Food Chemistry 113 (2009) 96–102

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Food Chemistry

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Glucosinolate degradation products and other bound and free volatilesin the leaves and roots of radish (Raphanus sativus L.)

I. Blazevic *, J. MastelicDepartment of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, N. Tesle 10/V, HR-21000 Split, Croatia

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 April 2008Received in revised form 5 May 2008Accepted 9 July 2008

Keywords:Raphanus sativus L.RadishBrassicaceaeGlucosinolatesO-glycosidesVolatile aglyconesIsothiocyanatesNitrilesGC–MS

0308-8146/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.foodchem.2008.07.029

* Corresponding author. Tel.: +385 21 329 434; faxE-mail address: ivica.blazevic@ktf-split.hr (I. Blaze

The volatile constituents from the leaves and roots of Raphanus sativus var. black, white and red were iso-lated by hydrodistillation in a Clevenger-type apparatus and were analysed by gas chromatography andgas chromatography–mass spectrometry. The major components found in the oil of all leaves were phytol(65.3–69.7%), hexadecanoic acid (2.5–14.3%), methyl linolenate (2.1–11.1%) and (Z)-3-hexen-1-ol (0.7–6.9%). Corresponding aliphatic or aromatic glucosinolate compounds identified in the leaves constitutedonly 0.3–5.7% of the isolated volatiles. They were 5-(methylthio)-4-pentenenitrile (0.3–2.2%), 4-(methyl-thio)butyl isothiocyanate (0.3–1.7%), 2-phenylethyl isothiocyanate (0.4%), 4-(methylthio)-3-butenyl iso-thiocyanate, 4-methylpentyl isothiocyanate, benzyl isothiocyanate and benzenepropanenitrile. Themajor components in the oil of all roots were hexadecanoic acid (30.3–49.9%), 4-(methylthio)butyl iso-thiocyanate (17.9–25.7%), methyl linolenate (8.5–21.7%), 5-(methylthio)-4-pentenenitrile (1.5–6.9%),dimethyl trisulfide (1.1–3.8%), 5-(methylthio)pentyl isothiocyanate (0.5–2.0%), 4-(methylthio)-3-butenylisothiocyanate (0.2–1.8%) and 2-phenylethyl isothiocyanate (0.1–1.5%).

O-Glycosidically-bound volatiles were isolated and hydrolysed by b-glucosidase from almonds. Nine-teen volatile aglycones were identified for the first time in the leaves of all varieties of radish. The mainaglycones were eugenol (29.2–37.1%), 2-phenylethanol (2.0–23.5%), 4-vinyl-2-methoxyphenol (2.4–12.4%), (Z)-3-hexen-1-ol (2.6–10.4%), methyl vanillate (6.3–10.0%), 1H-indole (0.7–6.8%), benzyl alcohol(1.4–4.2%), vanillin (3.5–6.3%) and methyl salicylate (0.8–3.4%). However, no volatile aglycones weredetected after isolation in different varieties of radish root.

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1. Introduction

The radish is a root vegetable of the Brassicaceae family, com-monly seen as a small-rooted, short-season vegetable, normally con-sumed in salads. The skin comes in a variety of colours. Mostcommonly known variety is the round, red-skinned variety. Othervarieties may have pink, white or grey–black skin. The most popularpart for eating is the napiform taproot, although the entire plant isedible and the tops can be used as a leaf vegetable. Roots are eatenraw or cooked or preserved by storage, pickling, canning or drying(Curtis, 2003). Radish is not only a vegetable crop but also an impor-tant source of medicinal compounds. Radish is used by people withdifferent gastrointestinal, biliary, hepatic, urinary and respiratorydisorders, and in cardiovascular diseases such as hypertension.

Consumption of cruciferous vegetables is more strongly associ-ated with cancer protection than vegetable consumption in general(Verhoeven, Verhagen, Goldbohm, Brandt, & Poppel, 1997). Cruci-fers contain many bioactive components; among the most-studiedbioactive compounds in crucifers associated with cancer protection

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: +385 21 329 461.vic).

are glucosinolates. Glucosinolates (GLS) are compounds that occurin many agronomically important crops (radish, cabbage, cauli-flower, broccoli, horseradish, mustard, turnip, oilseed rape) (Fahey,Zalcmann, & Talalay, 2001). The content of GLS in plants varies be-tween cultivars, plant individuals and parts of the plant, due to fac-tors such as genetics, environment and plant nutrients. GLS can befound in the roots, seeds, leaf and stem of the plant, while youngesttissues contain the highest amount. GLS have several functions inthe plant: plant defence against fungal diseases and pest infesta-tion, sulphur and nitrogen metabolism, and growth regulation.They may also play a role in plant defence against the effects ofstress from extreme temperatures in cold or warm climates (Ludv-ick-Müller, Krishna, & Forreiter, 2000) and may enhance plantseedling (Kelly, Bones, & Rossiter, 1998).

