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Changes in glucoraphanin content and quinone reductase activity in broccoli (Brassica oleracea var. italica) florets during cooling and controlled atmosphere storage
Postharvest Biology and Technology 42 (2006) 176–184
Changes in glucoraphanin content and quinone reductase activity in broccoli (Brassica oleracea var. italica) florets during cooling and controlled atmosphere storage Chao-Jiong Xu, De-Ping Guo, Jing Yuan, Gao-Feng Yuan, Qiao-Mei Wang ∗ Department of Horticulture, Zhejiang University, The State Agriculture Ministry, Laboratory of Horticultural Plant Growth, Development & Biotechnology, Hangzhou 310029, China Received 6 January 2006; accepted 19 June 2006
Abstract The effects of cooling and controlled atmosphere (CA) treatments on glucoraphanin content and induction of quinone reductase (QR) activity in broccoli florets were investigated. The results showed that both the glucoraphanin content and QR activity of broccoli florets increased during the initial 6 day of storage, and then decreased markedly at different temperatures of 0, 5, and 10 ◦ C. Immediate cooling of broccoli florets at 0 and 5 ◦ C could preserve the glucoraphanin content and QR activity for 12 days. Keeping broccoli florets at 20 ◦ C for 6 h before cooling at 5 ◦ C had little effects on shelf-life, glucoraphanin content and QR activity. CA treatments with elevated CO2 (21% O2 + 10% CO2 , and 21% O2 + 20% CO2 ) and an air treatment (21% O2 ) were found to increase the glucoraphanin content and the QR activity over the first 5 days of storage at 5 ◦ C, while CA treatments with reduced O2 concentrations (1% O2 , 1% O2 + 10% CO2 ) led to a steady decrease of glucoraphanin content and the QR activity during 20 days of storage at 5 ◦ C. The highest content of glucoraphanin and QR activity was found in florets stored under 21% O2 + 10% CO2 at 5 ◦ C. These conditions were able to maintain the visual quality, glucoraphanin content and QR activity of the broccoli florets for 20 days. © 2006 Elsevier B.V. All rights reserved. Keywords: Broccoli; Glucoraphanin; Quinone reductase; Controlled atmosphere (CA); Cooling
1. Introduction Glucosinolates are a group of sulfur- and nitrogencontaining secondary metabolites in cruciferous vegetables (Poulton and Møller, 1993). Upon tissue damage, glucosinolates are released from plant vacuoles and rapidly hydrolyzed by myrosinase (-thioglucoside glucohydrolase, EC 3.2.3.1) to glucose and unstable thiohydroximate-O-sulfonate intermediates which, as dictated by chemical conditions, spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles. Production of isothiocyanates is usually favored by neutral conditions (Fenwick et al., 1983; Bones and Rossiter, 1996; Fahey et al., 2001). Broccoli contains high
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levels of glucoraphanin, the glucosinolate precursor of sulforaphane. It has been reported that sulforaphane is an extremely potent monofunctional inducer of quinone reductase [NAD(P)H: (quinine-acceptor) oxidoreductase, EC 1.6.99.2, QR] in murine hepatoma cells (Zhang et al., 1992), and is the most potent, naturally occurring, monofunctional phase 2 enzyme inducer identified thus far (Talalay et al., 1995). The direct assay of quinone reductase activity in murine Hepa 1c1c7 cells provides a rapid and reliable indicator of the ability of vegetable extracts to induce enzymes that detoxify carcinogens (Prochaska and Santamaria, 1988; Prochaska et al., 1992), and hence of putative anticarcinogenic activity via a blocking mechanism (Wattenberg, 1985). The QR assay system has also been used to detect inducers of anticarcinogenic enzymes in human diet (Tawfiq et al., 1994), and assess the potential of extracts from cruciferous vegetables (Prochaska et al., 1992) and chemo-
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preventive glucosinolates (Gross et al., 2000; Wang et al., 2002). Broccoli is a highly perishable product and its visual and organoleptic qualities greatly depend on the storage conditions (Makhlouf et al., 1989). The effects of different storage methods on shelf-life and visual quality of broccoli heads have been widely investigated (Makhlouf et al., 1989; Izumi et al., 1996; Hansen et al., 2001a,b). During the last decade, storage of broccoli florets has also been studied with the increasing demand for minimally processed vegetables (Bastrash et al., 1993; Izumi et al., 1996). As with the storage of broccoli heads, low temperatures and controlled atmospheres are usually recommended for the storage of broccoli florets (Izumi et al., 1996). Even though glucosinolate is one of the main health-promoting secondary metabolites in broccoli, changes in glucosinolate levels of broccoli under various storage conditions have not been systematically studied until recently, and reports on the effect of postharvest storage on glucosinolate contents of broccoli are sparse, and sometimes contradictory. Storage of broccoli in modified atmosphere packaging (MAP) and refrigeration at 4 ◦ C has been shown to maintain glucoraphanin contents and visual quality of the broccoli heads for at least 10 days, and controlled atmosphere storage (1.5% O2 + 6% CO2 ) at 4 ◦ C could maintain glucoraphanin contents up to 25 days after harvest with an increase in glucoraphanin content compared to storage at air (Rangkadilok et al., 2002). However, a reduction of 48% of glucoraphanin content in broccoli after 7 days of storage under MAP (17% O2 + 3% CO2 ) at 1 ◦ C has been observed (Vallejo et al., 2003). Controlled atmospheres may exert an influence on the content of glucosinolates. Hansen et al. (1995) reported that both total glucosinolates and methylsulfinylalkyl glucosinolate (glucoiberin and glucoraphanin) contents of broccoli florets increased at day 7 and followed by a slight decline at day 9 as the broccoli deteriorated when stored under air and controlled atmospheres (0.5% O2 , 0.5% O2 + 20% CO2 , and 20% CO2 ). Similarly, a significant rise of glucoraphanin was also found in broccoli heads stored under a controlled atmosphere (1.5% O2 + 6% CO2 ) at 4 ◦ C (Rangkadilok et al., 2002). As more attention is paid to the effect of storage conditions on visual quality, limited information is available on the effects of different storage conditions on chemopreventive glucosinolates such as glucoraphanin and phase 2 enzyme (QR) induction ability of broccoli produce, especially minimally processed broccoli florets. It is still an open question if the storage conditions which keep broccoli florets with good visual quality can also preserve the glucoraphanin levels and QR induction ability of broccoli florets. The present study was undertaken with the objective of developing a QR assay for measuring the QR induction ratio of extracts, and investigating the effects of different cooling and controlled atmosphere conditions on glucoraphain contents and the induction of QR activity of broccoli florets.
