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L.F. DI CESARE1, Influence of heat treatments on C. MIGLIORI1,D.VISCARDI1, in italian V. FERRARI2

PROGRESS IN NUTRITION Summary VOL. 11,N.2, 100-109, 2009 Organic (ITCs), and particularly sulforaphane, are wi- dely studied as potential inhibitors carcinogenesis in animal models in- TITOLO cluding of lung, esophagus, forestomach, colon, mammary glands Influenza dei trattamenti and pancreas. Sulforaphane, enzymatic breakdown product of glucora- termici sul sulforafane nelle phanin, was extracted by solvent from homogenates and quali- di brassica coltivate in quantitatively analysed by GC/MS. This method, modified from VanEt- Italia ten procedure, was used to evaluate sulforaphane in fresh and cooked (microwave, boiling water, steam, pan cooking) , KEY WORDS (“Romanesco”, “verde di Macerata”, “violetto di Catania”, “Jesino” and Broccoli, , , commercial hybrid) and white cabbage. The highest content in sulfora- cooking methods (microwave, phane was noted in the raw broccoli, respect of the other examinated boiling water, steam, pan cooking), fresh brassica cultivars. When all these samples were cooked, the reten- sulforaphane; GC/MS analysis tion of sulforaphane, the most potent activator of phase II enzymes among all ITCs, were higher in the microwave cooked samples (61- PAROLE CHIAVE 86%), little lower in the boiling water (38-74%) and strongly lower in Broccoli, cavolfiore, cavolo, metodi the steam (18-52%) and pan cooking (14-48%). The microwave seems di cottura (microonde, acqua to be the most efficient cooking method for all these because bollente, vapore, frittura), it shows the highest retention in sulforaphane. sulforafane, analisi GC/MS Riassunto Gli isotiocianati, ed in particolare il sulforafane, sono largamente studiati in quanto hanno la capacità di inibire alcune forme tumorali che interes- sano la lingua, esofago, stomaco, colon, ghiandole mammarie e pancreas, in animali da laboratorio e nell’uomo. Il sulforafane, prodotto di idrolisi enzimatica delle glucorafanina, è stato estratto con solventi dai vegetali 1 C.R.A.- I.A.A.- Unità di Ricerca omogeneizzati ed analizzato quali-quantitativamente per via GC/MS. per i Processi dell’Industria Questo metodo, ottenuto in seguito a modifiche di quello originale di Agroalimentare, Milano 2 C.R.A.- O.R.A.- Unità di Ricerca VanEtten, è stato usato per la determinazione del sulforafane nei cam- per l’Orticoltura, Stella di pioni freschi e cotti (microonde, acqua bollente, vapore, frittura) di broc- Monsampolo del Tronto (AP) colo, cavolfiore (“Romanesco”, “verde di Macerata”, “violetto di Catania”, “Jesino” ed un ibrido commerciale) e cavolo-cappuccio bianco. Nei cam- Indirizzo per corrispondenza: pioni freschi di broccolo è stato accertato il più alto contenuto in sulfora- Dr. Luigi Di Cesare Tel. 02/239557215; fane, rispetto alle altre cultivar allo stato fresco. Quando tutti i campioni e-mail: [email protected] sono stati cotti, la ritenzione percentuale del sulforafane, il più potente

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attivatore degli enzimi della fase II tra tutti gli isotiocianati, è stata più elevata nei campioni cotti con microonde (61-86 %), un po’ più bassa nella cottura in acqua bollente (38-74 %) ed ancora più bassa nella cottu- ra a vapore (18-52 %) e frittura (14-48 %). Le microonde si sono dimo- strate il più efficiente sistema di cottura per questi vegetali, poiché esso mostra le più alte ritenzioni in sulforafane per tutti i vegetali presi in esa- me.