The odour and taste typical of Brassica vegetables are mainlydue to GLS and their breakdown products such as isothiocyanates,organic cyanides, oxazolidinethiones, and thiocyanate. Disruptingcellular structure of Brassica vegetables by cutting, chewing, cook-ing, fermenting or freezing, results in glucosinolate hydrolysis orthermal degradation and formation of several breakdown products(Oerlemans, Barrett, Suades, Verkerk, & Dekker, 2006; Rosa & Hea-ney, 1993; Rungapamestry, Duncan, Fuller, & Ratcliffe, 2007).

I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102 97

Cooking influences the glucosinolates and their hydrolysis prod-ucts. Water-soluble compounds leach into the cooking water (Rosa& Heaney, 1993) and myrosinase activity decreases due to thermalinactivation. In vitro and in vivo studies have reported that isothio-cyanates affect many steps of cancer development including mod-ulation of phase I and II detoxification enzymes, functioning as adirect or as an indirect antioxidant by phase II enzyme induction,modulating cell signalling, induction of apoptosis, control of thecell cycle and reduction of heliobacter infections (Barillari et al.,2005; Fahey et al., 2001; Hanlon, Webber, & Barnes, 2007; Takaya,Kondo, Furukava, & Niwa, 2003). The most characterised GSL com-pounds are sulforaphane, phenylethyl isothiocyanate, allyl isothio-cyanate and indole-3-carbinol, but many other isothiocyanatesthat are present in lower quantities also may contribute to theanti-carcinogenic properties of crucifers.

O-Glycosidically-bound volatile compounds (aliphatics, phe-nols, benzene derivatives, monoterpenes, norisoprenoids), havebeen reported in more than 170 plants belonging to 50 families(Crouzet & Chassagne, 1999). They are reported in Brassicaceaefamily in Iberis sempervirens L. (Mastelic, Blazevic, & Jerkovic,2006). These compounds have not been investigated in radish,which belongs to this important family. Usually, plant leaves, fol-lowed by flowers, stems, and roots show the highest variabilityof aglycons (Stahl-Biskup, Holthuizen, Stengele, & Schulz, 1993).O-Glycosidic bond cleavage occurs during enzymatic/chemicalhydrolysis or by pyrolysis (Matsumura, Takahashi, & Nishikitano,1997). The initial studies on the volatile precursors have mostlylooked at natural extraction, structure identification and enzymehydrolysis (Boulanger & Crouzet, 2001; Stahl-Biskup et al., 1993).

The objective of this work was on one hand to isolate and iden-tify the volatile compounds in leaves and root of different varietiesof radish and on the other hand to isolate and identify O-glycosid-ically-bound volatiles.

2. Materials and methods

2.1. Reagents

Solvents and b-glucosidase were purchased from Fluka Chemie,Buchs, Switzerland. Octyl- b-D-glucopyranoside, silica gel for col-umn chromatography (Kieselgel 60, 0.040–0.063 mm), precoatedsilica plates (Kieselgel 60, thickness 0.2 mm) for thin layer chroma-tography, ammonia, and sodium sulphate were obtained fromMerck, Darmstadt, Germany.

2.2. Plant material

Raphanus sativus (L.) var. black, white and red was cultivated inthe submediterranean region of south Croatia (near Sinj) inNovember 2006 and was used for analysis.

2.3. Isolation of volatiles

Volatiles were isolated from fresh leaves and roots (200 g) byhydrodistillation in a Clevenger-type apparatus for 3 h using 3 mlpentane/ether (1:1 v/v) for trapping. Hydrodistillation in a Cleveng-er-type apparatus consists of immersing the ground plant materialdirectly in a flask filled with 800 mL of water that is then broughtto the boil. Vapour carries volatile compounds and the condensatedrops on the pentane/ether trap in the inner tube of the apparatuswhere the volatiles are retained. After hydrodistillation, the pen-tane/ether extract was separated, condenser and inner tube werewashed with 5 ml of pentane two more times, extracts were com-bined and dried over anhydrous sodium sulphate. The extract wasconcentrated by careful fractional distillation to a small volume

(ca. 1 ml) and 1 ll of this solution was used for each GC and GC–MS analyses.