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2. Material and methods 2.1. Plant material Broccoli heads (Brassica oleracea var. italica cv. Luling) of prime quality were harvested in the early morning from the greenhouse of Zhejiang University (Hangzhou, China), topiced and then transported to the postharvest laboratory of the Vegetable Institute, Zhejiang University within 10 min. Broccoli heads with a diameter ranging from 15 to 20 cm were chosen, and the inner branches (with florets having stalks of approximately 2 cm) were then cut from these heads for experimentation. The florets were surface-sterilized by washing with a solution of 50 ppm NaOCl for 1 min and dried using a household model spin drier for 2 min. The florets were then randomly distributed into the required number of treatment units, each with four florets (roughly 100 g). 2.2. Postharvest treatments 2.2.1. Cooling treatments Broccoli florets were divided into three groups, and stored in dark rooms at 95% RH and temperatures of 0, 5, and 10 ◦ C, respectively. There were three replicates per temperature treatment at each sampling and three treatment units per replicate. The sampling times were 3, 6, 9, and 12 days after harvest. 2.2.2. Cooling delay treatments The florets from freshly harvested broccoli heads were divided into five groups, immediately transferred to 5 ◦ C storage or transferred to 5 ◦ C storage after being kept at 20 ◦ C for 3, 6, 12, and 24 h, respectively. There were three replicates per cooling delay treatment at each sampling time and three treatment units per replicate. The sampling times were 3, 6, 9, and 12 days after treatment. 2.2.3. Controlled atmosphere treatments at 5 ◦ C The florets were stored in 3.8 l glass jars, closed with a neoprene rubber stopper fitted with inlet and outlet polyethylene tubes. The jars were placed in a room at 5 ◦ C for 20 days and ventilated with humidified gas at 2.8 l h−1 . The humidified gas mixtures were established by controlling air, nitrogen, and carbon dioxide flows through different size capillary tubes and verified by gas chromatography. The gas mixtures for controlled atmosphere treatments were as follows: 21% O2 + 10% CO2 , 21% O2 + 20% CO2 , 1% O2 , and 1% O2 + 10% CO2 . There were three replicates per controlled atmosphere treatment at each sampling time and three treatment units per replicate. The sampling times were 5, 10, 15, and 20 days after harvest. 2.3. Determination of shelf-life Shelf-life was determined according to the method of Ku and Wills (1999) with minor modification. The time for qual-
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ity to decline to 30% yellowing or slight rotting was defined as the end of shelf-life for the florets. 2.4. Sample preparation and freeze-drying Freshly harvested and stored broccoli florets were frozen in liquid N2 and kept in polyethylene bags at −70 ◦ C until freeze-drying. Freeze-dried broccoli samples were stored in sealed polyethylene bags at 4 ◦ C until analysis. The samples were weighed fresh and re-weighed after being freeze-dried to determine the ratio of fresh weight to dry weight. 2.5. Glucosinolate assay Glucosinolates were extracted and analyzed as previously described with minor modifications (Wang et al., 2002). Freeze-dried samples (25 mg) were boiled in 1 ml water for 10 min. After recovery of the liquid, the residues were washed with water (1 ml), and the combined aqueous extract was applied to a DEAE-Sephadex A-25 (40 mg) column (pyridine acetate form). The column was washed three times with 20 mM pyridine acetate and twice with water. The glucosinolates were converted into their desulpho analogues by overnight treatment with 100 l of 0.1% (1.4 units) aryl sulphatase, and the desulphoglucosinolates were eluted with 2 × 0.5 ml water (Hogge et al., 1988). HPLC analysis of desulphoglucosinolates was carried out using a Shimadzu (Tokyo, Japan) mode VP liquid chromatograph with a dual wavelength spectrophotometer. Samples (100 l) were separated at 30 ◦ C on a Waters Spherisorb C18 column (150 mm × 4.6 mm i.d.; 5 m particle size) (Milford, MA 01757, USA) using acetonitrile and water at a flow rate of 1.0 ml/min. The procedure employed isocratic elution with 1.5% acetonitrile for the first 5 min; a linear gradient to 20% acetonitrile over the next 15 min followed by isocratic elution with 20% acetonitrile for the final 10 min. Absorbance was detected at 226 and 280 nm. Ortho-nitrophenyl--dgalactopyranoside (Sigma) was used as an internal standard for HPLC analysis. Concentrations of individual glucosinolate were determined according to published response factors (Haughn et al., 1991). The integrated area of the desulpho4-methylsulphinylbutyl glucosinolate peak was converted to a molar amount under the assumption that this compound has a molar extinction coefficient at 226 nm equal to that of sinigrin (Wang et al., 2002). 2.6. Quinone reductase assay 2.6.1. Preparation of extracts for quinone reductase assay Extracts from the freeze-dried samples were assayed for their induction activity in murine hepatoma Hepa 1c1c7 cells. A sample of 20 mg was homogenized for 1–2 min with a polypropylene pestle in 1.5 ml plastic tubes containing 200 l extraction buffer (5 mM K2 HPO4 –KH2 PO4 , 1 mM EDTA, pH 7.6). The pestle was rinsed with 300 l extraction buffer
and the homogenate was centrifuged twice for 10 min at 16,000 × g to recover the supernatant. The resulting supernatants were filtered through sterile non-pyrogenic filters (0.22 m) and stored at −70 ◦ C until use. 2.6.2. Induction of cultured Hepa 1c1c7 murine hepatoma cells Hepa 1c1c7 murine hepatoma cells were grown in a minimal essential medium supplemented with 10% foetal calf serum in a humidified incubator in 5% carbon dioxide at 37 ◦ C as described previously (Prochaska and Santamaria, 1988; Prochaska et al., 1992; Grubb et al., 2002). In order to monitor the inducer potency of plant extracts, Hepa 1c1c7 murine hepatoma cells were grown in 96-well microtiter plates. Typically, 10,000 Hepa 1c1c7 cells were seeded into each well and grown for 24 h in 200 l medium per well, and then induced for 24 h by the addition of 200 l medium containing serial dilutions of the floret extract to be assayed. Usually, the floret extract was diluted to the concentrations needed with cell culture medium, and the concentrations of each extract were expressed as dry weight (DW) of original material for each milliliter of cell culture medium. The two-fold serial dilutions were prepared in the microtiter plate using an octapipet. 2.6.3. Quinone reductase assay After exposure of Hepa 1c1c7 cell cultures to floret extracts, QR activity was assayed in hepatoma cell lysates, using menadione, MTT, and an NADPH-generating system (Prochaska and Santamaria, 1988). The cell culture medium was decanted and 50 l of lysing solution (0.8% digitonin in 2 mM EDTA, pH 7.8) was added to each well. Cells were lysed for 10 min at 37 ◦ C followed by shaking (100 rpm) for 10 min at room temperature. To each well was added 200 l of the QR assay solution containing 25 mM Tris buffer (pH 7.4), 1 mM glucose-6-phosphate, 50 M menadione, 30 M NADP, 5 M FAD, 0.07% (w/v) bovine serum albumin, 0.03% (w/v) MTT, 0.01% (v/v) Tween-20, and 1 unit/ml of yeast glucose-6-phosphate dehydrogenase (Gross et al., 2000). The reaction mixtures were incubated at room temperature and reactions were terminated after 10 min by the addition of 50 l of 0.1 M HCl. A reagent blank was prepared by adding stop solution to one column of each plate before the addition of assay solution (zero time control). Absorbances were measured by scanning the microtitre plates at 595 nm, and QR inducer potency was calculated as the QR activity (A595 ) ratio of treated to untreated cell cultures. Cell densities were determined by staining with crystal violet as previously described (Fahey et al., 1997; Gross et al., 2000). 2.7. Statistical analyses Means and standard errors of shelf-life were calculated. Data of glucoraphanin contents and QR induction ratios were analyzed by analysis of variance (ANOVA model one-way). Sources of variation were storage duration and treatments. The means were compared by the least significant differences
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(LSD) test at a significance level of 0.05. STATISTICA Base (a stand-alone product), STATISTICA 6.0 (StatSoft Inc.) was used in data tests.
