Purchased by U.S. Department of Agriculture for Official Use Reprint from Vol. 105(5), September 1980 Joumal of the American Society for Horticultural Science Alexandria, Virginia 22314

1. Amer. Soc. Hart. Sci. 105(5):710-714. 1980. 4670 : Potential Toxicants in Cultivars 1 C. H. VanEtten, M. E. Daxenbich1er, and H. L. Tookey Northern Regional Research Center, Agricultural Research, Science and Education Administration, u.s. Department ofAgriculture, Peoria, IL 61604 W. F. Kwolek North Central Region, Agricultural Research, Science and Education Administration, U.S Department ofAgriculture, Northern Regional Research Center, Peoria, IL 61604 P. H. Williams Department ofPlant Pathology, College ofAgricultural and Life Sciences and Agricultural Experiment Station, University of Wisconsin, iVladison, WI 53706 O. C. Yoder2 New York State College ofAgriculture and Life Sciences, Department ofPlant Pathology, Cornell University, Ithaca, NY 14853 Additional index words. Brassica oleracea (Capitata group) Abstract. Seventy-nine cultivars and lines of cabbage Brassica oleracea L. (Capitata group) were analyzed for 11 glucosinolates to provide a data base of the levels of these potential toxicants. Aglucon hydrolytic products of glucosinolates from fresh cabbage (mean of 79 cultivars) include 24 ppm allyl , 4S ppm 3-methyl­ sulfinylpropyl isothiocyanate, 18 ppm SCN ion, 17 ppm 4-methylsulfinylbutyl isothiocyanate, and 4 ppm goitrin. Composition of the cultivars are summarized by type (red, white, savoy) and by end use (market, storage, kraut). Glucosinolates with a 3-carbon aglucon (excluding the sinolate carbon) predominate over 4-carbon glucosinolates in white and savoy types. Four-carbon glucosinolates (including goitrin precursor) predominate in red . Savoy cabbages are high in glucosinolates yielding SCN ion. Distinctions between market, storage, and kraut cultivars are less well defined. No differences could be seen between open pollinated and hybrid cultivars. Year­ to-year variation for 12 cultivars is discussed.

The glucosinolates of cabbage are probably involved in insect which glucosinolates should be avoided in breeding. However, predation (9) and make a significant contribution to flavor. The a store of information on the glucosinolates present in cabbage flavor is attributed not to the intact glucosinolates but to the is required in order to select appropriate compounds for further enzymatic hydrolytic products from the aglucon of the glu­ study of toxic effects and to study the inheritance patterns of cosinolate, e.g., . However, a number of glucosinolates. these aglucon products are toxicants: goitrin and Selected cultivars of cabbage grown in Europe have been ion (SCN ion) are goitrogenic, and certain nitriles suppress the analyzed by Josefsson (7) for the products: growth of rats or are liver toxins (10, 13, 14). iSQthiocyanates, goitrin, and SCN ion. More recently, we re­ Recently there has been concern that the levels of natural ported the glucosinolate cohtent of the heads of 22 cultivars toxicants might be inadvertently raised during plant breeding of cabbage grown in the United States (12); we now wish to to improve yields, to raise insect resistance, or to enhance other report a summary of 79 cabbage cultivars and inbreds grown desirable traits of horticultural crops (6). Because of this con­ in the United States. Twelve cultivars were grown two or more cern, more information is needed on the amounts of gluco­ crop years. sinolates in crops from the Cruciferae and on the biological effects of the aglucon hydrolytic products. Materials and Methods Cabbage contains at least II glucosinolates that may be Source. Most cultivars were grown near Madison, Wisconsin, hydrolyzed by a co-occurring enzyme, thioglucosidase, to form for fall harvest from 1974 through 1978. Twelve cultivars either or organic nitriles as aglucon products were grown in both 1974 and 1975 and 'Market Prize' was also (Fig. I). Glucosinolates with a 3-carbon chain (R group) hy­ grown in 1978. Of the storage cultivars, 8 were harvested in drolyze to yield 3-carbon aglucon products. Certain of the Ithaca, N. Y., in 1973 and 1974 and were sampled at intervals isothiocyanates may further react to form oxazolidinethiones throughout 6 months ofcommercial cold storage. Kraut cultivars (Fig. 2). Other isothiocyanates, e.g., those containing the in­ dolyl group, are unstable and decompose to form SCN ion and other products (5). Crushing of fresh plant material may lead to the formation of sulfur-containing epithionitriles as shown in Fig. 2 (2, 4, 10). ll S Because the biological effects of many of these nitriles and )-CSH O H 0 ~ + Glucose R-C R-Ci-J isothiocyanates are as yet unknown, it is not possible to state ~ Thioglucosidase2. ~ + KHS04 N-O-S020- K+ N- lReceived for publication January 19, 1980. The mention of fIrm / ~ names or trade products does not imply that they are endorsed or rec­ R-N==C==S R-C==N omended by the U.S. Department of Agriculture over other firms or similar products not mentioned. Isothiocyanate Nitrile The cost of publishing this paper was defrayed in part by the payment + of page charges. Under postal regulations, this paper must therefore be S hereby marked advertisemen t solely to indicate this fact. 2The authors thank G. Rose and W. Schroeder for technical assistance Fig. 1. General scheme of enzymatic hydrolysis of glucosinolates. Nitriles and W. Bailey for calculations required in assembling the data. are favored by crushing of fresh plant material (10).

