Analiza glukozynolanów i produktów ich rozpadu.

Jean-Paul Wathelet

Agricultural University, Gembloux, Belgium

Analysis of glucosinolates

and their breakdown compounds

Analiza glukozynolanów i produktów ich rozpadu

Key words: glucose released method, HPLC, desulfoglucosinolates, ISO Słowa kluczowe: oznaczanie uwolnionej glukozy, HPLC, desulfoglukozynolany, ISO

Glukozynolany stanowią klasę ok. 120 drugorzędnych metabolitów roślinnych występujących generalnie w Brassicaceae. Związki te mają podobną strukturę charakteryzującą się wspólną dla wszystkich częścią zawierającą beta-tioglukozę związaną z grupą sulfonowaną oksymowi oraz z róż-nymi podstawnikami organiczróż-nymi zawierającymi grupy alkenylowe, alkilowe, hydroksyl alkenylo-we, aryloalkenylo-we, indolyloalkenylo-we, sulfinyloalkenylo-we, sulfonylowe oraz tio pochodne. Obecne w wysokim lub niskim stężeniu (100 odmian) w nasionach rzepaku/rzepiku i ich śrutach molekuły te łatwo się rozpadają podczas trawienia w organizmach zwierzęcych powodując powstanie produktów o charakterystyce antyżywieniowej i szkodliwych. Znane jest ich oddziaływanie na tarczycę i zdolność uszkadzania innych organów (Fenwick et al. 1983).

Znane są różne metody oznaczania pojedynczych glukozynolanów oraz ich sumy. Ważne jest poznanie korzystnych i niekorzystnych ograniczeń tych metod. Tanią i szybką metodą oznaczania całkowitej zawartości glukozynolanów jest oznaczanie uwalnianej glukozy bez etapu oczyszczania. Nowoczesną metodą równoczesnego oznaczania glukozynolanów, białka, tłuszczu i wilgotności jest NIRS. Metodą referencyjną (ISO 9167-1) do ilościowego i jakościowego oznaczania pojedynczych glukozynolanów w nasionach rzepaku jest HPLC desulfoglukozynolanów. Szereg laboratoriów na całym świecie stosuje tę metodę szczególnie do kontroli jakości podwójnie ulepszonych odmian rzepaku (00).

Introduction

Glucosinolates are a class of ca. 120 secondary plant metabolites generally found in Brassicaceae. These compounds have a similar structure characterised by a common part containing a beta thioglucose coupled with a sulfonated oxim group and by a variable organic side chain constituted with alkyl, alcenyl, hydroxy-alcenyl, aryl, indolyl, sulfinyl, sulfonyl or thio residues. Present in high or low level (00 varieties) in rapeseed/canola seeds and in rapeseed/canola meals, these molecules are easily broken down during animal digestion, leading to compounds with detrimental and antinutritional characteristics. They are known to interfere with the thyroid and to damage vital organs (Fenwick et al. 1983).

Enzymatic hydrolysis of glucosinolates

Myrosinase

Glucosinolates (GLs) are easily hydrolysed into isothiocyanates (ITC), oxazo-lidinethiones (OT), cyanides, D-glucose, sulphate ion by an endogenous enzyme (glycoprotein) called myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1).

Sulfatase and

β-O-glucosidase

Glucosinolates are also substrates of a sulfatase (EC 3.1.6.1) extracted from the snail Helix pomatia, which brings about easily their transformation into desulfo-glucosinolates (DS-glucosinolates) (Thies 1979, Leoni et al. 1998).

Desulfo-glucosinolates formed could also be hydrolysed with the recombinant

β-O-glucosidase from Caldocellum saccharolyticum into pure nitriles (Wathelet et al. 2001). HSO4 -2 S R'' R' C O C N-H CH Oxazolidine-2-thiones Glucosinolate Thiohydroxamate-O-sulphonate MYROSINASE H2O HO HO O OH OH S C R N -_O 3SO D-Glucose + Cyano-epithioalkanes R-N=C=S Isothiocyanates Nitriles S CH2-CH-CH-(CH2)n -R' +S R-S-C N R-C N C N -_O 3SO N C S H R Thiocyanates S-C N

Helix pomatia Sulfatase Sulfate R C Glucosinolate N O3SO -OH HO S HO O R C N HO OH HO S HO O OH Transgenic β-O-Glucosidase (70° C) D-Glucose Pure Nitriles: R-C N +S