3. Results and discussion 3.1. Induction of quinine reductase in murine hepatoma cells by broccoli floret extracts Methylsulfinylalkyl isothiocyanates are potent and specific inducers of mammalian phase 2 detoxification enzymes such as QR or glutathione S transferase (Zhang and Talalay, 1994). Since several methylsulfinylalkyl glucosinolates are synthesized in broccoli, and glucoraphanin is the most abundant glucosinolate in florets (Fahey et al., 1997; Brown et al., 2002), we tested the potency of broccoli floret extracts in inducing QR activity in cultured Hepa 1c1c7 murine hepatoma cells. As indicated by the colorimetric QR bioassay, broccoli florets contain readily detectable levels of phase 2 enzyme inducers (Figs. 1 and 2). Maximal induction (about 3.6-fold) of QR specific activity by floret extracts was achieved at a concentration of 2 mg DW ml−1 medium. Examination of detectable cytotoxicity showed that broccoli extract at 2 mg DW ml−1 medium reduced cell density by 16%. However, the cytotoxic effect was negligible at and below 1 mg DW ml−1 medium (Figs. 1 and 2). In our study, the extracts of broccoli floret samples from different treatments were diluted to the concentration of 1 mg DW ml−1 medium for the QR assay, and eight wells were used for
Fig. 2. Induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. Data are expressed as ratios of treated cells to control cells for QR activity and cell density. Specific QR activity is the ratio of QR activity and the cell density at the same concentration.
every sample in 96-well microtiter plates. In order to reach sufficient reproducibility of the QR assay, the florets were cut from top-quality heads at a uniform size to minimize the variation between different heads. To assess the head to head variation of the assay, we measured QR induction of floret extracts prepared from the different newly harvested heads at the concentration of 1 mg DW ml−1 medium in the same container (data not shown). Here, the QR inducer potency was expressed as the QR activity ratio of floret extract in treated versus untreated hepatoma cells. The standard deviation of the mean QR inducer potency was found to be 4%. The data demonstrate that the QR bioassay of broccoli florets we developed was reliable. 3.2. Effect of storage temperatures on glucoraphanin content and QR activity
Fig. 1. Colorimetric assay of QR activity (A) and of cell density (B). Hepa 1c1c7 murine hepatoma cells were grown in 96-well microtiter plates and induced with serial two-fold dilutions of broccoli floret extract (only one half of each assay plates is shown). Each column of wells received the same dilution of a single plant extract. A more intense color denotes higher QR activity and higher cell density.
Different cooling temperatures had a great influence on the shelf-life of broccoli florets. The shelf-life of florets stored at 0, 5, and 10 ◦ C was found to be 34.7, 17.5, and 9.0 days, respectively (data not shown). In the present experiment, a significant reduction of both glucoraphanin content and QR activity was observed in broccoli florets during 9 days storage at 10 ◦ C compared to 0 and 5 ◦ C (Fig. 3). Glucoraphanin content was increased by cooling treatments during the first 6 days of storage, and the highest content was observed in broccoli florets stored at 0 ◦ C (32% increase), followed by 5 ◦ C (20% increase) and 10 ◦ C (2% increase) at day 6 (Fig. 3). After day 6, glucoraphanin content decreased in broccoli florets stored at 0 and 5 ◦ C, by 2% and 23%, respectively, at day 12, although the broccoli florets were still in good visual condition. A great decrease in glucoraphanin content was observed in broccoli florets stored at 10 ◦ C by day 9 (60% loss), which coincided with a rapid loss in visual quality of the broccoli florets (Fig. 3). These results were similar to those of Hansen et al. (1995) who observed an increase in methylsul-
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C.-J. Xu et al. / Postharvest Biology and Technology 42 (2006) 176–184 Table 1 Effects of cooling delay on shelf-life of broccoli at 5 ◦ C Hours at 20 ◦ C
Shelf-life at 5 ◦ C (days)
0 3 6 12 24
17.5 17.3 16.5 15.3 8.7
± ± ± ± ±
0.69 a 0.53 a 0.66 a 0.42 a 0.3 b
The broccoli florets were transferred to 5 ◦ C storage after they have been kept in 20 ◦ C for 3, 6, 12, or 24 h. Values represent the means of three replicate samples with standard error of the means. Values followed by the same letter are not significantly different at P = 0.05.
Fig. 3. Effects of storage temperature on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were stored at 0, 5, or 10 ◦ C. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96-well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate). The vertical bars represent the LSD at a significance level of 0.05.
phinylalkyl glucosinolates content in broccoli florets during 7 days storage under air at 10 ◦ C followed by a slight decline at day 9 as the broccoli deteriorated. The trend in activity of quinone reductase was similar to that of glucosinolate content (Fig. 3). A steady increase in QR induction ratio was observed in broccoli florets stored at 0, 5, and 10 ◦ C during the first 6 days of storage, and the QR induction ratio increased by 33%, 32%, and 13% respectively, at day 6. However, the QR induction ratio decreased dramatically after day 6 (Fig. 3). Storage of broccoli florets at 0 and 5 ◦ C could maintain the glucoraphanin content and QR activity for 12 days. 3.3. Effects of cooling delay on glucoraphanin content and QR activity Although “cool as soon as possible” is the general recommendation for broccoli handling, delays of several hours
before cooling may occur in postharvest handling of broccoli, especially when the broccoli is treated with forced air or hydrocooling instead of immediate liquid-ice cooling in field. Our results showed that keeping broccoli florets at 20 ◦ C for 3 and 6 h before cooling at 5 ◦ C did not significantly affect the shelf-life, glucoraphanin content, and QR activity compared to the controls (immediate cooling at 5 ◦ C), but 24 h at 20 ◦ C significantly decreased the shelf-life glucoraphanin content, and QR activity (Table 1, Fig. 4). The increase in glucoraphanin content and QR induction ratio was observed during the first 6 days storage in treatments with 12 or less hours at 20 ◦ C. However, the glucoraphanin content and QR induction ratio decreased in all treatments from day 6. Only slight reductions of glucoraphanin content and QR activity were observed in broccoli florets during 9 days of storage at 5 ◦ C, when they were treated with 6 or less hours at 20 ◦ C before cooling at 5 ◦ C. The glucoraphanin content and QR induction ratio decreased steadily over 24 h at 20 ◦ C (Fig. 4). The above results suggest that keeping florets at 20 ◦ C should be for less than 6 h in order to maintain visual quality, glucoraphanin content and QR activity. 3.4. Effect of controlled atmospheres on glucoraphanin content and QR activity The glucoraphanin content and QR induction ratio in broccoli florets fluctuated moderately during 20 days storage under air and CA treatments at 5 ◦ C (Fig. 5). Significant increases in glucoraphanin content during 20 days storage and in the QR induction ratio during 15 days of storage were observed in broccoli florets under the 21% O2 + 10% CO2 treatment compared to air, while significant reductions in glucoraphanin content and QR induction ratio were observed in the 1% O2 treatment compared to air during 20 days storage (Fig. 5). During the initial 5 days of storage, the glucoraphanin content and QR induction ratio increased in all three treatments with 21% O2 , and the greatest increase was observed in the 21% O2 + 10% CO2 treatment, with the 21% O2 treatment next and the 21% O2 + 20% CO2 the least (Fig. 5). After CA treatment, the glucoraphanin content and QR induction ratio decreased in all these treatments (Fig. 5). The glucoraphanin content and QR induction ratio decreased steadily during 20 days storage under 1% O2 and 1% O2 + 10% CO2 , causing 63% and 54% losses in gluco-
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Fig. 4. Effects of cooling delay on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were transferred to 5 ◦ C storage after they have been kept in 20 ◦ C for 3, 6, 12, or 24 h. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate). The vertical bars represent the LSD at a significance level of 0.05.