710 J. Amer. SOC. Hart. Sci. 105(5):710-714. 1980. Table 1. Cultivars of cabbage analyzed.

Red, for market Open pollinated Hybrid Mammoth Red Rock Red Head Red Acrez Red Man / ~ Red Danish Ruby Ball . z CHz==CH--CHOH--CHz--C==N + S LCHz==CH--CHOH--CHz--N==C==~ Red Hollander 1·cyano·2 ·hydroxy·3·butenes Red, for storage or market Open pollinated + t Red Winter S Savoy, for market / 'v • Open pollinated Hybrid CHz--CH--CHOH--CHz--C==N Chieftain Savoy Savoy King 1·cyano·2 ·hydroxy·3 ,4·epithiobutanes Savoy Perfected Drumheadz IEpithionitriles) Vanguard 5· vin yI0xaz olid in e·2· th iones White, for market IGoitrins) Open pollinated Open pollinated Oenotes centers of asymmetry Badger MarketZ Jersey Queen Copenhagen Mkt. Earlyz Premium Late Flat Dutch Fig. 2. Enzymatic products from progoitrins. Isothiocyanates containing Danish Ballhead Marion Market ,,-hydroxyl groups form -2-thiones (14). Nitriles contain­ Ferry's Round Dtuch Penn State Ballhead ing terminal double bonds may form epithionitriles (4,10, 14). Eastern Ballhead Wisconsin Golden Acrez Globelle Wisconsin Hollander Golden Acre Hybrid Hybrid z were grown in 1976: 'Roundup' in New York and Oregon, Badger Hybrid 15 Market Prize Sanibel, and 'TBR Globe' in Wisconsin. Blue Crown Market Topper Preparation and analyses. Samples of the edible or leafy Emerald Cross Market Victor Excelz Prime Pak portion of cabbage heads were prepared as previously described Gourmet Princess (12) from at least 3 heads of each cultivar. Total glucosinolates Green BoyZ Resistant Danish were determined by an estimation of glucose released upon Hancock Rio Verde enzymatic hydrolysis of the glucosinolates (11). Individual Harvest Queenz Stonehead glucosinolates (except indolymethyl glucosinolates) were Head StartZ Sun Up estimated by gas-liquid chromatography of aglucon products Hercules Superdane z (organic isothiocyanates and goitrin) released under specified Jet Pack Supperette conditions (1). Indolylmethyl glucosinolates were estimated King Cole Tastie Little Rock Wizard from the SCN ion released upon hydrolysis of these gluco­ Hybrid 15 sinolates essentially as previously described (8). A concise outline of the analytical methodology may be found in Daxen­ White, for storage bichler et al. (3). In most cases, estimation of SCN ion from Open pollinated Hybrid Dolme Amager Stonehead 80 red cabbage heads was not made because of interference by the Dural 173 Supergreen Hybrid red coloration. Dural 453 Green Winter 206 Results and Discussion Green Winter 274 Presentation of data. The cultivars analyzed are listed in Houston Evergreen Table 1. The analytical data may be expressed as ppm of the Lovet aglucon products isothiocyanate, goitrin, and SCN ion. This White, for kraut manner of presentation should be of optimum usefulness to the Open pollinated Hybrid nutritionist or toxicologist. However, the plant geneticist may Globe Roundup wish to know the relative proportions of the components in TBR Globe Sanibel terms of the parent glucosinolates that are the inherited char­ White, experimental acters. The various nitriles, isothiocyanates, and goitrin are Hybrid Inbred derived from glucosinolates having molecular weights 2.7 to 4 A Badger inbred 2 times that of the component analyzed, but 3-indolylmethyl Inbred 4 glucosinolate is 8.4 times the molecular size of SCN ion. To 452 (B 1863, USDA) 5 provide a more satisfactory comparison among the gluco­ 453 (B 1863, USDA) 8 sinolates, the values are also presented in molar concentrations: 527 (1975 Wis.) 10 micromoles per 100 g. 536 (1975 Wis.) 13 14 Composition of the leafy portion of cabbage heads is sum­ 16 marized by type: red, savoy, and white in Tables 2 (gluco­ 18 sinolates, J,Lmol/l00 g) and 3 (aglucons, ppm). Data from 67 cultivars of white cabbage are summarized for each category ZGrown in Wisconsin in successive years 1974, 1975. (market, storage, kraut) and by genetic background (open pollinated, hybrid, inbred) in Table 4. Variation of gluco­ sino1ate with year of growth is shown in Table 5. cosinolates in the savoy and white types. In the red cabbages, Glucosinolate pattems of cabbage types. The glucosino1ate 4-carbon glucosinolates predominate. The red type contains, patterns for red, savoy, and white cabbage differ from one in general, greater total glucosinolates than does the white. another (Table 2). G1ucosinolates with a 3-carbon chain (ex­ Savoy cultivars contain large amounts of 3-methylsulfinyl­ cluding the sinolate carbon) predominate over 4-carbon glu- propyl and 3-indolylmethyl glucosinolates. Total glucosinolates