Fig. 2. Hydrolysis of glucosinolates with sulfatase and desulfo-glucosinolates with a β-O-glucosidase

Hydroliza glukozynolanów za pomocą sulfatazy i desulfoglukozynolanów za pomocą β-O-glukcozydazy Table 1 Main glucosinolates in Brassica and Sinapis oilseeds

Podstawowe glukozynolany w nasionach Brassica i Sinapis Aliphatic GLs — Glukozynolany alifatyczne

progoitrin (PRO) (2R)-2-hydroxybut-3-enyl GLs

epiprogoitrin (ePRO) (2S)-2-hydroxybut-3-enyl GLs

sinigrin (SIN) prop-2-enyl GLs or allyl GLs

napoleiferin (GNL) (2R)-2-hydroxypent-4-enyl GLs

gluconapin (GNA) but-3-enyl GLs

glucobrassicanapin (GBN) pent-4-enyl GLs

Aromatic GLs — Glukozynolany aromatyczne

sinalbin (SNB) 4-hydroxybenzyl GLs

gluconasturtin (GST) phenethyl GLs

Indolyl GLs — Glukozynolany indolowe

4-OH glucobrassicin (4-OH GBS) 4-hydroxyindol-3-ylmethyl GLS

glucobrassicin (GBS) indol-3-ylmethyl GLs

4-methoxyglucobrassicin (4-Me GBS) 4-methoxyindol-3-ylmethyl GLs neoglucobrassicin (Neo GBS) N-methoxyindol-3-ylmethyl GLs

Table 2 Methods for determination of the total glucosinolate content

Metody oznaczania całkowitej zawartości glukozynolanów Method

Metoda Main advantages Główne zalety Main disadvantages Główne wady

Palladium fast method repeatability

interferences

Sulfate fast method interferences with free sulfate

Glucose without a purification step fast method interferences with free glucose Glucose with a purification step no interference

cheap material

NIRS fast indirect method calibration

XRF fast indirect method interferences

ELISA fast method to be more experimented

The glucose method with a purification step to avoid free glucose seems to be a good method. NIRS needs some time for calibration, however, this method could give, in a short time, both the glucosinolate, protein, lipid contents and moisture.

Table 3 Methods for determination of the individual glucosinolate contents

Metody oznaczania zawartości poszczególnych glukozynolanów Method Metoda Main advantages Główne zalety Main disadvantages Główne wady

GC of ITC and OT partial results (indolyl ?)

GC of DS-GLs repeatability drying and silylation steps problems with sulphur GLs HPLC of intact GLs each GLs quantify

HPLC of DS-GLs each GLs quantify

retention times stable excepted cinnamoyl derivatives selectivity sulfatase step

repeatability no problem in routine

Capillary electrophoresis high selectivity to be more experimented

ELISA fast method to be more experimented

HPLC of desulfated glucosinolates is at present the reference method (ISO 9167-1) for qualitative and quantitative analysis of individual glucosinolates in rapeseed/canola seeds. Several laboratories all over the world use now this method, especially in order to control the 00 varieties.

ISO 9167-1 method: results and comments

Principle of the ISO method

The glucosinolates are extracted from grinded seeds with hot methanol/water (70/30), desulfated with Helix pomatia sulfatase (H1, EC 3.1.6.1), purified on a DEAE A-25 resin and separated by HPLC using a C8 or C18 stationary phase

column with a water/acetonitrile gradient.

Sampling

In the ISO method it is first recommended to grind the seeds (moisture < 10%) with a microgrinder and to weight 200 mg of ground seeds. The variability of the total glucosinolate content according to the sample amount has been evaluated in the laboratory. A minimum of 200 mg of sample amount is recommended (Fig. 3).

0 1 2 3 4 5 6 0,05 0,1 0,2 0,3 0,4 0,5 Sample amount (g) Variability (% )

Fig. 3. Variability of the glucosinolate level according to the sample amount — Zmienność poziomu

glukozynolanów w odniesieniu do wielkości próbki

Extraction

A single extraction (against 2 in the ISO method) is possible in a 10 ml test tube. 200 mg of ground seeds are stirred with 10 ml of 75°C methanol/water (70/30) with an internal standard. The mixture is permanently agitated with a magnetic stirrer for 10 min (Figure 4) and then centrifuged. Using this system it is possible to extract quickly a lot of samples.

Sinigrin or glucotropaeolin can be used as internal standard but it is necessary to pay attention to their natural absence in the sample.