Fig. 5. Effects of controlled atmosphere storages on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were stored under low O2 (1% O2 ), high CO2 (21% O2 + 10% CO2 , 21% O2 + 20% CO2 ), or low O2 plus high CO2 (1% O2 + 10% CO2 ) at 5 ◦ C. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate) The vertical bars represent the LSD at a significance level of 0.05.
raphanin content, and 28% and 15% losses in QR induction ratio under 1% O2 and 1% O2 + 10% CO2 respectively at 5 ◦ C by day 20 (Fig. 5). All four CA treatments (21% O2 + 10% CO2 , 21% O2 + 20% CO2 , 1% O2 , and 1% O2 + 10% CO2 ) maintained the visual quality of broccoli florets for more than 20 days at 5 ◦ C (data not shown), but 21% O2 + 10% CO2 was by far the best treatment for marinating glucoraphanin contents and QR activity at 5 ◦ C. Broccoli florets suffer a series of stresses after harvesting (washing/pre-cutting) and during storage (cooling and CA conditions), which might trigger complex metabolism of glucosinolates, and change the levels of glucoraphanin and its breakdown products, including sulforaphane. Cutting of broccoli heads to broccoli florets brings myrosinase in contact with glucoraphanin, which might lead to a high degree of glu-
coraphanin hydrolysis. On the other hand, it might also induce the biosynthesis of glucoraphanin during cooling and controlled atmosphere storage. The glucoraphanin level of stored broccoli florets is a reflection of two opposing mechanisms, hydrolysis of glucoraphanin by myrosinase, and induction of glucoraphanin biosynthesis by an unknown mechanism (Mithen et al., 2000). An increase of glucoraphanin contents during 6 days storage at 0, 5, and 10 ◦ C (Fig. 3) and 5 days of storage under controlled atmosphere treatments with elevated CO2 concentration at 5 ◦ C (Fig. 5) was observed in the present experiment. Similarly, Hansen et al. (1995) also reported a rise in methylsulfinylalkyl glucosinolates (glucoiberin and glucoraphanin) in broccoli florets during the first 7 days storage under air and controlled atmospheres. Furthermore, a significant rise of glucoraphanin was also found in broc-
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coli heads during the first 3 days storage under a controlled atmosphere (1.5% O2 + 6% CO2 ) at 4 ◦ C (Rangkadilok et al., 2002). The predominance of the induction of glucoraphanin biosynthesis might be the cause of the increase of glucoraphain content under these cases during storage, though the specific mechanism involved remains to be further elucidated. With respect to changes in glucoraphanin contents of broccoli during CA and MAP storage, some contradictory results have been reported with different broccoli cultivars and under different storage temperatures combined with different O2 and CO2 concentrations. Glucoraphanin content could be maintained up to 25 days in broccoli heads stored under 1.5% O2 + 6% CO2 at 4 ◦ C, or for at least 10 days stored in MAP and refrigeration at 4 ◦ C (Rangkadilok et al., 2002), whereas, a reduction of 48% in glucoraphanin content after 7 days of storage under MAP (17% O2 + 3% CO2 ) at 1 ◦ C has also been reported (Vallejo et al., 2003). Our results showed that the glucoraphanin content was significantly higher in broccoli florets under 21% O2 + 10% CO2 treatment compared to air (Fig. 5), suggesting that the elevated CO2 concentration might favor the induction of glucoraphanin biosynthesis or/and reduction of glucoraphanin degradation by myrosinase. Inactivation of myrosinase by elevated CO2 concentrations (Dunford and Temelli, 1996) might explain the decreased glucoraphanin degradation in controlled atmosphere conditions with elevated CO2 concentrations. However, with regard to the decreased glucoraphanin contents in broccoli florets under 21% O2 + 20% CO2 at 5 ◦ C compared to 21% O2 + 10% CO2 during the 20 days storage (Fig. 5), extremely high concentrations of CO2 (20%) should be avoided to prevent accelerated hydrolysis of glucoraphanin. In our study, the reduced O2 concentration treatments (1% O2 , 1% O2 + 10% CO2 ) led to a steady decrease of glucoraphanin content and the QR induction ratio during the 20 days storage (Fig. 5), which was consistent with the observation of Hansen et al. (1995) who reported that the content of methylsulphinylalkylglucosinolate (glucoiberin and glucoraphanin) content in broccoli florets during 7 days of storage under CA with low concentrations or absence of O2 (0.5% O2 + 20% CO2 , 0.5% O2 , and 20% CO2 in the absence of O2 ) was lower than that under air. These results suggest that low concentrations or absence of O2 might result in the reduced biosynthesis of glucoraphanin or/and promoted breakdown of glucoraphanin. CYP79F1, a cytochrome P450-dependent monooxygenase, is responsible for aldoxime production, a key step in biosynthesis of glucoraphanin (Hansen et al., 2001a,b; Grubb and Abel, 2006), and the oxygen dependence of CYP79F1 action might explain the reduction of glucoraphanin biosynthesis under the conditions of extremely low concentrations or absence of O2 . The relatively higher glucoraphanin content in broccoli florets under 1% O2 + 10% CO2 compared to 1% O2 during the 20 days storage (Fig. 5) might be due to reduction of glucosinolate degradation by inactivation of myrosinase under elevated CO2 concentrations (Dunford and Temelli, 1996).
Great progress has been achieved in recent years in understanding glucosinolate biosynthesis (Du et al., 1995; Du and Halkier, 1996; Halkier and Du, 1997; Du and Halkier, 1998; Li et al., 2001; Wittstock and Halkier, 2002; Grubb and Abel, 2006). The main pathway of glucosinolate biosynthesis has been elucidated (Halkier and Du, 1997; Wittstock and Halkier, 2002; Grubb and Abel, 2006), and most of the enzymes involved in biosynthesis of glucoraphain, such as MAM1 (BoGSL-ELONG), CYP79F1, CYP83A1, UDPG: thiohydroximate glucosyltransferase (S-GT), PAPS: desulfoglucosinolate sulfotransferase, AOP2 (BoGSL-ALK) and AOP3 (BoGSL-OH) have been identified in Arabidopsis thaliana or Brassica species (Wittstock and Halkier, 2002; Mikkelsen et al., 2002; Li et al., 2001; Li and Quiros, 2002, 2003). Recently, IQD1 (At3g09710), the first regulatory gene in glucosinolate biosynthesis which stimulates glucoraphanin accumulation is identified in A. thaliana (Levy et al., 2005). It will be interesting to investigate further the effects of different storage methods on the activity of enzymes involved in glucoraphanin biosynthesis and expression of the related genes. These studies, together with the information of effects of storage methods on glucoraphanin hydrolysis, will shed light on the impact of storage methods on glucoraphanin content and anticarcinogenic activity of broccoli. From our results, the glucoraphanin content of broccoli florets was found to be correlated with the QR induction ratio in most cases during cooling and CA storage conditions, although this was not always true in our study. Such a situation is probably due to the different extent of hydrolysis of glucoraphanin to sulforaphane, which may be influenced by the activity of myrosinase after storage. Other methylsulfinylalkyl glucosinolates, including glucoiberin (3methylsulphinylpropyl glucosinolate) and glucoalyssin (5methylsulphinylpentyl glucosinolate) also contribute to QR activity in broccoli florets. As an anticarcinogenic marker enzyme, QR induction is widely used as a biomarker for cancer chemoprevention (Grubb et al., 2002; Cuendet et al., 2006). In our study, the QR induction ratio by broccoli extract of suitable concentration provides a sensitive and high-throughput method for measuring the detoxified phase 2 enzyme inducer activity of broccoli produce. Our results indicate that the QR assay system can be used in elucidating the effects of storage and processing on induction of phase 2 enzymes in carcinogenic metabolism.
4. Conclusions Immediate cooling of broccoli florets at 0 and 5 ◦ C can preserve the glucoraphanin content and QR induction ratio for 12 days. Both are good storage methods of broccoli florets in marinating visual quality, glucoraphanin content and QR activity. Controlled atmosphere conditions with relative higher CO2 concentrations (10%) and normal O2 concentrations facilitated the maintenance of glucoraphanin content and the QR induction ratio at 5 ◦ C, which can maintain visual
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quality and anticarcinogenic glucosinolate levels of broccoli florets for up to 20 days.
Acknowledgements The authors thank Dr. Marita Cantwell (University of California, Davis) for helpful suggestions in experiment design and sample preparation, Dr. Steffen Abel (University of California, Davis) for advice on QR assay, and Dr. Liangcheng Du (University of Nebraska, Lincoln) for critical reading of the manuscript. This study was supported by Natural Science Foundation of Zhejiang Province (R304103), National Natural Science Foundation of China (30320974), Fok Ying Tong Education Foundation (104034), and NCET-05-0516.