Introduction gregated from and is prevention. These ITCs are potent released after cellular damage. inhibitors of tumorigenesis in va- The natural organic isothiocyana- Myrosynase catalyzes at pH=7 the rious animal models (10, 11, 12). tes (ITCs) are synthesized and following reaction (4, 5) (Fig. 1). The dual action of ITCs is to in- stored as glucosinolates (β-thio- It has long been known that ITCs hibit phase I enzymes that are re- N-hydroxy sulfates) in have different biological effects sponsible for the bioactivation of consumed due to their chemical reactivity (2, carcinogenesis, reducing the pro- by humans, such as broccoli, cab- 6, 7). More recently, ITCs such as duction of electrophilic interme- bage, cauliflower, , 4-methylsulfinylbutyl NCS or sul- diates (13) and to enhance the ac- and (1-3). foraphane, 3-methylsulfinylpropyl tivity of phase II enzymes such as The glucosinolates are hydrolyzeed NCS, 3(methylthio)propyl NCS, glutathione transferases, epoxide to isothiocyanates by 2-phenylethyl NCS, allyl NCS hydrolases, NAD(P)H: quinone (β-thioglucoside glucohydrolase; and methyl NCS (8, 9), have been reductases and glucoronosyl trans- EC 3,2. 3-1), that is normally se- studied for their effects in cancer ferases, that increase the detoxifi- cation and clearance of carcino- Figura 1 - Production of isothiocyanates from glucosinolates gens (14-16). The effects on phase I and II enzymes, and consequently a re- duced cancer risk, have been de- monstrated in humans that consu- me a large quantity of cruciferous vegetables (12, 17-22). Recent works have also shown that ITCs act as suppressing agents during the promotion of neoplastic process and as signal transduction pathways within the cell, induce apoptosis and inhibit cell grown (23-26).

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A large number of brassica vegeta- glucosinolates in four type of portu- hybrid ( var. bles are consumed after cooking guese cabbage and in one hybrid of Botrytis) were grown in the and the amounts of glucosinolates, white cabbage before and after co- C.R.A.-ISOR, Monsampolo del and consequently of the corrispec- oking for 5-10 min in boiling water. Tronto (AP) fields. tive isothiocyanates, are usually re- Analysis of fresh cabbage, cooked Broccoli (Brassica oleracea var. Ita- duced in the cooked vegetables leaves and cooking water showed lica) and white cabbage (Brassica (27). that the glucosinolates content of oleracea var. Capitata) were pur- Jiao et al (28) determined the con- was reduced by more than chased at the Milan general horti- tent in total isothiocyanates in 102 50%. Almost all of this loss was ac- cultural market. cooked vegetables consumed in counted as intact glucosinolates in The heads of broccoli and cauli- Singapore. They used an HPLC the cooking water. Even in Italy a flowers were decored to florets be- method to quantify the cyclic pro- lot of papers were published about fore using. The heads of white ducts of ITCs and 1,2-benzenedi- the content of glucosinolates in cabbage were cut as a wedge with thiol after treatment of vegetable fresh crucifers (31-33). the edge parallel to the core. Than juices with myrosinase. The con- The aim of this paper is to evalua- the samples were cut into 3-5 cm tent of total ITCs in 9 type of cru- te the influence of different heat cube before using. ciferous vegetables ranged bet- treatments (microwave, boiling ween 4.9 µmol 100 g-1 d.m. in water, steam and pan cooking) on Cooking methods bokchoi and 81.3 µmol 100 g-1 the sulforaphane content in some d.m. in watercress. brassica cultivar (broccoli, cabbage, a) Microwave: 300 g of each sam- Wennberg et al (29) studied the cauliflower) that are frequently ple were cooked in 1L special effect of blanching and treatment consumed in Italy. glass becker, containing 800 mL with white vinegar on glucosinola- In the present research, to deter- of water with 0.3% of NaCl, co- tes of white cabbage (cv Heckla, minate the sulforaphane content vered by a Petri glass. Microwa- Predikant). Glucosinolates content in the brassica samples it was used ve power applied was 900-1000 decreased substancially in both a GC/MS procedure, modifying watt for 5-6 min for all samples. blanched cultivars, although the the original method of VanEtten b) Boiling water: 300 g of samples total loss was higher in Predikant (34, 35). Sulforaphane was also were cooked for 6-8 min in (74%) than in Heckla (50%). The determined with a similar proce- 1300 mL of boiling water with individual glucosinolates were ef- dures by Chang et al (36) and 0.3% of NaCl for all samples. fected to different degrees (15- Omary et al (37). c) Steam: 300 g of each sample 91%). During souring with acetic were cooked in a steam cooker acid, total content of Multigourmet (Braun) for 20- was not effected in Heckla but Material and methods 25 min, for all sample. In the was further reduced in Predikant. lying container were put 1500 A substantial increase in 4-me- Plant source mL of water. thoxyglucobrassicin was noted in d) Pan cooking: 300 g of each both cultivars. Cauliflower “Romanesco”, “verde sample were cooked in steel pan Rosa et al (30) measured the con- di Macerata”, “violetto di Cata- with 15-20 g of olive oil and 3 centrations of individual and total nia”, “Jesino” and commercial g of NaCl for 18-22 min.