2.4. O-Glycosides isolation

One hundred grams of fresh plant material were immersed for20 min in boiling water (400 ml) to inactivate enzymes and simul-taneously to extract water-soluble compounds and partially re-move volatile compounds by evaporation. Thereafter, theaqueous extract was separated, and the residual plant materialwas ground and extracted one more time with boiling water(300 ml). The pooled aqueous extracts were concentrated to50 ml in a rotary evaporator under reduced pressure. Ballast com-ponents were removed by precipitation with ethanol (300 ml). Thefinal precipitation of acidic ballast compounds was performed inethanol solution (almost non-aqueous) with several drops of con-centration ammonia. Finally, the purification was performed byflash chromatography on a silica gel column applying ethyl acetate,ethanol: ammonia (6: 3: 1). TLC analysis showed the absence offree carbohydrates in the glycosidic fraction. The obtained glyco-sidic fraction was concentrated to dryness and dissolved in a cit-rate buffer (pH 5.5, 5 ml); octyl- b-D-glucoside (500 lg; internalstandard for glycosides) was added. Before adding enzyme, pen-tane–dichloromethane extraction was needed to remove remain-ing trace volatile compounds. The last extract was concentrated,and TLC and GC–MS analyses were performed to confirm that novolatiles were present in the extract-‘‘negative proof”.

2.5. O-Glycosides hydrolysis

b-Glucosidase from almonds (20 mg, 5–8 U/mg) was added to theglycosidic solutions along with 3 ml pentane for trapping the liber-ated aglycones. The hydrolysis was carried out for 72 h at 30 �C withthe mixture being shaken occasionally. After the hydrolysis, the pen-tane layer was separated and the remaining aglycones were ex-tracted from the aqueous layer with pentane (10 � 2 ml). Thecombined pentane extract were dried (Na2SO4) and concentratedto a final volume of 0.5 ml, and 1 ll was used for GC and GC–MSanalysis.

2.6. Gas chromatography (GC-FID)

Gas chromatography analysis was performed on a Hewlett-Pack-ard Model 5890 Series II gas chromatograph equipped with flameionisation detector and capillary column HP-101 (methyl siliconefluid; 25 m � 0.2 mm i.d., coating thickness 0.2 lm). Chromato-graphic conditions were as follows: helium as carrier gas at 1.0 ml/min; injector and detector temperatures, 250 �C and 300 �C. Oventemperature was isothermal at 70 �C for 2 min, then increased to200 �C, at a rate of 3 �C min�1 and then held at 200 �C for 15 min.The volume injected was 1 ll, with a split ratio of 1:50.

2.7. Gas chromatography–mass spectrometry analysis

Analysis was performed on a Hewlett–Packard GC (model 5890)with a mass selective detector (model 5971A; Hewlett Packard,Vienna, Austria) using two columns with different stationaryphases. GC operating conditions: column HP-20 M, (50 m � 0.2 mmi.d., film thickness 0.2 lm) temperature programmed from 70 �C for4 min, to 180 �C at a rate of 4 �C min�1; column HP-101, (25 m �0.2 mm i.d., film thickness 0.2 lm); temperature programmed from70 �C for 2 min, to 200 �C at a rate of 3 �C min�1; carrier gas: heliumat a flow rate of 1 ml min�1; injector temperature: 250 �C; volumeinjected: 1 ll; split ratio of 1:50. MS conditions: ionisation voltage:70 eV; ion source temperature: 280 �C; mass range: 35–300 massunits.

Table 1Content and composition of volatile compounds in the leaves and roots of different varieties of Raphanus sativus (L.) isolated from fresh plant materials by hydrodistillation

No. Compound name RI1 RI2 Radish (Raphanus sativus L.)

var. black var. white var. red

1 2 1 2 1 2(%) (%) (%) (%) (%) (%)

Alkanes1. undecanea 1100 1100 tr 0.1 0.2 0.3 0.2 tr2. dodecanea 1200 1200 0.3 – 1.1 1.1 0.8 1.43. tridecanea 1300 1300 0.1 0.4 0.3 0.5 0.3 1.24. tetradecanea 1400 1400 tr tr tr – 0.1 0.75. heneicosanea 2100 2100 – – – 2.5 – –6. tricosaneb,c >2200 >2200 0.2 – 0.3 2.3 0.5 –7. tetracosaneb,c >2200 >2200 0.1 – 0.2 1.2 0.2 –Group sum (%) 0.7 0.5 2.1 7.9 2.4 3.3