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Changes in glucoraphanin content and quinone reductase activity in broccoli (Brassica oleracea var. italica) florets during cooling and controlled atmosphere storage Chao-Jiong Xu, De-Ping Guo, Jing Yuan, Gao-Feng Yuan, Qiao-Mei Wang ∗ Department of Horticulture, Zhejiang University, The State Agriculture Ministry, Laboratory of Horticultural Plant Growth, Development & Biotechnology, Hangzhou 310029, China Received 6 January 2006; accepted 19 June 2006
Abstract The effects of cooling and controlled atmosphere (CA) treatments on glucoraphanin content and induction of quinone reductase (QR) activity in broccoli florets were investigated. The results showed that both the glucoraphanin content and QR activity of broccoli florets increased during the initial 6 day of storage, and then decreased markedly at different temperatures of 0, 5, and 10 ◦ C. Immediate cooling of broccoli florets at 0 and 5 ◦ C could preserve the glucoraphanin content and QR activity for 12 days. Keeping broccoli florets at 20 ◦ C for 6 h before cooling at 5 ◦ C had little effects on shelf-life, glucoraphanin content and QR activity. CA treatments with elevated CO2 (21% O2 + 10% CO2 , and 21% O2 + 20% CO2 ) and an air treatment (21% O2 ) were found to increase the glucoraphanin content and the QR activity over the first 5 days of storage at 5 ◦ C, while CA treatments with reduced O2 concentrations (1% O2 , 1% O2 + 10% CO2 ) led to a steady decrease of glucoraphanin content and the QR activity during 20 days of storage at 5 ◦ C. The highest content of glucoraphanin and QR activity was found in florets stored under 21% O2 + 10% CO2 at 5 ◦ C. These conditions were able to maintain the visual quality, glucoraphanin content and QR activity of the broccoli florets for 20 days. © 2006 Elsevier B.V. All rights reserved. Keywords: Broccoli; Glucoraphanin; Quinone reductase; Controlled atmosphere (CA); Cooling
1. Introduction Glucosinolates are a group of sulfur- and nitrogencontaining secondary metabolites in cruciferous vegetables (Poulton and Møller, 1993). Upon tissue damage, glucosinolates are released from plant vacuoles and rapidly hydrolyzed by myrosinase (-thioglucoside glucohydrolase, EC 3.2.3.1) to glucose and unstable thiohydroximate-O-sulfonate intermediates which, as dictated by chemical conditions, spontaneously rearrange to isothiocyanates, thiocyanates, or nitriles. Production of isothiocyanates is usually favored by neutral conditions (Fenwick et al., 1983; Bones and Rossiter, 1996; Fahey et al., 2001). Broccoli contains high
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levels of glucoraphanin, the glucosinolate precursor of sulforaphane. It has been reported that sulforaphane is an extremely potent monofunctional inducer of quinone reductase [NAD(P)H: (quinine-acceptor) oxidoreductase, EC 1.6.99.2, QR] in murine hepatoma cells (Zhang et al., 1992), and is the most potent, naturally occurring, monofunctional phase 2 enzyme inducer identified thus far (Talalay et al., 1995). The direct assay of quinone reductase activity in murine Hepa 1c1c7 cells provides a rapid and reliable indicator of the ability of vegetable extracts to induce enzymes that detoxify carcinogens (Prochaska and Santamaria, 1988; Prochaska et al., 1992), and hence of putative anticarcinogenic activity via a blocking mechanism (Wattenberg, 1985). The QR assay system has also been used to detect inducers of anticarcinogenic enzymes in human diet (Tawfiq et al., 1994), and assess the potential of extracts from cruciferous vegetables (Prochaska et al., 1992) and chemo-
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preventive glucosinolates (Gross et al., 2000; Wang et al., 2002). Broccoli is a highly perishable product and its visual and organoleptic qualities greatly depend on the storage conditions (Makhlouf et al., 1989). The effects of different storage methods on shelf-life and visual quality of broccoli heads have been widely investigated (Makhlouf et al., 1989; Izumi et al., 1996; Hansen et al., 2001a,b). During the last decade, storage of broccoli florets has also been studied with the increasing demand for minimally processed vegetables (Bastrash et al., 1993; Izumi et al., 1996). As with the storage of broccoli heads, low temperatures and controlled atmospheres are usually recommended for the storage of broccoli florets (Izumi et al., 1996). Even though glucosinolate is one of the main health-promoting secondary metabolites in broccoli, changes in glucosinolate levels of broccoli under various storage conditions have not been systematically studied until recently, and reports on the effect of postharvest storage on glucosinolate contents of broccoli are sparse, and sometimes contradictory. Storage of broccoli in modified atmosphere packaging (MAP) and refrigeration at 4 ◦ C has been shown to maintain glucoraphanin contents and visual quality of the broccoli heads for at least 10 days, and controlled atmosphere storage (1.5% O2 + 6% CO2 ) at 4 ◦ C could maintain glucoraphanin contents up to 25 days after harvest with an increase in glucoraphanin content compared to storage at air (Rangkadilok et al., 2002). However, a reduction of 48% of glucoraphanin content in broccoli after 7 days of storage under MAP (17% O2 + 3% CO2 ) at 1 ◦ C has been observed (Vallejo et al., 2003). Controlled atmospheres may exert an influence on the content of glucosinolates. Hansen et al. (1995) reported that both total glucosinolates and methylsulfinylalkyl glucosinolate (glucoiberin and glucoraphanin) contents of broccoli florets increased at day 7 and followed by a slight decline at day 9 as the broccoli deteriorated when stored under air and controlled atmospheres (0.5% O2 , 0.5% O2 + 20% CO2 , and 20% CO2 ). Similarly, a significant rise of glucoraphanin was also found in broccoli heads stored under a controlled atmosphere (1.5% O2 + 6% CO2 ) at 4 ◦ C (Rangkadilok et al., 2002). As more attention is paid to the effect of storage conditions on visual quality, limited information is available on the effects of different storage conditions on chemopreventive glucosinolates such as glucoraphanin and phase 2 enzyme (QR) induction ability of broccoli produce, especially minimally processed broccoli florets. It is still an open question if the storage conditions which keep broccoli florets with good visual quality can also preserve the glucoraphanin levels and QR induction ability of broccoli florets. The present study was undertaken with the objective of developing a QR assay for measuring the QR induction ratio of extracts, and investigating the effects of different cooling and controlled atmosphere conditions on glucoraphain contents and the induction of QR activity of broccoli florets.