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For all the sample, all the tests we- Figure 2 - GC/MS chromatogram of sulforaphane obtained from hydrolysis re repeated three times. of prepared according to Rochfort et al (31). The mass-spec- Isolation and purification of glu- trum of sulforaphane was in agreement with previously published by Spencer coraphanin from broccoli seeds. at al (39) Glucoraphanin was prepared from broccoli seeds with boiling water, purified and concentrated by SPE and preparative scale HPLC, ac- cording to Rochfort et al (38), using method 3 for the concentra- tion of the glucoraphanin. To evaluate the purity of glucora- phanin, the aqueous fraction was submitted to modified method, following described, for determi- nation of relative breakdown sul- foraphane, that was quali-quanti- tatively analysed by GC/MS. The chromatogram in figure 2 shows that the substance content in the fraction with 22-25 min of reten- by GC/MS was per- The combined filtrates were redu- tion time is glucoraphanin, becau- formed according to VanEtten et ced to about 10 mL under vacuum se its breakdown product is sulfo- al (34, 35). rotavapor at T< 60°C. The con- raphane. In fact the mass spec- centrate was centrifuged at 4000 trum is characteristic of sulfora- Determination of sulforaphane rpm for 10 min and the superna- phane compounds and the reten- (modified method) tant diluted to 25 mL with distil- tion time is the same of the com- led water. mercial standard. 15 g of fresh or 1.5 g of frozen dry Enzymatic conversion of glucora- GC/MS determination of iso- samples were homogenized with phanin in sulforaphane was done as breakdown pro- 50 mL of boiling methanol. The in a 100 ml teflon tube, adding ducts of glucosinolates by VanEt- mixture was boiled for 15 min in a 12.5 mL of phosphate buffer solu- ten et al. (original method) flask covered with a watch glass. tion pH=7 (30.5 ml of Na2HPO4·

The extraction of glucosinolates After cooling, the mixture was 2H2O 0.2 M and 19.5 mL of from brassica samples, their ab- centrifuged at 4000 rpm for 10 NaH2PO4·1H2O 0.2 M were dilu- sorption on the anion exchange re- min and than filtered on glass ted to 100 mL with distilled wa- sin (DOWEX1x2), their hydroly- wool. The residue was dispersed in ter) and 40 mg of thioglucosidase sis with aqueous myrosinase prepa- 30 mL of 70:30 methanol:water (E.C. 3.2.3.1. Sigma Aldrich, Mi- ration (39) to isothiocyanates in solution, homogenized for 5 min lano) to 12.5 mL of homogenate. methylene chloride and finally the and than performed as described The tube was tapped and shacked quali-quantitative analysis of iso- hereafter. for 60 min horizontally in a