Aliphatic alcohols and carbonyls8. (E)-2-hexenala,b 1190 860 tr – tr – 0.8 –9. (Z)-3-hexene-1-ola,b 1351 869 0.7 tr 1.4 0.7 6.9 –10. nonanala,b 1365 1091 – – tr – 0.2 –11. 2-nonen-4-onea,b 1446 1111 – – – – – –12. (E,E)-2,4-heptadienalb,c 1453 – 0.1 0.1 – – – 0.113. benzaldehydea,b 1465 – tr – – – – –14. 2-phenylacetaldehydea,b 1588 1049 tr 0.1 0.1 tr 0.2 –15. 4-ethylbenzaldehydea,b 1658 - – – – – tr 0.116. 6,10,14-trimethyl- 2-pentadecanonea,b 2106 1837 – 0.1 – – – –17. isobutylphthalateb,c >2200 1845 – – 0.3 0.1 – 0.318. dibutylphthalatea,b >2200 1946 0.4 1.0 1.1 0.7 1.0 1.1Group sum (%) 1.1 1.3 2.9 1.4 9.1 1.6

Fatty acids and esters19. (Z)-3-hexenyl acetatea,b – 999 – tr – tr – tr20. dodecanoic acid (lauric acid)a,b >2200 1645 – tr – – – 0.521. benzyl benzoatea,b – 1724 – 0.1 – – – –22. tetradecanoic acid (myristic acid)a,b >2200 1826 tr 1.9 – 0.5 – –23. methyl palmitatea,b >2200 1909 0.1 – – – – –24. pentadecanoic acida,b >2200 1935 – 0.2 – – – –25. hexadecanoic acid (palmitic acid)a,b >2200 2059 14.3 32.2 2.5 30.3 8.5 49.926. methyl linolenatea,b >2200 2133 11.1 21.7 8.9 13.7 2.1 8.5Group sum (%) 25.4 59.1 11.4 44.5 10.6 58.9

Isothiocyanates and nitriles27. 4-methylpentyl isothiocyanatea,b,d 1492 1139 – 0.1 0.1 0.1 – –28. 5-(methylthio)-4-pentenenitriled,t,* 1869 1195 – 1.5 1.2 – – –29. 5-(methylthio)-4-pentenenitriled,t,* 1953 1250 tr 6.9 2.2 5.3 0.3 tr30. benzenepropanenitrilea,b,d 1968 1261 – tr 0.1 tr – –31. 3-(methylthio)propyl isothiocyanate (iberverin)a,b,c,d – 1305 – tr tr 0.5 – 0.732. benzyl isothiocyanatea,b,d 2021 1359 tr tr tr 0.5 – tr33. 4-(methylthio)-3-butenyl isothiocyanateb,c,d,* – 1408 – 0.2 tr 1.7 – 0.734. 4-(methylthio)butyl isothiocyanate (erucin)a,c,d 2041 1419 0.3 21.5 1.7 25.7 tr 17.935. 4-(methylthio)-3-butenylisothiocyanateb,c,d,* 2129 1433 – 0.9 tr 1.8 – 0.536. 2-phenylethyl isothiocyanatea,b,c,d 2142 1451 tr 1.0 0.4 1.5 – 0.137. 5-(methylthio)pentyl isothiocyanate (berteroin)a,c,d 2181 1524 – 0.5 – 2.0 – 0.6Group sum (%) 0.3 32.6 5.7 36.9 0.3 20.5

Other sulphur and/or nitrogen compounds38. dimethyl disulfidea,b 1067 – – 1.8 – tr – 1.339. dimethyl trisulfidea,b 1347 958 tr 1.3 0.4 1.1 0.2 3.840. 2-acetylthiazoleb – 1029 – tr tr – – –41. 1-(methylthio)-3-pentanoneb 1554 1073 – 0.1 tr 0.3 1.1 –42. dimethyl tetrasulphidea,b – 1193 – – – – – 0.5Group sum (%) tr 3.2 0.4 1.4 1.2 5.6

Terpenes43. b-caryophyllenea,b 1575 1377 – tr – – – tr44. b-cyclocitrala,b,c 1579 – – – tr – tr –45. b-damasconea,b 1768 1394 – – 0.1 – – –46. (E)-b-iononea,b 1887 1465 0.5 – 1.3 – 1.4 –47. neophytadienea,b,c 1912 1814 1.5 0.1 1.1 tr 1.3 –48. phytola,b,c – 2138 65.3 – 69.7 – 65.9 –Group sum (%) 67.3 0.1 72.2 tr 68.6 tr

Other compounds49. 4-vinyl-2-methoxyphenola,b 2100 1368 0.5 tr 1.3 – 1.0 tr50. 2,3-dihydrobenzofuran (coumaran)a,b – 1404 0.1 – tr – 0.4 –51. 1H-Indolea,b >2200 1426 0.1 – 1.8 – 0.2 –Group sum (%) 0.6 tr 3.1 – 1.6 tr

(continued on next page)

98 I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102

Table 1 (continued)

No. Compound name RI1 RI2 Radish (Raphanus sativus L.)

var. black var. white var. red

1 2 1 2 1 2(%) (%) (%) (%) (%) (%)