J. Amer. Soc. Hart. Sci. 105(5):710-714. 1980. 711 Table 2. Glucosinolate content of fresh cabbage.z

Glucosinolate content (umoles per 100 g) 3-carbon GSY 4-carbon GS 3- 4- 4- 3- Methyl- 2- 4- Methyl- Methyl- Methyl- sul- Hydroxy- Methyl- sul- sul- 2- Total Number thio- finyl- 3- 3- thio- finyl- fonyl- Phenyl- Indolyl- gluco- of Allyl propyl propyl Butenyl butenyl butyl butyl butyl Benzyl ethyl methyl sino- Type cultivars GS GS GS GS GS x GS GS GS GS GS GS'sw lateV

Red 8 10.5 0.1 14.5 9.9 8.3 1.9 52.3 2.5 0.1 0.9 16804 Savoy 4 14.2 004 46.7 0.3 0.5 0.1 5.3 5.5 0.9 0.9 80.5 169.2 White 67 2604 1.7 28.3 1.7 2.9 0.5 4.9 1.9 0.1 1.0 31.2 112.1 All types 79 24.1 104 27.8 2.5 3.3 0.6 9.6 2.2 0.2 1.0 31.7 120.7

ZData for individual cultivars are available upon request: HSC Laboratory, NRRC, USDA, 1815 North University, Peoria, Illinois 61604. YGS =glucosinolate. xPrecursor of goitrin. wPrecursors of SCN ion. vCalculations for total glucosinolates based on molecular weight of 455.

Table 3. Glucosinolate aglucon content of fresh cabbage.