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box–typer shaker at 80-100 oscil- Figure 3 - Regression curve obtained by comparison VanEtten and modified lations for minute at room tempe- methods, using standard (ST) solutions of Allyl NCS, sulforaphane and bras- rature. The mixture was added of sica extracts (SAMPLES). 25 mL of methylene chloride, – – – – = regression curve of Allyl NCS; shacked for 5 min and centrifuged — – – — = regression curve of sulforaphane. for 10 min at 4000 rpm. Data are expressed as µmol g-1. The methylene chloride fraction was filtered through a paper filter in a 250 mL glass flask. The ho- mogenate was extracted a second time as described above. Both methylene chloride fractions were pooled, dried over anidrous

Na2SO4 and then filtered trough a paper filter. The filtrate was con- centrated until 1 mL under redu- ced pressure at 35°C using a rotary evaporator. The methylene chlori- de extract was injected into GC/MS (6890 N network GC (MSD). Compounds were identi- (3.37-23.61 µmol g-1, purchased system; 5973Mass Selective detec- fied by comparing spectra of stan- from SIGMA) and some fresh tor, Agilent Technologies). dard and spectra contained in the brassica samples (broccoli, cauliflo- For the qualitative analysis, 1 µL instrument library, or by compa- wer “Romanesco”, “violetto di Ca- of the extract was injected at ring retention time of standard. tania” and “verde di Macerata”). In 200°C into a DB-1 capillary co- The calculation of sulforaphane figure 3 is reported the regression lumn (60m x 0.25 mm i.d., 0.25 and allyl NCS concentrations we- curve of breakdown products of µL film thickness, J & W Scienti- re based on the response factor, glucoraphanin (sulforaphane), raw fic, Folsom, CA). The column using solutions standard of sulfo- brassica samples (sulforaphane) temperature was kept at 50°C for raphane and allyl NCS (SIGMA) and (allyl NCS). Linear 5 min and than raised to 240°C at to encompass three series of con- regression of concentrations of 2°C/min heating rate; finally it centrations. standard solution and raw samples was kept at 240°C for 20 min. The obtained with two methods give a inlet flow and the split of the car- Methods comparison correlation coefficient of 0.9675 rier (He) were set at 2 mL/min for allyl NCS and 0.9871 for sul- and 10 mL/min, respectively. The The modified method was compa- foraphane both in the standard so- temperature of the transfer-line red with the VanEtten method lution and in the brassica samples. was 240°C. The MS spectra was using four standard solutions of In the modified method, the generated at 70 eV, and the 10- glucoraphanin, (2.95-20.63 µmol quantities of starting samples and 400 Amu mass range was selected g-1, prepared following the Roch- solvents (methanol, methylene for mean square displacement fort et al (38) method), sinigrin chloride) are reduced and the