0 200 400 600 800 1000 1200 0 2 4 6 8 10 12 14 Extraction time(min) Sig n al ( arbit ra ry unit s) Progoitrin_{Epiprogoitrin} Sinigrin * Napoleiferin Gluconapin 4-OH glucobrassicin Glucobrassicin Gluconasturtiin 4-MeOH glucobrassicin

Fig. 4. Kinetic of the glucosinolate extraction — Kinetyka ekstrakcji glukozynolanów

Desulfatation and purification

One ml of the crude extract are directly put on a DEAE A-25 resin (25 mg) prepared according to the ISO method. Glucosinolates are desulfated by addition of 100 µl of Helix pomatia sulfatase (H1, EC 3.1.6.1). The sulfatase is quickly prepared in the laboratory by fractionated ethanol precipitation (75 mg in 40% ethanol, centrifugation, recover the upper phase and precipitation of sulfatase with 70% ethanol, take up the precipitate in 5 ml of distilled water and dilution 10 before use). The kinetic of desulfatation with diluted sulfatase (activity at pH 5.8 and 30°C: 0,05 µ/ml; one activity unit corresponds to the desulfatation of 1 µmol of sinigrin per min) is represented in Figure 5.

A minimum of 11 hours is necessary for complete desulfatation in these operating conditions. If necessary, the desulfatation time can be reduced to 2 hours with a ten times concentrated sulfatase solution (activity: 0,5 µ/ml) with no problems with any of glucosinolates (alcenyl, benzyl, indolyl, methylthio, methylsulfinyl, methylsulfonyl...).

Elution of the DS-glucosinolates is realised with 4 × 0,5 ml distilled water (Figure 6). The desulfo-4-hydroxyglucobrassicin is eluted later than the other glucosinolates.

0 200 400 600 800 1000 1200 1400 1600 -1 1 3 5 7 9 11 13 15 Time (h)

Signal (arbitrary units)

DS-progoitrin DS-epiprogoitrin DS-sinigrin * DS-napoleiferin DS-gluconapin DS-4-OH glucobrassicin DS-gluconasturtiin DS-4-MeOH glucobrassicin DS-glucobrassicin

Fig. 5. Kinetic of the glucosinolates desulfatation (sulfatase activity: 0,05 µ/ml) — Kinetyka

desulfa-tacji glukozynolanów (aktywność sulfatazy: 0,05 µ/ml)

0 500 1000 1500 2000 2500 3000 3500 4000 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 Elution volume (µl) Si gnal (arbi trary uni ts) DS-progoitrin DS-epiprogoitrin DS-sinigrin * DS-napoleiferin DS-gluconapin DS-4-OH glucobrassicin DS-gluconasturtiin DS-glucobrassicin DS-4-MeOH glucobrassicin

HPLC analysis

Desulfo-glucosinolates are separated in the laboratory by HPLC using an Inertsil 3 ODS-3 column (100 × 3 mm, 3 µm) with a water/acetonitrile gradient (from 2 to 25% in 35 min).

Resolution is high (Figure 7) and the limited flow (0.4 ml/min) reduces simultaneously the consumption and the elimination of solvent.

In our chromatographic conditions, the retention time and response factors of 24 DS-glucosinolates have been determined. The response factor for sinalbin is equal to 0.5 while the ISO method recommend 1.

0

50

100

150

200

250

300

350

400

0

10

20

30

40

Fig. 7. HPLC chromatogram of rapeseed desulfo-glucosinolates — Chromatogram HPLC

desulfoglu-kozynolanów rzepakowych

Identification of glucosinolates

Identification of glucosinolates could be realised by comparison of retention times. Different reference materials are available: pure glucosinolates/desulfo-glucosinolates (Visentin et al. 1992, Barillari et al. 2001, Iori et al. 2003) or rape seeds with a certified content in total glucosinolates (BCR-366R: 11,9 ± 1,3 µmol/g, BCR-190R: 23,0 ± 4 µmol/g, BCR-367R: 99,0 ± 9 µmol/g) and indicative values for all the individual glucosinolates (IRMM, U.E. http://www.irmm.jrc.be/mrm.html) (Linsinger et al. 2001).

Comparison of UV spectra of desulfo-glucosinolates could also be useful for identification, especially if the HPLC is connected to a diode array detector. GC-MS, LC-MS, NMR are helpful if necessary.