Glucosinolate aglucon content (ppm) 3-carbon aglucons 4-carbon aglucons 3- 4- 4- Glucose 3- Methyl- 4- Methyl- Methyl- calc. Methyl- sul- Methyl- sul- sul- 2- Total from Number thio- finyl- 3- thio- finyl- fonyl- Phenyl- gluco- aglucons, of Allyl propyl propyl Butenyl butyl butyl butyl Benzyl ethyl SCN sino- (% of Type cultivars NCSz NCS NCS NCS Goitrin NCS NCS NCS NCS NCS ion lateY total)x

Red 8 Mean lOA 0.2 23.7 11.2 10.7 3.1 92.6 4.9 0.2 104 766 Sow 8.1 004 15.5 4.2 5.1 3.9 31.3 2.8 0.3 0.7 240 Savoy 4 Mean 14.1 0.6 76.1 0.3 0.6 0.2 904 10.7 104 1.5 46.7 770 95 SO 17.5 0.6 51.4 004 0.8 0.3 6.3 2.2 1.9 1.9 1l.5 336 White 67 Mean 26.1 2.5 46.1 1.9 3.7 0.8 8.6 3.7 0.2 1.6 18.1 510 91 SO 18.3 3.3 20.5 204 4.0 104 12.9 3.1 004 1.3 8.9 187 All types 79 Mean 23.9 2.1 45.4 2.8 4.3 1.0 17.0 4.2 0.3 1.6 1804 549 91 SO 17.6 3.1 22.1 2.6 4.1 1.7 15.3 3.1 0.5 1.3 9.6 201 zNCS =isothiocyanate. YCalculation based on average molecular weight of 455. xPercent recovery relative to glucose that was obtained as a measure of total glucosinolate. WStandard deviation between cultivars. are also high in this type. Table 3 persents the data in terms of butyl glucosinolate. No changes in glucosinolates occurred aglucon products and includes standard deviations as a measure during 6 months storage. The 13 inbreds were low in total of variability between cultivars. glucosinolate, but of similar pattern to that commonly found in Seven cultivars of white cabbage have an atypical pattern white cabbage. similar to that of red cabbage. Of the 67 cultivars of white Glucosinolate pattem in open pollinated and hybrid culti­ cabbage examined, the atypical cultivars are 'Market Prize', vars. One of the objectives of our research is to accumulate 'Harvest Queen', 'Houston Evergreen', 'Premium Late Flat data on current cultivars of cabbage so that newly developed Dutch', 'Lovet', and experimental inbreds 452 and 453. Glu­ cultivars may be examined for greater amounts or different cosinolate patterns of 'Market Prize' will be described under the glucosinolates than are present in existing cultivars. New culti­ section on glucosinolate variation with year of growth. vars remain to be tested, but the data on white cabbage (Table Glucosinolate pattems of white cabbage: market, storage, 4) shows there is no difference between the open pollinated kraut, and experimental. Market cultivars of white cabbage are and the hybrids for total glucosinolates or glucosinolate pattern. higher in thiocyanate ion-forming glucosinolates and methyl­ Glucosinolate variation with year ofgrowth. Twelve cultivars sulfonylbutyl glucosinolates than are cultivars used for storage were grown in Wisconsin in both 1974 and 1975 (for names, see and kraut (Table 4). Also, the cultivars for market use are lower footnote z, Table 1). The data suggested that larger heads in allyl, methylthiopropyl, methylsulfinylpropyl, and butenyl might have a lower glucosinolate concentration: in 1974, aver­ glucosinolates. The storage cultivars tend to be high in the age head size was 0.8 kg and total glucosinolate was 161 /1mole/ methylthio glucosinolates and quite low in methylsulfonyl- 100 g; in 1975, these values were 1.5 kg and 117 /1mole/ 100 g,

712 J. Amer. Soc. Hart. Sci. 105(5):710-714. 1980. Table 4. Glucosinolate aglucon content of 67 cultivars of white cabbage.