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anion exchange resin is not used Figure 4 - Time course of sulforaphane formation when 12,5 mL of extract to separate glucosinolates from are incubated with 40 mg of thioglucosidase and 12,5 mL of phosphate buf- free sugars to the original method. fer solution pH=7 at room temperature. Mean values of three replicates for For our purposes is not important each solution to know the content of total glu- cosinolates, determined by the glucose hydrolyzed from glucosi- nolates. Besides, to establish the optimum yield of sulforaphane the enzyme hydrolysis of relative glu- cosinolate is performed for various time (15, 30, 60, 120 min) at pH=7 and room temperature on a pool of examined raw extracts. Fi- gure 4 shows that the optimum yield is obtained after 30-60 min of incubation. For the determination of reprodu- values were considered signifi- vegetables. These decreases de- cibility, a sample of each raw culti- cantly different when ρ≤0.05. pend on the cooking method used var is extracted and analysed four and on the type of cultivar. The times. The relative standard devia- sulforaphane quantity retained in- tion of sulforaphane concentration Results to the vegetables after cooking, is ranged between 4.91 and 7.25%. expressed as % retention. Among the Italian brassica culti- In the figure 5 are reported the % Dry matter vars, the higher levels of sulfora- retentions of sulforaphane in all phane is found in fresh broccoli the treated vegetables. The samples Four samples of each cultivar, raw (25 µmol g-1) (Tab. 1), while in the cooked by microwave have the hi- and treated, were dehydrated in a cauliflower “Jesino”, white cabba- ghest retentions respect of the laboratory oven kept at 85°C until ge, cauliflower “verde di Macerata” other cooking methods, except the a constant weight. The moisture and white cauliflower commercial cauliflower “verde di Macerata”, content was calculated from the hybrid show the lowest values, where the retention of Mw and difference between the wet and ranged among 0.25-1.60 µmol g-1. boiling water cooking are similar. the dry weight divided for the wet Levels between 4-15 µmol g-1 are The retentions of boiling water are weight (40). respectively ascertained in the cau- superior than the other methods liflower “violetto di Catania” and in broccoli, cauliflower “violetto di Statistical analysis “Romanesco”. From the same ta- Catania”, “verde di Macerata” and ble it is noted that all the cooking white cabbage commercial hybrid. The Tukey HSD test was used to methods cause a decrease on sul- The retention of boiling water are evaluate the difference between foraphane content in all the exa- similar in white cabbage cooked fresh and treated samples. Mean mined cultivars respect of the raw by steam, inferior in cauliflower

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Table 1 - Sulforaphane content (µmol g-1 d.m.) in raw and cooked cultivars. Mean values of three replicates for each cultivar.

Raw Mw Boiling water Steam Pan Broccoli 24.62 a A 17.98 a B 12.26 a C 9.87 a C 5.93 a D C. Romanesco 14.76 b A 10.13 b B 5.77 b C 6.42 b C 5.17 a C C. violetto di Catania 4.44 c A 3.84 c AB 3.33 c B 0.82 c D 1.84 b C C. verde di Macerata 1.02 de A 0.61 d B 0.62 d B 0.52 c B 0.26 c C C. Jesino 0.25 e A 0.17 d B 0.09 d C 0.05 d D 0.12 c C C. Commercial hybrid 1.60 d A 1.14 d B 0.95 d C 0.77 c D 0.22 c E White cabbage 0.89 de A 0.67 d B 0.41 d C 0.41 c C 0.23 c C Small letters indicate statistically significant difference among cultivars in the same cooking method (ρ≤0,05). Capital letters indicate stati- stically significant difference among cooking methods in the same cultivar (ρ≤0,05). C = cauliflower

Figure 5 - % retention of sulforaphane in the cooked samples. Mean values of three replicates for each cultivar. Small letters indicate statistically significant difference among cultivars in the same cooking method (ρ≤0,05). Capital let- ters indicate statistically significant difference among cooking methods in the same cultivar (ρ≤0,05). C = cauliflower