Total identified (%) 95.4 96.8 97.8 92.1 93.8 89.9Number of compounds identified (No.) 27 33 35 28 25 25Yield (mg/kg) 1877.9 2441.2 1538.5 1323.6 509.8 358.7

1-leaves; 2-roots; RI1-retention indices on HP-20M column; RI2-retention indices on HP-101 column;-not detected; tr-trace < 0.01 mg/kg; �both (E, Z)-isomers are identified,but not differentiated.

a Compound identified by mass spectra and RI comparison with homemade library.b Compound identified by mass spectra comparison with Wiley library.c Compound identified by mass spectra comparison with literature values (Adams, 1995; Vaughn & Berhow, 2005).d Obtained MS spectral data, m/z (relative intensity) are given in Table 2.e Tentatively identified compound.

I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102 99

2.8. Identification and quantitative determination of components

Individual peaks were identified by comparing their retentionindices (RI) and mass spectra (MS) with spectra in our homemadelibrary, as well as by computer matching against the Wiley 275-li-brary spectra database and comparison of the mass spectra with

Table 2Structure and MS spectral data of identified isothiocyanates and nitriles in different varie

Compound name Structure

4-Methylpentylisothiocyanate

NC

S

5-(Methylthio)-4-pentenenitrilea

CS

NBenzenepropanenitrile

C N3-(Methylthio)propyl

isothiocyanate(iberverin)

S NC

S

Benzyl isothiocyanate

N C S

4-(Methylthio)-3-butenylisothiocyanatea

SN

CS

4-(Methylthio)butylisothiocyanate (erucin) S

NC

S

2-Phenylethylisothiocyanate

NC

S

5-(Methylthio)pentylisothiocyanate(berteroin)

NC

S

a both (E, Z)-isomers are identified, but not differentiated.

literature data (Adams, 1995; Vaughn & Berhow, 2005). Spectraand RI values were compared with those of authentic compoundsobtained commercially and from the main components of manyessential oils using non-polar (HP-101) and/or polar (HP-20M) col-umns. Solvent was removed by careful fractional distillation andthe oil yields were determined by gravimetry. The yields of vola-

ties of Raphanus sativus L.

Obtained MS spectral data, m/z (relative intensity)

143 (M+, 18%), 128 (33%), 115 (10%), 110 (24%), 101 (12%), 100 (14%), 82 (12%),72 (68%), 69 (29%), 56 (36%), 43 (94%), 41 (100%)

127 (M+, 29%), 87 (100%), 71 (17%), 59 (14%), 53 (15%), 45 (66%)

131 (M+, 15%), 91 (100%), 77 (5%), 65 (12%), 63 (7%), 51 (10%), 40 (20%)

147 (M+, 8%), 101 (100%), 85 (16%), 72 (52%), 61 (38%), 41 (36%)

149 (M+, 17%), 121 (3%), 91 (100%), 77 (4%), 65 (19%), 63 (9%), 51 (8%), 44 (14%),40 (24%)

159 (M+, 18%), 87 (100%), 85 (16%), 72 (29%), 59 (14%), 53 (16%), 45 (51%)

161 (M+, 11%), 146 (5%), 115 (50%), 85 (30%), 72 (47%), 61 (100%)

163 (M+, 21%), 105 (8%), 91 (100%), 77(12%), 72 (8%), 65 (14%), 63 (6%), 51 (10%)

S

175 (M+, 19%), 160 (5%), 142 (9%), 129 (21%), 114 (7%), 101 (23%), 85 (8%), 72(53%), 69 (32%), 61 (100%), 55 (15%), 47 (16%), 45 (26%), 41 (42%)

100 I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102

tiles were expressed as mg/kg, relative to the masses of startingfresh plant material. The component percentages in Tables 1 and3 were calculated as the mean value of component percentageson column HP-20M and HP-101 of duplicate analyses.

3. Results and discussion

Hydrodistillation in a Clevenger-type apparatus insures isola-tion and concentration of free volatile compounds and volatileaglycones obtained by degradation of glucosinolates during isola-tion. O-Glycosidically-bound volatiles were water extracted andpurified by flash chromatography.

Volatile compounds, isolated from the leaves and roots of Raph-anus sativus var. black, white and red, as well as O-aglycones ob-tained by hydrolysis with b-glucosidase from O-glycosides, weresubjected to a detailed GC and GC–MS analyses. Structure and massspectral data of isothiocyanates and nitriles formed by glucosinolatedegradation identified in the leaves and/or roots of different varie-ties of Raphanus sativus L. are given in Table 2. Roots were foundnot to contain volatiles bound as O-glycosides. The yields of obtainedvolatiles were from fresh leaves 510–1880 mg/kg with identifica-tion from 91.7% to 97.8%; from root 359–2440 mg/kg with identifi-cation from 89.9% to 96.8%; from leaves as O-glycosidically boundvolatiles 69–112 mg/kg with identification from 80.5% to 84.5%.