Glucosinolate aglucon content (ppm) Glucose 3- Methyl- 4- Methyl- Methyl- calc. Number Aver- Methyl- sul- Methyl- sul- sul- 2- Gluco- from of Number age thio- finyl- 3- thio- finyl- fonyl- Phenyl- sino- aglucons, culti- of head Allyl- propyl- propyl- Butenyl- Goi- butyl- butyl- butyl- Benzyl- ethyl- SCN late, (% of Category vars samples wt, kg NCS z NCS NCS NCS trin NCS NCS NCS NCS NCS ion total total)y

Market a.p. 13 80 1.1 Mean 25.4 1.1 48.5 1.9 3.1 0.4 8.0 4.7 0.2 2.1 24.1 541 93 SDx 8.6 1.1 19.2 1.9 1.9 0.6 10.0 3.7 0.3 1.5 14.1 230 10 Hybrid 27 129 1.4 Mean 25.7 1.6 43.7 1.5 3.0 0.3 6.1 4.0 0.2 1.5 18.5 477 92 SD 25.5 2.3 16.4 2.0 4.3 0.4 8.5 1.9 0.3 1.2 7.5 161 9

Storage a.p. 7 64 2.6 Mean 31.6 6.2 65.5 5.2 7.9 3.6 20.9 1.3 0.06 1.7 16.6 675 90 SD 13.1 2.7 22.6 3.7 5.2 2.2 14.1 2.0 0.03 1.1 4.2 174 3 Hybrid 2 9 2.4 Mean 30.8 12.3 52.5 1.6 2.4 1.5 3.7 0.1 0.03 2.8 9.7 508 89 SD 13.4 10.8 29.5 0.8 1.0 0.6 1.2 0.1 0.04 3.2 2.2 258 4

Kraut a.p. 2 13 2.0 Mean 54.4 2.0 67.3 3.4 5.1 0.1 6.9 2.2 0.08 2.7 18.9 732 89 SD 3.3 2.3 18.4 0.7 2.8 0.1 5.7 2.5 0.1 0.8 4.3 160 Hybrid 2 33 3.8 Mean 39.5 5.5 67.6 2.2 4.0 1.1 6.6 2.5 0.3 2.4 16.1 678 84 SD 6.9 0.7 21.8 1.0 2.5 1.0 4.6 0.9 0.1 0.9 1.1 117 4

Experi- mental Hybrid 3 1.6 Mean 26.1 6.2 36.0 3.6 2.4 1.6 8.8 2.9 0.4 1.4 11.7 508 79 SD Inbred 13 42 1.3 Mean 19.3 1.1 31.6 0.7 3.7 0.5 7.8 4.5 0.5 0.8 13.7 402 83 SD 9.7 1.0 18.3 1.4 4.0 1.0 20.6 4.6 0.6 0.9 3.4 125 8

zNCS =isothiocyanate. YPercent relative to glucose obtained as a measure of total glucosinolate. XStandard deviation between cultivars.