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“Romanesco” cooked by steam and Glucosinolates are relatively stable presence of olive oil. In this case, cauliflower “Jesino” by pan-co- at high temperature (≤100°C) and the high losses of sulforaphane oking. water soluble (29). Microwave, can be also depended on the ther- The retentions of the steam are boiling water and steam cooking mal degradation of glucoraphanin. higher respect of pan cooking in are performed around 100°C. In Oerlemans et al (43) studied the broccoli, cauliflower “Romanesco”, this condition, glucosinolates are thermal degradation of glucosino- “verde di Macerata”, commercial stable and the decrease of glucosi- lates in red cabbage submitted to hybrid and white cabbage. While nolates and consequently of sulfo- three different heat treatments they are inferior only in cauliflo- raphane is due to removal of intact (blanching, cooking and canning). wer “violetto di Catania” and “Jesi- glucosinolates from vegetable tis- They noted that the glucosinolates no” by pan-cooking. sues in the cooking water. This be- are not influenced during blan- So, when the vegetables are co- haviour is according to Slominski ching (3 min at 95°C); while its oked, we can see that in Mw the et al (42). They demonstrated that losses during boiling water co- retentions of sulforaphane content 90% of intact glucosinolates or oking (40 min at 100°C) and can- are the highest as concern all the their breakdown products, were ning (40 min at 120°C) were re- cultivars and range 61-86%. The found in the vegetable tissues spectively 10 and 85%. However, retentions of sulforaphane content (40%) and in the cooking water some ITCs such as sulforaphane decrease between 38-74% in the (50%), when the cabbage was co- in the first step of cooking, may boiling water, lower values are oked in water for 40 min. The ex- escape into the air or degrade to showed respectively in steam (18- planations of the differences in the volatile sulphur compounds 52%) and pan cooking (14-48%). losses of sulforaphane and other (44). Recent researches have de- ITCs after heat treatments, could monstred indigenous myrosinase depend on the cultivar, leaf thick- activity in the gut microflora (45). Discussion ness and waxes, size of florets, fi- However, little is known about the bre content, diffusivity of the glu- amounts of ITCs produced by the The most of sulforaphane content cosinolates in vegetable tissues human microflora from glucosino- decrease in all the examinated (Rosa et al, Sones et al) (24, 41). lates. Krul et al (45), in an intetsti- brassica cultivars, when the vegeta- The loss of glucosinolates from nal model system with a pooled bles are submitted to cooking vegetable depends on the quantity and culturated human microflora, treatments. of the processing water. Higher is found that the conversion of sini- As ITCs are breakdown products the quantity of water and higher is grin into allyl NCS ranged bet- of glucosinolates, the decrease of their losses. In our experiments ween 10 and 30% and allyl NCS is ITCs depended on the corrispecti- the quantity of water used for mi- converted further in other unk- ve decrease of glucosinolates. Los- crowave (800 mL) is smaller re- nown metabolites. ses of glucosinolates were reported spect of the boiling water (1300 More recently, Iori et al (46) have in previous studies by Sones et al mL) and steam (1500 mL). This studied the in vitro gastrointestinal (41). They noted that in cabbage can explain the better recoveries of digestion of broccoli, using as eva- and cauliflower cooked in boiling sulforaphane during Mw cooking. luation index glucosinolates, phe- water for 10 min, glucosinolates In the pan cooking, the tempera- nolic compounds and . were reduced about 20-30%. ture is higher (160-180°C) for the