3.1. Volatiles obtained by hydrodistillation

3.1.1. LeavesThe major components found in the oil of all different varieties

of Raphanus sativus leaves were compounds without sulphur andnitrogen such as phytol (65.3–69.7%), hexadecanoic acid (2.5–14.3%), methyl linolenate (2.1–11.1%) and (Z)-3-hexen-1-ol (0.7–6.9%). Alcohols and aldehydes are common volatiles in processedvegetables, and are formed by oxidative breakdown of free fattyacids by the action of lipoxygenase (Schreier, 1984).

Corresponding aliphatic or aromatic volatiles formed by knowndegradation of glucosinolates found in leaves constituted only 0.3–5.7% of the isolated volatiles (Table 1). They were 4-methylpentylisothiocyanate, benzyl isothiocyanate, 4-(methylthio)-3-butenylisothiocyanate, 4-(methylthio)butyl isothiocyanate, 5-(methyl-thio)-4-pentenenitrile, 2-phenylethyl isothiocyanate and benzene-propanenitrile. Obtained mass spectral data are given in Table 2. 4-(Methylthio)-3-butenyl isothiocyanate, was previously reported tobe a volatile component of radish leaf (Wei, Zhang, & Zhang, 2002).

It was reported, previously, that radish leaves contain gluco-brassicin (3-indolylmethyl GLS) (Sang, Minchinton, Johnstone, &Truscott, 1984). It was shown that indole glucosinolates showedhigher degradation rates than aliphatic glucosinolates (Oerlemanset al., 2006). However, volatile indole degradation products of cor-responding glucosinolate after 3 h of hydrodistillation were not de-tected among volatiles isolated from the leaves. Also, thiocyanates,epithionitriles and oxazolidine-2-thiones, which can originatefrom glucosinolate degradation, were not identified in these oils.

Dimethyl trisulfide (tr.-0.4%), found in the leaf oils, according toChin and Lindsay (1994), originates from (+)-S-methyl-L-cysteinesulfoxide, an a-amino acid found in Brassica vegetables (Marks,Hilson, Leichtweis, & Stoewsand, 1992).

3.1.2. Roots4-(Methylthio)-3-trans-butenyl isothiocyanate, a breakdown

product of glucodehydroerucin, was reported to be the main vola-tile constituent of radish root responsible for its pungency (Coogan,Wills, & Nguyen, 2001; Friis & Kjaer, 1966; Nakamura et al., 2001;Visentin, Tava, Iori, & Palmieri, 1992). However, it was reportedpreviously that radish roots can contain several glucosinolates

such as 4-methylpentyl GLS, hexyl GLS, 5-hexenyl GLS, 4-(methyl-thio)butyl GLS (glucoerucin), 5-(methylthio)pentyl GLS (glucober-teroin) and 4-(methylthio)-3-butenyl GLS (glucodehydroerucin)(Kjaer, Madsen, Maeda, Ozawa, & Uda, 1978; Visentin et al., 1992).

We found, after volatile isolation in a Clevenger-type apparatus,that major components in the oil of all roots, were sulphur and/ornitrogen containing compounds, such as 4-(methylthio)butyl iso-thiocyanate (17.9–25.7%), 5-(methylthio)pentyl isothiocyanate(1.5–2.0%), dimethyl trisulfide (0.8–3.8%), 4-(methylthio)-(E, Z)-3-butenyl isothiocyanate (0.2–1.8%), 2-phenylethyl isothiocyanate(0.1–1.5%), 5-(methylthio)-(E, Z)-4-pentenenitrile (0–6.9%). Othersulphur and/or nitrogen containing compounds detected were 4-methylpentyl isothiocyanate, dimethyl disulfide, dimethyl tetra-sulfide, 2-acetylthiazole and 1-(methylthio)-3-pentanone.

In contrast with previously reported data, we found 4-(methyl-thio)butyl isothiocyanate (erucin) to be the most abundant com-pound isolated from roots by hydrodistillation in the Clevengerapparatus. It is formed by Loosen rearrangement of an unstablethiohydroximate intermediate that originates from glucoerucindegradation, a glucosinolate previously reported in radish root.No corresponding nitrile that could be formed from the same gluc-osinolate was found in oils after isolation.

Among isolated volatiles from ground roots after hydrodistilla-tion without prior autolysis, 4-(methylthio)-3-butenyl isothiocya-nate (E, Z)-isomers (0.2–1.8%) were also detected. Previously,4-(methylthio)-3-(E)-butenyl isothiocyanate, produced by hydroly-sis of 4-(methylthio)-3-(E)-butenyl glucosinolate by the enzymemyrosinase, was reported as the compound primarily responsiblefor the characteristic sulphurous, pungent flavour and aroma of rad-ish root (Friis & Kjaer, 1966).