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2. Material and methods 2.1. Plant material Broccoli heads (Brassica oleracea var. italica cv. Luling) of prime quality were harvested in the early morning from the greenhouse of Zhejiang University (Hangzhou, China), topiced and then transported to the postharvest laboratory of the Vegetable Institute, Zhejiang University within 10 min. Broccoli heads with a diameter ranging from 15 to 20 cm were chosen, and the inner branches (with florets having stalks of approximately 2 cm) were then cut from these heads for experimentation. The florets were surface-sterilized by washing with a solution of 50 ppm NaOCl for 1 min and dried using a household model spin drier for 2 min. The florets were then randomly distributed into the required number of treatment units, each with four florets (roughly 100 g). 2.2. Postharvest treatments 2.2.1. Cooling treatments Broccoli florets were divided into three groups, and stored in dark rooms at 95% RH and temperatures of 0, 5, and 10 ◦ C, respectively. There were three replicates per temperature treatment at each sampling and three treatment units per replicate. The sampling times were 3, 6, 9, and 12 days after harvest. 2.2.2. Cooling delay treatments The florets from freshly harvested broccoli heads were divided into five groups, immediately transferred to 5 ◦ C storage or transferred to 5 ◦ C storage after being kept at 20 ◦ C for 3, 6, 12, and 24 h, respectively. There were three replicates per cooling delay treatment at each sampling time and three treatment units per replicate. The sampling times were 3, 6, 9, and 12 days after treatment. 2.2.3. Controlled atmosphere treatments at 5 ◦ C The florets were stored in 3.8 l glass jars, closed with a neoprene rubber stopper fitted with inlet and outlet polyethylene tubes. The jars were placed in a room at 5 ◦ C for 20 days and ventilated with humidified gas at 2.8 l h−1 . The humidified gas mixtures were established by controlling air, nitrogen, and carbon dioxide flows through different size capillary tubes and verified by gas chromatography. The gas mixtures for controlled atmosphere treatments were as follows: 21% O2 + 10% CO2 , 21% O2 + 20% CO2 , 1% O2 , and 1% O2 + 10% CO2 . There were three replicates per controlled atmosphere treatment at each sampling time and three treatment units per replicate. The sampling times were 5, 10, 15, and 20 days after harvest. 2.3. Determination of shelf-life Shelf-life was determined according to the method of Ku and Wills (1999) with minor modification. The time for qual-
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ity to decline to 30% yellowing or slight rotting was defined as the end of shelf-life for the florets. 2.4. Sample preparation and freeze-drying Freshly harvested and stored broccoli florets were frozen in liquid N2 and kept in polyethylene bags at −70 ◦ C until freeze-drying. Freeze-dried broccoli samples were stored in sealed polyethylene bags at 4 ◦ C until analysis. The samples were weighed fresh and re-weighed after being freeze-dried to determine the ratio of fresh weight to dry weight. 2.5. Glucosinolate assay Glucosinolates were extracted and analyzed as previously described with minor modifications (Wang et al., 2002). Freeze-dried samples (25 mg) were boiled in 1 ml water for 10 min. After recovery of the liquid, the residues were washed with water (1 ml), and the combined aqueous extract was applied to a DEAE-Sephadex A-25 (40 mg) column (pyridine acetate form). The column was washed three times with 20 mM pyridine acetate and twice with water. The glucosinolates were converted into their desulpho analogues by overnight treatment with 100 l of 0.1% (1.4 units) aryl sulphatase, and the desulphoglucosinolates were eluted with 2 × 0.5 ml water (Hogge et al., 1988). HPLC analysis of desulphoglucosinolates was carried out using a Shimadzu (Tokyo, Japan) mode VP liquid chromatograph with a dual wavelength spectrophotometer. Samples (100 l) were separated at 30 ◦ C on a Waters Spherisorb C18 column (150 mm × 4.6 mm i.d.; 5 m particle size) (Milford, MA 01757, USA) using acetonitrile and water at a flow rate of 1.0 ml/min. The procedure employed isocratic elution with 1.5% acetonitrile for the first 5 min; a linear gradient to 20% acetonitrile over the next 15 min followed by isocratic elution with 20% acetonitrile for the final 10 min. Absorbance was detected at 226 and 280 nm. Ortho-nitrophenyl--dgalactopyranoside (Sigma) was used as an internal standard for HPLC analysis. Concentrations of individual glucosinolate were determined according to published response factors (Haughn et al., 1991). The integrated area of the desulpho4-methylsulphinylbutyl glucosinolate peak was converted to a molar amount under the assumption that this compound has a molar extinction coefficient at 226 nm equal to that of sinigrin (Wang et al., 2002). 2.6. Quinone reductase assay 2.6.1. Preparation of extracts for quinone reductase assay Extracts from the freeze-dried samples were assayed for their induction activity in murine hepatoma Hepa 1c1c7 cells. A sample of 20 mg was homogenized for 1–2 min with a polypropylene pestle in 1.5 ml plastic tubes containing 200 l extraction buffer (5 mM K2 HPO4 –KH2 PO4 , 1 mM EDTA, pH 7.6). The pestle was rinsed with 300 l extraction buffer
and the homogenate was centrifuged twice for 10 min at 16,000 × g to recover the supernatant. The resulting supernatants were filtered through sterile non-pyrogenic filters (0.22 m) and stored at −70 ◦ C until use. 2.6.2. Induction of cultured Hepa 1c1c7 murine hepatoma cells Hepa 1c1c7 murine hepatoma cells were grown in a minimal essential medium supplemented with 10% foetal calf serum in a humidified incubator in 5% carbon dioxide at 37 ◦ C as described previously (Prochaska and Santamaria, 1988; Prochaska et al., 1992; Grubb et al., 2002). In order to monitor the inducer potency of plant extracts, Hepa 1c1c7 murine hepatoma cells were grown in 96-well microtiter plates. Typically, 10,000 Hepa 1c1c7 cells were seeded into each well and grown for 24 h in 200 l medium per well, and then induced for 24 h by the addition of 200 l medium containing serial dilutions of the floret extract to be assayed. Usually, the floret extract was diluted to the concentrations needed with cell culture medium, and the concentrations of each extract were expressed as dry weight (DW) of original material for each milliliter of cell culture medium. The two-fold serial dilutions were prepared in the microtiter plate using an octapipet. 2.6.3. Quinone reductase assay After exposure of Hepa 1c1c7 cell cultures to floret extracts, QR activity was assayed in hepatoma cell lysates, using menadione, MTT, and an NADPH-generating system (Prochaska and Santamaria, 1988). The cell culture medium was decanted and 50 l of lysing solution (0.8% digitonin in 2 mM EDTA, pH 7.8) was added to each well. Cells were lysed for 10 min at 37 ◦ C followed by shaking (100 rpm) for 10 min at room temperature. To each well was added 200 l of the QR assay solution containing 25 mM Tris buffer (pH 7.4), 1 mM glucose-6-phosphate, 50 M menadione, 30 M NADP, 5 M FAD, 0.07% (w/v) bovine serum albumin, 0.03% (w/v) MTT, 0.01% (v/v) Tween-20, and 1 unit/ml of yeast glucose-6-phosphate dehydrogenase (Gross et al., 2000). The reaction mixtures were incubated at room temperature and reactions were terminated after 10 min by the addition of 50 l of 0.1 M HCl. A reagent blank was prepared by adding stop solution to one column of each plate before the addition of assay solution (zero time control). Absorbances were measured by scanning the microtitre plates at 595 nm, and QR inducer potency was calculated as the QR activity (A595 ) ratio of treated to untreated cell cultures. Cell densities were determined by staining with crystal violet as previously described (Fahey et al., 1997; Gross et al., 2000). 2.7. Statistical analyses Means and standard errors of shelf-life were calculated. Data of glucoraphanin contents and QR induction ratios were analyzed by analysis of variance (ANOVA model one-way). Sources of variation were storage duration and treatments. The means were compared by the least significant differences
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(LSD) test at a significance level of 0.05. STATISTICA Base (a stand-alone product), STATISTICA 6.0 (StatSoft Inc.) was used in data tests.
3. Results and discussion 3.1. Induction of quinine reductase in murine hepatoma cells by broccoli floret extracts Methylsulfinylalkyl isothiocyanates are potent and specific inducers of mammalian phase 2 detoxification enzymes such as QR or glutathione S transferase (Zhang and Talalay, 1994). Since several methylsulfinylalkyl glucosinolates are synthesized in broccoli, and glucoraphanin is the most abundant glucosinolate in florets (Fahey et al., 1997; Brown et al., 2002), we tested the potency of broccoli floret extracts in inducing QR activity in cultured Hepa 1c1c7 murine hepatoma cells. As indicated by the colorimetric QR bioassay, broccoli florets contain readily detectable levels of phase 2 enzyme inducers (Figs. 1 and 2). Maximal induction (about 3.6-fold) of QR specific activity by floret extracts was achieved at a concentration of 2 mg DW ml−1 medium. Examination of detectable cytotoxicity showed that broccoli extract at 2 mg DW ml−1 medium reduced cell density by 16%. However, the cytotoxic effect was negligible at and below 1 mg DW ml−1 medium (Figs. 1 and 2). In our study, the extracts of broccoli floret samples from different treatments were diluted to the concentration of 1 mg DW ml−1 medium for the QR assay, and eight wells were used for
Fig. 2. Induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. Data are expressed as ratios of treated cells to control cells for QR activity and cell density. Specific QR activity is the ratio of QR activity and the cell density at the same concentration.