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Conclusions well RG. Glucosinolate composition Kelloff G J, Eds. CRC Press, Boca Ra- of seeds from 297 species of wild ton, FL, 1992: 227-45. plants. Phytochemistry 1991; 30: 11. Chung FL, Morse LA, Eklind KI, Le- Brassica cultivars are of great inte- 2623-38. wis J. Quantitation of human uptake of rest in the field of human nutrition 2. Fenwick GR, Heaney RK, Mullin WJ. the anticarcinogen phenethyl isothioc- and disease prevention. These ca- Glucosinolates and their breakdown yanate after a watercress meal. Cancer products in and plants. CRA Crit Epidemiol. Biomarkers Prev 1992; 1: pacities are due to the ITCs, such Rev Food Sci Nutr 1983; 18: 123-201. 383-8. as sulforaphane, produced by the 3. VanEtten CH, Tookey HL. Glucosi- 12. Hecht SS. Chemoprevention by iso- enzymatic hydrolysis of glucora- nolates in cruciferous plants. In Herbi- thiocyanates. J Cell Biochem 1995; 22: phanin. The used GC/MS me- vores, Their Interaction in Secondary 195-209. Plant Metabolites; Rosenthal GA, 13. Guo Z, Smith TJ, Wang E, et al. Ef- thod, is sensitive, reproducible and Janzen DH, Eds; Academic Press: fects of phenethyl , a car- suitable for routine analysis and is New York, 1979: 507-520. cinogenesis inhibitor, on xenobiotic- applied to determine sulforaphane 4. Uda Y, Kurata T, Arakawa N. Effects metabolizing enzymes and nitrosamine in some fresh and treated brassica of pH and ferrous ion on the degrada- metabolism in rats. Carcinogenesis tion of glucosinolates by myrosinase. 1992; 13: 2205-10. cultivars. There is a substantial lea- Agric Biol Chem.1986; 50: 2735-40. 14. Sparnis VL, Venegas PL, Wattenberg ching of sulforaphane during co- 5. Iori R, Barillari J, Tatibouet A, Rollin P, LW. Glutathione S-transferase activity: oking processes. If the heat treat- Riva A, Morazzoni P. Estrazione di enancement by compounds inhibiting ment is performed around 100°C glucosinolati da e loro tra- chemical carcinogenesis and by dietary sformazione enzimatica in isotiocianati, constituents. J Natl Cancer Inst 1982; as boiling water, steam and micro- potenziali agenti chemiopreventivi e/o 68: 493-6. wave cooking, almost all these los- agenti chemioterapici. XVI Conv. Naz. 15. Mithen RF, Dekker M, Verkerk R, Ra- ses are accounted for as intact glu- Chimica Farmaceutica, 18-22/09/2002, bot S, Johnson IT. The nutritional si- cosinolates in cooking water. Abo- Sorrento (I); atti del convegno L42, 76. gnificance, biosynthesis and bioavailabi- 6. Nishie K, Daxenbichler ME. Toxico- lity of glucosinolates in human . J ve 100°C (pan cooking) its losses logy of glucosinolates, related com- Sci.Food Agric 2000; 80: 967-84. can depend on the thermal degra- pounds (nitriles, R-, isothiocya- 16. Bogaards JJ, van Ommen B, Falke HE, dation. Among the cooking me- nates) and vitamin U found in crucife- Willems ML, van Bladeren PJ. Gluta- thods, microwave is thought to be rae. Food Cosmet Toxicol 1980; 18: thione S-transferase subunit induction 1191-4. pattern of Brussels sprouts, allyl iso- an efficient alternative for cooking 7. Barillari J, Rollin P, Iori R. Isolation of thiocyanate and goitrin in rat liver and these vegetables due to the low dietary glucosinolates and their derived small intestinal mucosa: a new approach amount of cooking water required isothiocyanates involved in cancer pre- for the identification of inducing xeno- and shorter cooking times. The co- vention. NutraCos Nov/Dec 2002: 6-9. biotics. Food Chem Toxicol 1990; 28: 8. Faulkner K, Mithen R, Williamson G. 81-8. oking water can be used as base in Selective increase of the potential anti- 17. Bogaards J, Verhagen H, Willems ML, soup or gravy, so that glucorapha- 4-methylsulfinylbutyl gluco- van Bladeren PJ. Consuption of brussels nin or its breakdown product (sul- sinolate in broccoli. Carcinogenesis sprouts results in elevated α-class gluta- foraphane) may enter the body 1998; 19: 605-9. thione S-transferase levels in human 9. Knasmuller S, Schwab CE, Land SJ, et blood plasma. Carcinogenesis 1994; 15: with the part remaining in the co- al. Genotoxic effect on the therocyclic 1073-5. oked vegetables. amines in human derived eparome 18. Negri E, La Vecchia C, Franceschi S, (Hep 42). Cells Mutagenesis 1999; 14: D'Avanzo B, Parazzini F. Vegetable and 533-40. fruits consumption and cancer risk. Int References 10. Chung FL. Chemoprevention of lung J Cancer 1990; 48: 350-4. carcinogenesis by aromatic isothiocya- 19. Nijhoff WA, Mulder TPJ, van Poppel 1. Daxenbichler ME, Spencer GF, Carl- nates. In Cancer Chemoprevention: G, Peters WHM. Effects of consuption son DG, Rose GB, Brinker AM, Po- Wattenberg L, Lipkin M, Boone C W, of brussels sprouts on plasma and uri-

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