Hydrolyse of intact glucosinolates with a commercial myrosinase induces the formation of isothiocyanates and nitriles. Enzymatic transformation of DS-GLs with a recombinant β-O-glucosidase gives pure nitriles without isothiocyanates. Analysis of these breakdown compounds by GC-MS is another way for indirect identification of GLs.

Analysis of glucosinolates breakdown compounds

Isothiocyanates, nitriles

After myrosinase hydrolysis, isothiocyanates could be easily extracted with diethylether and injected in the GC or the GC-MS for identification. GC column could be, for example, a CP WAX 52 CB (50 m × 0.25 mm, 0.2 µm dp) and an oven temperature gradient from 35°C to 265°C (Figure 8).

0 5 0 . 0 0 0 1 0 0 . 0 0 0 1 5 0 . 0 0 0 2 0 0 . 0 0 0 2 5 0 . 0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 min Pent-4-ene nitrile But-3-enyl ITC Pent-4-enyl ITC 4,5-epithiopentane nitrile 3-hydroxypent-4-ene nitrile Phenylethyl ITC Abundance

Fig. 8. Chromatogram of isothiocyanates and nitriles — Chromatogram izotiocjanianów i nitryli

5-vinyl-1,3-oxazolidine-2-thione (5-VOT)

The 5-vinyl-1,3-oxazolidine-2-thione, one breakdown compound produced by progoitrin hydrolysis with myrosinase could be measured by HPLC (column: Lichrospher C18 125 × 4 mm, 5 µm; solvent: water/acetonitrile 90/10 v/v;

wave-lenght: 240 nm). The 4,4 dimethyl-1,3-oxazolidine-2-thione could be used as internal standard.

When it is necessary to purify the 5-VOT solution before HPLC injection, a purification by complexation with phenyl mercury acetate followed by a decomplexation with sodium thiosulfate is needed (Quinsac et al. 1992).

Conclusions

Different kinds of methods are available for determination of the total or the individual content of glucosinolates. It is important to know well the limits, the advantages or disadvantages of each method. The glucose released method with a purification step seems to be a cheap and fast method for the determination of the total glucosinolate content. NIRS is also a good method for the simultaneous determination of glucosinolates, proteins, lipids, moisture contents.

HPLC of desulfated glucosinolates is, at present, the reference method (ISO 9167-1) for qualitative and quantitative analysis of individual glucosinolates in rapeseed/canola seeds. Several laboratories, all over the world use this method, especially in order to control the 00 varieties.

References

Fenwick R.G., Heaney R.K., Mullin W. 1983. Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr., 18: 123-201.

Iori R., Barillari J., Galletti S., Venturi G., Marotti M., Rollin P. 2003. Production of glucobrassicin, a phytochemical of major interest, through jasmonic acid treatment of woad (Isatis tinctoria L.) leaves. Agroindustria, 2, (2/3): 69-72.

ISO 9167-1:1992. Rapeseed-Determination of glucosinolates content. Part 1: Method using gradient elution high-performance liquid chromatography.

Leoni O., Iori R., Haddoum T., Marlier M., Wathelet J-P., Rollin P., Palmieri S. 1998. Approach to the use of immobilised sulfatase for analytical purposes and for the production of desulfo-glucosinolates. Ind. Crops Prod., 7: 335-343.

Linsinger T., Kristiansen N., Beloufa N., Schimmel H., Pauwels J. 2001. The certification of the total glucosinolate and sulphur contents of three rapeseed (colza) materials. European Commission, Joint Research Center, Institute for Reference Materials and Measurements, Geel, Belgium, EUR 19764 EN, 1-27.

Quinsac A., Ribaillier D., Rollin P., Dreux M. 1992. Analysis of 5-vinyl-1,3-oxazolidine-2-thione by Liquid Chromatography. J. Assoc. Off. Anal. Chem., 75 (3): 529-536.

Thies W. 1979. Detection and utilisation of a glucosinolate sulfohydrolase in the edible snail Helix pomatia. Naturwiss., 66: 364-365.

Visentin M., Tava A., Iori R., Palmieri S. 1992. Isolation and identification of trans-4-(methylthio)-3-butenyl glucosinolate from radish root (Raphanus sativus). J. Agric. Food Chem., 40: 1687-1691. Wathelet J-P., Iori R., Leoni O., Rollin P., Mabon N., Marlier M., Palmieri S. 2001. A recombinant beta-O-glucosidase from Caldocellum saccharolyticum to hydrolyse desulfo-glucosinolates. Biotechnology Letters, 23: 443-446.