every sample in 96-well microtiter plates. In order to reach sufficient reproducibility of the QR assay, the florets were cut from top-quality heads at a uniform size to minimize the variation between different heads. To assess the head to head variation of the assay, we measured QR induction of floret extracts prepared from the different newly harvested heads at the concentration of 1 mg DW ml−1 medium in the same container (data not shown). Here, the QR inducer potency was expressed as the QR activity ratio of floret extract in treated versus untreated hepatoma cells. The standard deviation of the mean QR inducer potency was found to be 4%. The data demonstrate that the QR bioassay of broccoli florets we developed was reliable. 3.2. Effect of storage temperatures on glucoraphanin content and QR activity
Fig. 1. Colorimetric assay of QR activity (A) and of cell density (B). Hepa 1c1c7 murine hepatoma cells were grown in 96-well microtiter plates and induced with serial two-fold dilutions of broccoli floret extract (only one half of each assay plates is shown). Each column of wells received the same dilution of a single plant extract. A more intense color denotes higher QR activity and higher cell density.
Different cooling temperatures had a great influence on the shelf-life of broccoli florets. The shelf-life of florets stored at 0, 5, and 10 ◦ C was found to be 34.7, 17.5, and 9.0 days, respectively (data not shown). In the present experiment, a significant reduction of both glucoraphanin content and QR activity was observed in broccoli florets during 9 days storage at 10 ◦ C compared to 0 and 5 ◦ C (Fig. 3). Glucoraphanin content was increased by cooling treatments during the first 6 days of storage, and the highest content was observed in broccoli florets stored at 0 ◦ C (32% increase), followed by 5 ◦ C (20% increase) and 10 ◦ C (2% increase) at day 6 (Fig. 3). After day 6, glucoraphanin content decreased in broccoli florets stored at 0 and 5 ◦ C, by 2% and 23%, respectively, at day 12, although the broccoli florets were still in good visual condition. A great decrease in glucoraphanin content was observed in broccoli florets stored at 10 ◦ C by day 9 (60% loss), which coincided with a rapid loss in visual quality of the broccoli florets (Fig. 3). These results were similar to those of Hansen et al. (1995) who observed an increase in methylsul-
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C.-J. Xu et al. / Postharvest Biology and Technology 42 (2006) 176–184 Table 1 Effects of cooling delay on shelf-life of broccoli at 5 ◦ C Hours at 20 ◦ C
Shelf-life at 5 ◦ C (days)
0 3 6 12 24
17.5 17.3 16.5 15.3 8.7
± ± ± ± ±
0.69 a 0.53 a 0.66 a 0.42 a 0.3 b
The broccoli florets were transferred to 5 ◦ C storage after they have been kept in 20 ◦ C for 3, 6, 12, or 24 h. Values represent the means of three replicate samples with standard error of the means. Values followed by the same letter are not significantly different at P = 0.05.
Fig. 3. Effects of storage temperature on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were stored at 0, 5, or 10 ◦ C. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96-well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate). The vertical bars represent the LSD at a significance level of 0.05.
phinylalkyl glucosinolates content in broccoli florets during 7 days storage under air at 10 ◦ C followed by a slight decline at day 9 as the broccoli deteriorated. The trend in activity of quinone reductase was similar to that of glucosinolate content (Fig. 3). A steady increase in QR induction ratio was observed in broccoli florets stored at 0, 5, and 10 ◦ C during the first 6 days of storage, and the QR induction ratio increased by 33%, 32%, and 13% respectively, at day 6. However, the QR induction ratio decreased dramatically after day 6 (Fig. 3). Storage of broccoli florets at 0 and 5 ◦ C could maintain the glucoraphanin content and QR activity for 12 days. 3.3. Effects of cooling delay on glucoraphanin content and QR activity Although “cool as soon as possible” is the general recommendation for broccoli handling, delays of several hours
before cooling may occur in postharvest handling of broccoli, especially when the broccoli is treated with forced air or hydrocooling instead of immediate liquid-ice cooling in field. Our results showed that keeping broccoli florets at 20 ◦ C for 3 and 6 h before cooling at 5 ◦ C did not significantly affect the shelf-life, glucoraphanin content, and QR activity compared to the controls (immediate cooling at 5 ◦ C), but 24 h at 20 ◦ C significantly decreased the shelf-life glucoraphanin content, and QR activity (Table 1, Fig. 4). The increase in glucoraphanin content and QR induction ratio was observed during the first 6 days storage in treatments with 12 or less hours at 20 ◦ C. However, the glucoraphanin content and QR induction ratio decreased in all treatments from day 6. Only slight reductions of glucoraphanin content and QR activity were observed in broccoli florets during 9 days of storage at 5 ◦ C, when they were treated with 6 or less hours at 20 ◦ C before cooling at 5 ◦ C. The glucoraphanin content and QR induction ratio decreased steadily over 24 h at 20 ◦ C (Fig. 4). The above results suggest that keeping florets at 20 ◦ C should be for less than 6 h in order to maintain visual quality, glucoraphanin content and QR activity. 3.4. Effect of controlled atmospheres on glucoraphanin content and QR activity The glucoraphanin content and QR induction ratio in broccoli florets fluctuated moderately during 20 days storage under air and CA treatments at 5 ◦ C (Fig. 5). Significant increases in glucoraphanin content during 20 days storage and in the QR induction ratio during 15 days of storage were observed in broccoli florets under the 21% O2 + 10% CO2 treatment compared to air, while significant reductions in glucoraphanin content and QR induction ratio were observed in the 1% O2 treatment compared to air during 20 days storage (Fig. 5). During the initial 5 days of storage, the glucoraphanin content and QR induction ratio increased in all three treatments with 21% O2 , and the greatest increase was observed in the 21% O2 + 10% CO2 treatment, with the 21% O2 treatment next and the 21% O2 + 20% CO2 the least (Fig. 5). After CA treatment, the glucoraphanin content and QR induction ratio decreased in all these treatments (Fig. 5). The glucoraphanin content and QR induction ratio decreased steadily during 20 days storage under 1% O2 and 1% O2 + 10% CO2 , causing 63% and 54% losses in gluco-
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Fig. 4. Effects of cooling delay on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were transferred to 5 ◦ C storage after they have been kept in 20 ◦ C for 3, 6, 12, or 24 h. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate). The vertical bars represent the LSD at a significance level of 0.05.
Fig. 5. Effects of controlled atmosphere storages on glucoraphanin content of broccoli florets and induction of QR activity in murine Hepa 1c1c7 cells by extracts of broccoli florets. The broccoli florets were stored under low O2 (1% O2 ), high CO2 (21% O2 + 10% CO2 , 21% O2 + 20% CO2 ), or low O2 plus high CO2 (1% O2 + 10% CO2 ) at 5 ◦ C. The extracts of samples from different treatments were diluted to a concentration of 1 mg DW ml−1 medium for QR assay, and eight wells were used for every sample in 96well microtiter plates. Each data point is the mean of three replicate samples (three units each of four florets in each replicate) The vertical bars represent the LSD at a significance level of 0.05.
