed in Table I.

Table I. Chemical shifts (δ, ppm) from 13 C and 1 H NMR of glucosinolates 15

Carbon

Compound 1

Compound 2

Compound

Compound

Compound

 

3

 

4

 

5

no.

13

C

1

H

13

C

1

H

13

C

1

H

13

C

1

H

13

C

1

H

1 62.5

 

3.74,

62.1

3.63,

62.8

 

3.69

62.6

 

3.63

61.2

 

3.66

3.76

4.12

 

3.90

 

3.87

 

3.69

2 79.5

 

3.56

82.5

3.16

79.5

 

3.62

82.1

 

3.56

81.3

 

3.38

3 71.1

 

3.54

71.0

3.09

71.3

 

3.50

71.2

 

3.47

70.1

 

3.20

4 82.1

 

3.57

79.4

3.18

74.4

 

3.61

79.3

 

3.56

78.3

 

3.34

5 74.1

 

3.54

74.1

3.16

73.1

 

3.52

74.1

 

3.47

73.1

 

3.18

6 83.6

 

5.04

83.3

5.01

82.4

 

4.83

83.4

 

5.09

82.8

 

5.10

7 161.6

   

156.5

 

163.6

   

162.5

   

161.6

   

8 33.0

 

2.65

35.8

3.04

35.8

 

2.84

32.9

 

2.80

31.1

 

2.83

9 32.9

 

2.41

32.3

2.32

23.2

 

2.67

22.8

 

1.89

20.7

 

2.49

10

138.2

5.85

32.4

2.65

32.6

 

2.28

26.9

 

2.21

52.9

 

3.00

 

116.0

5.05,

138.8

5.88

27.3

 

1.79

   

2.93

38.1

 

2.72

11

4.93

54.1

12

   

116.5

5.12,

25.7

 

1.85

38.4

 

2.68

   

5.08

13

       

23.0

 

2.81

       

14

       

54.4

 

2.86

       

15

       

38.1

 

2.68

       

Results and Discussion

With the solvent system n-butanol–acetonitrile–10% ammonium sulfate so- lution 1:0.5:2.2 (v/v) separation of 2 g crude extract was performed at a flow rate of 3 mL min 1 . The chromatogram (Fig. 2) contained seven peaks (A–G). The fractions corresponding to peaks A and B furnished more than two spots in TLC analysis whereas the fractions corresponding to peaks C–G gave one spot. The fractions corresponding to peaks C (886–1050 mL),

D (1231–1575 mL), E (1726–1980 mL), F (2101–2460 mL), and G (2671–

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3210 mL) were separately combined and evaporated to dryness at 70°C un- der vacuum. The residues were extracted with methanol (2 × 5 mL) The methanol solutions were combined and evaporated to yield 46.1 mg com- ponent I, 28.3 mg component II, 26.7 mg component III, 246.1 mg compo- nent IV, and 56.1 mg component V. Components I–V were desalted and de- colorized by MCI column chromatography to yield 33.6, 24.0, 22.4, 161.4,

and 29.6 mg of compounds 15, respectively. The purities of compounds 1

5 were >98% (by HPLC; Fig. 3). Results from ESI-MS, 1 H NMR (500 Hz),

and 13 C NMR (125 Hz) analysis of compounds 1, 2, 4, and 5 were consistent with 4-pentenyl glucosinolate (glucobrassicanapin), 3-butenyl glucosinolate (gluconapin), 4-methylsulfinylbutyl glucosinolate (glucoraphanin), and 3- methylsulfinylpropyl glucosinolate (glucoiberin) , respectively [9–11]. By comparison of the 1 H NMR and 13 C NMR spectra of compound 3 with those

of 4-methylsulfinylbutyl glucosinolate (4) (Table I), compound 3 was identi- fied as 7-methylsulfinylheptyl glucosinolate (glucoibarin), which was ob- tained from broccoli seeds for the first time. ESI-MS of compound 3 in nega- tive-ion mode gave a pseudomolecular ion at m/z 477 [M ], confirming that

3 was glucoibarin.

m / z 477 [M − ], confirming that 3 was glucoibarin. Fig. 2. HSCCC chromatogram

Fig. 2. HSCCC chromatogram obtained from 2 g crude extract of broccoli seeds. Solvent system: n-butanol–acetonitrile–10% ammonium sulfate solution, 1:0.5:2.2 (v/v); mobile phase, upper phase; flow rate, 3 mL min 1 ; the sample was dissolved in 50 mL upper phase; retention of the stationary phase was 55%. Peak elution was monitored at 233 nm by use of a variable-wavelength detector

HSCCC Isolation of Glucosinolates from Broccoli Seeds

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HSCCC Isolation of Glucosinolates from Broccoli Seeds 4 9 5 Fig. 3. HPLC analysis (233 nm)

Fig. 3. HPLC analysis (233 nm) of the crude extract of the broccoli seeds and the products from HSCCC: (a) crude extract; (b) 3-methylsulfinylpropyl glucosinolate; (c) 4-methylsulfinylbutyl glucosinolate; (d) 3-butenyl glucosinolate; (e) 4-pentenyl glucosinolate; (f) 7-methylsulfinylheptyl glucosinolate

An early method of isolation of glucosinolates from broccoli seeds was flash chromatography which yielded impure components except for 4- methylsulfinylbutyl glucosinolate, the most abundant glucosinolate in broc- coli seeds [5]. Recently, solid phase extraction (SPE) and preparative HPLC have been used for the isolation of glucoraphanin from broccoli seeds. This method was tedious and inefficient, however, because one preparative HPLC injection yielded only 17.6 mg 4-methylsulfinylbutyl glucosinolate from 98 mg semi-purified crude sample from SPE [6]. Our HSCCC method

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enables loading of 2 g crude sample and yields five glucosinolates. This has the advantages of simultaneous recovery of several glucosinolate mono- mers and high sample loading.

Conclusion

HSCCC enabled excellent separation of the glucosinolates of broccoli seeds, yielding five pure glucosinolate monomers – 7-methylsulfinylheptyl glu- cosinolate, 4-methylsulfinylbutyl glucosinolate, 3-methylsulfinylpropyl glu- cosinolate, 4-pentenyl glucosinolate, and 3-butenyl glucosinolate after de- salting and decolorizing by MCI column chromatography. This method can be used for rapid isolation of glucosinolate monomers from broccoli seeds, and is possibly suitable for preparation of the glucosinolates from other plant materials.

Acknowledgments

The authors thank the Department of Zhejiang Science and Technology for financial support (2005C12037 and 2004C12005).

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