The (E)-isomer of 4-(methylthio)-3-butenyl isothiocyanate inparticular has been reported to be labile enough to form a varietyof 3-substituted 2-thioxopyrrolidines and dithiocarbamates(Kosemura, Yamamura, & Hasegawa, 1993; Matsuoka, Yoshinori,Yoneyama, & Uda 1998; Uda, Ozawa, Ohshima, & Kawakishi,1990). Uda et al. (1990) reported formation of enolated 2-thioxo-3-pyrrolidinecarbaldehyde, a degradation product of radish 4-(methylthio)-3-butenyl isothiocyanate in aqueous media. They con-sidered that this compound can be formed in crushed or salted rad-ish root by enzymatic hydrolysis of its corresponding glucosinolateand can be stabilised by internal molecular hydrogen bonds be-tween the enolic hydroxyl group and sulphur atoms. Kosemura etal. (1993) suggested that some 4-(methylthio)-3-butenyl isothiocy-anate, released from 4-(methylthio)-3-butenyl glucosinolate viamyrosinase (thioglucosidase) spontaneously converts to raph-anusanins.Matsuoka et al. (1998) examined formation of raphanusa-nins using different extraction procedures and solvents anddemonstrated that raphantin or raphanusanin is an artefact formedduring extraction of radish with methanol. In our experiments,where volatiles were obtained by hydrodistillation, no artefacts thatcan be formed from (E)-4-(methylthio)-3-butenyl isothiocyanateunder certain conditions, were identified. Corresponding 5-(methylthio)-4-pentenenitrile (E, Z)-isomers (1.5–6.9%), formedfrom the same 4-(methylthio)-3-butenyl glucosinolate, were tenta-tively identified among isolated volatiles and their mass spectrafragments are given in Table 2. Although 4-(methylthio)-3-butenylglucosinolate decomposed to its corresponding isothiocyanate andnitrile (E, Z)-isomers, 4-(methylthio)butyl isothiocyanate (erucin)was still the most abundant volatile in all oils after hydrodistillation.

5-(Methylthio)pentyl isothiocyanate (berteroin) was isolated asa hydrolysis product of glucoberteroin, an analogue of glucoerucin.Thiocyanates, epithionitriles and oxazolidine-2-thiones were notidentified in these oils.

Dimethyl trisulfide (1.1–3.8%) and dimethyl disulfide (tr.–1.8%),as in leaves, can originate from (+)-S-methyl-L-cysteine sulfoxideduring crushing (and heating). However, sulfides, as well as other

Table 3O-Glycosidically-bound volatiles in the leaves of radish var. black, white and red

No. Compound name RI1 RI2 Radish (Raphanus sativus L.)

var black (%) var. white (%) var. red (%)

1 (Z)-3-Hexen-1-ola,b 1351 869 6.2 2.6 10.42 Benzaldehydea,b 1465 955 0.1 1.4 –3 2-Phenylacetaldehydea,b 1588 1049 – 0.9 –4 Methyl salicylatea,b 1700 1182 3.4 0.8 –5 1-Phenylethyl alcohola,b 1731 1217 0.2 0.1 0.26 2-Methoxyphenol (guaiacol)a,b 1775 1119 0.6 1.7 –7 Benzyl alcohola,b 1809 1095 4.2 1.4 1.88 2-Phenylethyl alcohola,b 1854 1185 23.5 2.0 11.89 2-methoxy-4-methylphenola,b 1889 – – 2.2 –10 Phenola,b 1923 1159 – 2.3 –11 4-Ethyl-2-methoxyphenola,b 1959 1289 2.6 2.3 –12 2,4-Dimethylphenola,b 2002 1311 – 3.2 0.313 Eugenola,b 2076 1396 29.2 30.2 37.114 4-Vinyl-2-methoxyphenol (vinyl guaiacol)a,b 2123 1365 2.7 12.4 2.415 1H-Indolea,b > 2200 1426 6.8 2.2 0.716 Vanillina,b > 2200 1525 3.7 3.5 6.317 Methyl vanillatea,b > 2200 1611 – 10.0 6.318 4-Keto-a-ionolb,t > 2200 1672 0.5 2.9 2.119 3-Hydroxy-b-iononeb,t > 2200 – – 2.4 1.1Total identified (%) 83.7 84.5 80.5Number of compounds identified (No.) 13 19 12Yield (mg/kg) 103.0 112.4 69.0

RI1-retention indices on HP-20M column; RI2-retention indices on HP-101 column;-not detected.a Compound identified by mass spectra and RI comparison with homemade library.b Compound identified by mass spectra comparison with Wiley library.t Tentatively identified compound.