respectively. The yield differences between these 2 years might Since 'Market Prize' is a major cabbage grown for the U.S. have been caused by stress induced by heavy application of a market, this cultivar was planted again in 1978. Six replicate herbicide in 1974. In spite of these differences, the glucosinolate plots were grown on a sandy soil from the same seed source pattern within most of the cultivars remained stable for the as 1974 and 1975. Irrigation, tillage, and herbicide treatments 2 years: correlations between years for 10 pairs of glucosinolates were the same as in 1975. In 1974, 'Market Prize' had a glu­ ranged from 0.94 to 1.00 within each cultivar except for 'Har­ cosinolate pattern similar to red cabbage - high in 4-carbon vest Queen' and 'Market Prize', which were 0.75 and 0.45, glucosinolates; but in 1975, its pattern was that of white cab­ respectively. bage - high in 3-carbon glucosinolates (Table 5). In 1978, cabbage from 2 plots resembled red cabbage, and from 4 plots resembled white (Table 5). This variability in glucosinolate pattern was unexpected since 10 of the 12 cultivars had re­ Table 5. Comparison of glucosinolate pattern of 'Market Prize' grown in tained their characteristic glucosinolate pattern through the 3 different years. 2 crop years 1974 and 1975. Investigations to assess the re­ 1978 sponse of cabbage to environmental stress are in progress. Variable 1974 1975 2 plots 4 plots Literature Cited Heads analyzed 5 4 6 12 1. Daxenbichler, M. E. and C. H. VanEtten. 1977. Glucosinolates and Head wt (kg) 1.1 1.4 2.0 2.0 derived products in : gas-liquid chromato­ 3-carbon GS z (/lmo!/100 g) 19.9 51.9 11.5 15.5 graphic determination of the aglucon derivatives from cabbage. 4-carbon GSY C/lmo!/100 g) 37.1 5.7 18.6 5.9 J. Assoc. Off Anal. Chem. 60:950-953. Indolyl-GSx (/lmol/l00 g) 50.3 32.8 12.8 12.1 2. , , and G. F. Spencer. 1977. Gluco- Aromatic-GSw C/lmol/l00 g) 0.6 0.2 1.1 1.0 sinolates and derived products in cruciferous vegetables: identifi­ Total GSv (/lmol/l00 g) 105 92 57 49 cation of organic nitriles from cabbage. J. Agr. Food Chenl. 25: 121-124. zGS =glucosinolate; for names, see Table 2. 3. , , and P. H. Williams. 1979. Gluco- sinolates and derived products in cruciferous vegetables. Analysis of YFor names, see Table 2. fourteen varieties of Chinese cabbage. J. Agr. Food Chem. 27 :34-37. xPrecursor of SCN ion. 4. , , and 1. A. Wolff. 1968. Diastereomeric wlncludes benzyl and phenylethyl glucosinolates. episulfides from epi- upon autolysis of crambe seed meal. vCalculated from glucose. Phytochemistry 7:989-996.

J. Amer. SOC. Hart. Sci. 105(5):710-714. 1980. 713 5. Gmelin, R. and A. 1. Virtanen. 1961. , the precursor plant foodstuffs, 2nd ed. Academic Press, New York. of 3-indolylacetonitrile, ascorbigen, and SCN- in Brassica oleracea 11. VanEtten, C. H. and M. E. Daxenbichler. 1977. Glucosinolates and species. Suomen Kemistilehti B34: 15-18. derived products in cruciferous vegetables: total glucosinolates by 6. Hanson, C. H. (ed.). 1974. The Effect of FDA Regulation (GRAS) on retention on an anion exchange resin and enzymatic hydrolysis to Plant Breeding and Processing, Special Pub!. 5, Crop Science Soc. of measure released glucose. J. Assoc. Off Anal. Chem. 60:946-949. America, Madison, Wise. 12. , , P. H. Williams, and W. F. Kwolek. 7. Josefsson, E. 1967. Distribution of thioglucosides in different parts 1976. Glucosinolates and derived products in cruciferous vegetables: ofBrassica plants. Phytochemistl}' 6:1617-1627. Analysis of the edible part from twenty-two varieties of cabbage. J. 8. . 1968. Method for quantitative determination of p- Agr. Food Chem. 24:452-455. hydroxybenzyl isothiocyanate in digests of seed meal of Sinapis 13. , W. E. Gagne, D. J. Robbins, A. N. Booth, M. E. alba L. J. Sci. Food Agr. 19:192-194. Daxenbichler, and 1. A. Wolff. 1969. Biological evaluation of crambe 9. Nair, K. S. S. and F. L. McEwen. 1976. Host selection by the adult seed meals and derived products by rat feeding. Cereal Chem. 46: cabbage maggot, Hylemya brassicae (diptera:anthomyiidae): effect of 145-155. glucosinolates and common nutrients on oviposition. Can. Entomol. 14. and H. L. Tookey. 1979. Chemistry and biological 108:1021-1030. effects of glucosinolates. p. 471-500. In G. A. Rosenthal and D. H. 10. Tookey, H. L., C. H. VanEtten, and M. E. Daxenbichler. 1980. Janzen (eds.) Herbivores: their interaction with secondary plant Glucosinolates. p. 103-142. In 1. E. Liener (ed.) Toxic constituents of metabolites. Academic Press, New York.

714 J. Amer. SOC. Hart. Sci. 105(5):714-717. 1980.