raphanin content, and 28% and 15% losses in QR induction ratio under 1% O2 and 1% O2 + 10% CO2 respectively at 5 ◦ C by day 20 (Fig. 5). All four CA treatments (21% O2 + 10% CO2 , 21% O2 + 20% CO2 , 1% O2 , and 1% O2 + 10% CO2 ) maintained the visual quality of broccoli florets for more than 20 days at 5 ◦ C (data not shown), but 21% O2 + 10% CO2 was by far the best treatment for marinating glucoraphanin contents and QR activity at 5 ◦ C. Broccoli florets suffer a series of stresses after harvesting (washing/pre-cutting) and during storage (cooling and CA conditions), which might trigger complex metabolism of glucosinolates, and change the levels of glucoraphanin and its breakdown products, including sulforaphane. Cutting of broccoli heads to broccoli florets brings myrosinase in contact with glucoraphanin, which might lead to a high degree of glu-
coraphanin hydrolysis. On the other hand, it might also induce the biosynthesis of glucoraphanin during cooling and controlled atmosphere storage. The glucoraphanin level of stored broccoli florets is a reflection of two opposing mechanisms, hydrolysis of glucoraphanin by myrosinase, and induction of glucoraphanin biosynthesis by an unknown mechanism (Mithen et al., 2000). An increase of glucoraphanin contents during 6 days storage at 0, 5, and 10 ◦ C (Fig. 3) and 5 days of storage under controlled atmosphere treatments with elevated CO2 concentration at 5 ◦ C (Fig. 5) was observed in the present experiment. Similarly, Hansen et al. (1995) also reported a rise in methylsulfinylalkyl glucosinolates (glucoiberin and glucoraphanin) in broccoli florets during the first 7 days storage under air and controlled atmospheres. Furthermore, a significant rise of glucoraphanin was also found in broc-
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coli heads during the first 3 days storage under a controlled atmosphere (1.5% O2 + 6% CO2 ) at 4 ◦ C (Rangkadilok et al., 2002). The predominance of the induction of glucoraphanin biosynthesis might be the cause of the increase of glucoraphain content under these cases during storage, though the specific mechanism involved remains to be further elucidated. With respect to changes in glucoraphanin contents of broccoli during CA and MAP storage, some contradictory results have been reported with different broccoli cultivars and under different storage temperatures combined with different O2 and CO2 concentrations. Glucoraphanin content could be maintained up to 25 days in broccoli heads stored under 1.5% O2 + 6% CO2 at 4 ◦ C, or for at least 10 days stored in MAP and refrigeration at 4 ◦ C (Rangkadilok et al., 2002), whereas, a reduction of 48% in glucoraphanin content after 7 days of storage under MAP (17% O2 + 3% CO2 ) at 1 ◦ C has also been reported (Vallejo et al., 2003). Our results showed that the glucoraphanin content was significantly higher in broccoli florets under 21% O2 + 10% CO2 treatment compared to air (Fig. 5), suggesting that the elevated CO2 concentration might favor the induction of glucoraphanin biosynthesis or/and reduction of glucoraphanin degradation by myrosinase. Inactivation of myrosinase by elevated CO2 concentrations (Dunford and Temelli, 1996) might explain the decreased glucoraphanin degradation in controlled atmosphere conditions with elevated CO2 concentrations. However, with regard to the decreased glucoraphanin contents in broccoli florets under 21% O2 + 20% CO2 at 5 ◦ C compared to 21% O2 + 10% CO2 during the 20 days storage (Fig. 5), extremely high concentrations of CO2 (20%) should be avoided to prevent accelerated hydrolysis of glucoraphanin. In our study, the reduced O2 concentration treatments (1% O2 , 1% O2 + 10% CO2 ) led to a steady decrease of glucoraphanin content and the QR induction ratio during the 20 days storage (Fig. 5), which was consistent with the observation of Hansen et al. (1995) who reported that the content of methylsulphinylalkylglucosinolate (glucoiberin and glucoraphanin) content in broccoli florets during 7 days of storage under CA with low concentrations or absence of O2 (0.5% O2 + 20% CO2 , 0.5% O2 , and 20% CO2 in the absence of O2 ) was lower than that under air. These results suggest that low concentrations or absence of O2 might result in the reduced biosynthesis of glucoraphanin or/and promoted breakdown of glucoraphanin. CYP79F1, a cytochrome P450-dependent monooxygenase, is responsible for aldoxime production, a key step in biosynthesis of glucoraphanin (Hansen et al., 2001a,b; Grubb and Abel, 2006), and the oxygen dependence of CYP79F1 action might explain the reduction of glucoraphanin biosynthesis under the conditions of extremely low concentrations or absence of O2 . The relatively higher glucoraphanin content in broccoli florets under 1% O2 + 10% CO2 compared to 1% O2 during the 20 days storage (Fig. 5) might be due to reduction of glucosinolate degradation by inactivation of myrosinase under elevated CO2 concentrations (Dunford and Temelli, 1996).
Great progress has been achieved in recent years in understanding glucosinolate biosynthesis (Du et al., 1995; Du and Halkier, 1996; Halkier and Du, 1997; Du and Halkier, 1998; Li et al., 2001; Wittstock and Halkier, 2002; Grubb and Abel, 2006). The main pathway of glucosinolate biosynthesis has been elucidated (Halkier and Du, 1997; Wittstock and Halkier, 2002; Grubb and Abel, 2006), and most of the enzymes involved in biosynthesis of glucoraphain, such as MAM1 (BoGSL-ELONG), CYP79F1, CYP83A1, UDPG: thiohydroximate glucosyltransferase (S-GT), PAPS: desulfoglucosinolate sulfotransferase, AOP2 (BoGSL-ALK) and AOP3 (BoGSL-OH) have been identified in Arabidopsis thaliana or Brassica species (Wittstock and Halkier, 2002; Mikkelsen et al., 2002; Li et al., 2001; Li and Quiros, 2002, 2003). Recently, IQD1 (At3g09710), the first regulatory gene in glucosinolate biosynthesis which stimulates glucoraphanin accumulation is identified in A. thaliana (Levy et al., 2005). It will be interesting to investigate further the effects of different storage methods on the activity of enzymes involved in glucoraphanin biosynthesis and expression of the related genes. These studies, together with the information of effects of storage methods on glucoraphanin hydrolysis, will shed light on the impact of storage methods on glucoraphanin content and anticarcinogenic activity of broccoli. From our results, the glucoraphanin content of broccoli florets was found to be correlated with the QR induction ratio in most cases during cooling and CA storage conditions, although this was not always true in our study. Such a situation is probably due to the different extent of hydrolysis of glucoraphanin to sulforaphane, which may be influenced by the activity of myrosinase after storage. Other methylsulfinylalkyl glucosinolates, including glucoiberin (3methylsulphinylpropyl glucosinolate) and glucoalyssin (5methylsulphinylpentyl glucosinolate) also contribute to QR activity in broccoli florets. As an anticarcinogenic marker enzyme, QR induction is widely used as a biomarker for cancer chemoprevention (Grubb et al., 2002; Cuendet et al., 2006). In our study, the QR induction ratio by broccoli extract of suitable concentration provides a sensitive and high-throughput method for measuring the detoxified phase 2 enzyme inducer activity of broccoli produce. Our results indicate that the QR assay system can be used in elucidating the effects of storage and processing on induction of phase 2 enzymes in carcinogenic metabolism.
4. Conclusions Immediate cooling of broccoli florets at 0 and 5 ◦ C can preserve the glucoraphanin content and QR induction ratio for 12 days. Both are good storage methods of broccoli florets in marinating visual quality, glucoraphanin content and QR activity. Controlled atmosphere conditions with relative higher CO2 concentrations (10%) and normal O2 concentrations facilitated the maintenance of glucoraphanin content and the QR induction ratio at 5 ◦ C, which can maintain visual
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quality and anticarcinogenic glucosinolate levels of broccoli florets for up to 20 days.
Acknowledgements The authors thank Dr. Marita Cantwell (University of California, Davis) for helpful suggestions in experiment design and sample preparation, Dr. Steffen Abel (University of California, Davis) for advice on QR assay, and Dr. Liangcheng Du (University of Nebraska, Lincoln) for critical reading of the manuscript. This study was supported by Natural Science Foundation of Zhejiang Province (R304103), National Natural Science Foundation of China (30320974), Fok Ying Tong Education Foundation (104034), and NCET-05-0516.
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