I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102 101

volatile products, can be formed by subsequent degradation ofsome volatiles derived from glucosinolate degradation (Jin, Wang,Rosen, & Ho, 1999).

This oil, except for the above mentioned compounds, containedcompounds without nitrogen and sulphur, mostly fatty acids andesters; the major components being: hexadecanoic acid (30.3–49.9%) and methyl linolenate (8.5–21.7%). Also aliphatic alcohols,carbonyls, alkanes, terpenes and other components were presentin the oil, but at a small percentage.

3.2. O-Glycosidically bound volatiles

Isolated and purified O-glycosides were hydrolysed by b-gluco-sidase, and liberated O-aglycones were analysed by GC and GC–MS.After enzymatic treatment, no volatile aglycones were detected inthe different varieties of roots. Volatile compounds released byenzymatic treatment of the glycosidic fraction isolated from leavesare given in Table 3. The contents of glycosidically-bound volatilecompounds in fresh leaves were 69.0–112 mg kg�1. Compoundsare listed in order of their elution from the HP-20 M column. Com-pounds with sulphur and/or nitrogen which are characteristic ofglucosinolate were not identified.

Nineteen aglycones were identified representing 80.5–84.5% ofthe total volatile O-aglycones. Aliphatic alcohols, derivatives ofphenylpropanes and C13-norisoprenoids were identified (Table3.). The main aglycones were eugenol (29.2–37.1%), 2-phenyletha-nol (2.0–23.5%), (Z)-3-hexen-1-ol (2.6–10.4%), 4-vinyl-2-methoxy-phenol (2.4–12.4%), 1H-indole (0.7–6.8%), methyl vanillate (6.3–10.0%), benzyl alcohol (1.4–4.2%) and methyl salicylate (0.8–3.4%).

Comparing the chemical composition of O-aglycones (Table 3)with the essential oil Table 1) only five identified compounds werecommon. They were 4-vinyl-2-methoxyphenol, (Z)-3-hexen-1-ol,benzaldehyde, 2-phenylacetaldehyde and 1H-indole. These resultsshow a small correlation between the chemical composition of ob-tained oil and volatile O-aglycones. The aglycones, such as eugenol,benzyl alcohol, 2-phenylethanol and aliphatic alcohols are ubiqui-tous in the aglycone fractions of many plant families (Stahl-Biskup

et al., 1993). Eugenol and other p-hydroxyphenylpropanes whichhave been identified in many plants as the main aglycones can beconnected with lignin biosynthesis via the peroxidase-hydrogen per-oxide system. The aliphatic volatiles (alcohols, carbonyls, acids) canoriginate from fatty acid catabolism and aromatic volatiles (alcohols,acids, carbonyls) from cinnamic acid catabolism (Schreier, 1984).

4. Conclusion

Sulphur and nitrogen-containing compounds, such as isothiocy-anates and nitriles, seem to play an important role in the character-istic aroma of Raphanus sativus oil (Table 2). It could be foreseenthat sulphur and nitrogen-containing compounds that originatefrom glucosinolate compounds should be generally found at great-er concentrations in roots than in leaves. The main volatile foundamong isolated volatiles by hydrodistillation of roots was 4-(methylthio)butyl isothiocyanate (erucin) in all three investigatedvarieties of radish, in contrast to reports where after hydrolysisand dichloromethane extraction 4-(methylthio)-trans-butenyl iso-thiocyanate was found to be the major pungent volatile. Duringisolation of volatiles by hydrodistillation most nitriles formed werefrom 4-(methylthio)-3-butenyl glucosinolate, while 4-methylbutylglucosinolate (glucoerucin) and 5-(methylthio)pentyl glucosino-late (glucoberteroin), gave only isothiocyanates. Formation of thefound sulphides can originate from (+)-S-methyl-L-cysteine sulfox-ide and subsequent degradation of the volatiles formed by gluco-sinolate degradation.

There were a number of O-glycosidically bound volatile com-pounds that have not been investigated previously (Table 3.).According to our research, we found nineteen O-glycosidically-bound volatiles in leaves, while roots of different varieties of Raph-anus sativus (L.) did not contain this compound class.

Acknowledgment

This work was supported by Ministry of Science, Education andSports, Republic of Croatia, project ‘‘Essential oils and flavours –

102 I. Blazevic, J. Mastelic / Food Chemistry 113 (2009) 96–102

biological active compounds and their modifications”, Grant 011-0982929-1329, in the framework of the program ‘‘Spectroscopyand Modelling of Bioactive Molecules”.

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