Sulforaphane and sulforaphene

CHAPTER 9

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates Li Cheng, Kai Wan, Hao Liang, Qipeng Yuan State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, P.R. China

Contents 9.1 9.2 9.3 9.4 9.5

Introduction Discovery and existence of sulforaphane and sulforaphene Analysis and recovery of sulforaphane and sulforaphene Procedures and analysis data Purification 9.5.1 Macroporous resin 9.5.2 Preparative high-performance liquid chromatography 9.5.3 High-speed countercurrent chromatography 9.6 Anticarcinogenic activity of sulforaphane and sulforaphene 9.6.1 In vitro inhibition of cancer cells 9.6.2 The anticarcinogenic mechanism of sulforaphane and sulforaphene 9.6.3 Acute toxicity testing in mice 9.6.4 Pharmacokinetics in mice 9.6.5 In vivo anticancer effects of sulforaphene 9.7 Conclusions References

281 282 284 286 288 290 292 293 296 296 298 300 302 302 305 306

9.1 Introduction Chemoprevention refers to the use of nontoxic substances, including many food factors, to interfere with the process of cancer development or carcinogenesis before invasion and metastasis can occur (Smith et al., 2005; Keum et al., 2005). Over the last decades, chemoprevention has been successfully studied in different in vitro and in vivo researches as well as validated in human intervention trials (Kwak et al., 2004; O’Shaughnessy et al., 2002; Talalay et al., 2003). According to epidemiological data, a diet rich in Glucosinolates: Properties, Recovery, and Applications ISBN 978-0-12-816493-8 https://doi.org/10.1016/B978-0-12-816493-8.00009-3

Copyright © 2020 Elsevier Inc. All rights reserved.

281

282

Glucosinolates: Properties, Recovery, and Applications

cruciferous vegetables (e.g., broccoli, cabbage, Brussels sprouts, cauliflower, and kale) can reduce the risk from a number of cancers (Lampe and Peterson, 2002; Kristal and Lampe, 2002). These results could be attributed to the fact that cruciferous vegetables have a high content of glucosinolates (Drewnowski and Gomez-Carneros, 2000; Fahey et al., 2001; Bianchini and Vainio, 2004). Today, at least 120 different glucosinolates with unique hydrolysis products have been identified in plants (Fahey et al., 2003). Plant’s myrosinase hydrolyze glucosinolates and transform them to biologically active compounds including indoles and isothiocyanates. Among these compounds, sulforaphane and sulforaphene have received much attention due to their antioxidant (Hong et al., 2005; Higgins et al., 2009), anticarcinogenic (Chiao et al., 2002), and antiinflammatory properties (Heiss et al., 2001). Both chemopreventive agents may be potential useful clinical dietderived substances. This chapter discusses this prospect by highlighting the formation, extraction, purification, and analytical methodologies of these compounds prior revealing their bioactivity.

9.2 Discovery and existence of sulforaphane and sulforaphene Sulforaphane was identified from savoy, red cabbage, and hoary cress identified in 1959 using differ (Procháska, 1959; Procháska and Komersová, 1959) prior merchandised worldwide as a range-land weed (McInnis et al., 1993). Thereafter, both sulforaphane and sulforaphene did not receive much attention until 1992 when they were recovered from different fruits and vegetables and investigated for the induction of phase 2 enzymes. The latest play an important role during electrophiles’ detoxification and protect animals’ cells against carcinogenesis and mutagenesis. According to the results of this study, many extracts exhibited significant inducer activities, whereas broccoli extract was found to be as one of the richest sources (Prochaska et al., 1992). Sulforaphane and sulforaphene are inherently present intact vegetables, but they are formed from glucoraphanin hydrolysis. This compound is their glucosinolate precursor that is subjected to hydrolysis by thioglucosidase enzyme. The action of the socalled myrosinase is accelerated when vegetable tissues are crushed or chewed. Glucoraphanin biosynthesis probably occurs by methionine’s elongation (step by step addition of methylene groups to form dihomomethionine), prior the addition of glycoside moiety. The latest reaction generates provide 4-methylthiobutyl glucosinolate that is subjected to

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

283

Figure 9.1 The biosynthesis pathway of glucoraphanin (Liang and Yuan, 2012).

side-chain modification for the generation of 4-methylsulfinylbutyl glucosinolate (Halkier and Du, 1997; Faulkner et al., 1998; Graser et al., 2001; Falk et al., 2004). Fig. 9.1 illustrates the proposed biosynthetic pathway of glucoraphanin that has been successfully engineered in tobacco. Glucoraphanin can be purified and analyzed by high-speed countercurrent chromatography (HSCCC) (Fahey et al., 2003) and

284

Glucosinolates: Properties, Recovery, and Applications

preparative high-performance liquid chromatography (HPLC) (Prestera et al., 1996; Rochfort et al., 2006), respectively. Gut microflora is able to hydrolyze glucoraphanin to sulforaphane and sulforaphene, but clinical trials have shown that the transformation rate in humans is very limited (Shapiro et al., 2006). On other hand, glucoraphanin is able to boost phase 1 enzymes (including activators of polycyclic aromatic hydrocarbons, nitrosamines, and olefins in rat liver), and to a lesser extent induces phase 2 enzymes. Phase 1 enzymes usually involve oxidation, reduction, and hydrolysis, resulting in more hydrophilic chemical molecules that are able to damage DNA. Concomitant with this phase 1 induction, glucoraphanin generates large amounts of reactive radical species. This fact suggests that the long-term uncontrolled administration of glucoraphanin could pose potential health hazards (Perocco et al., 2006).

9.3 Analysis and recovery of sulforaphane and sulforaphene In natural substrates, glucosinolates may occur in different concentrations as a function of different species, cultivars, type of tissues, and environmental factors (Fahey et al., 2001; Daxenbichler et al., 1991; Rosa et al., 1997; Ciska et al., 2000; Pereira et al., 2002; Holst and Williamson, 2004). The richest sources of glucoraphanin are cruciferous plants and particularly their seeds (Fahey et al., 2001, 2003; Daxenbichler et al., 1991; Pereira et al., 2002; West et al., 2004). Among the different sources, broccoli cultivars are the richest one. For instance, West and coworkers determined the glucoraphanin’s content of seeds from 59 cultivars of broccoli, rabe, kohlrabi, radish, cauliflower, Brussels sprouts, kale, and cabbage glucoraphanin contents in seeds and found the highest amounts in broccoli cultivars (West et al., 2004).Following this approach, sulforaphane and sulforaphene can be recovered from processed seeds using organic solvent extraction after hydrolysis of glucoraphanin. However, broccoli’s and other seeds contain also appreciable amounts of lipids that should be removed using a defatting step prior hydrolysis (Kore et al., 1993; Matusheski and Jeffery, 2001; Vaughn and Berhow, 2005). For convenience and cost purposes, hydrolysis of glucoraphanin in seed meals is usually conducted using endogenous myrosinase (Kore et al., 1993; Matusheski and Jeffery, 2001; Liang et al., 2007, 2008; Li et al., 2008). During glucoraphanin hydrolysis, sulforaphane and nitrile are produced (see Fig. 9.2). Nitrile, in contrast to sulforaphane, does not exhibit any

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

285

Figure 9.2 Enzymatic conversion of glucoraphanin into an aglucone intermediate by myrosinase and subsequent conversion to sulforaphane and nitrile (Liang and Yuan, 2012).

beneficial effects and in fact may be toxic to healthy cells (Matusheski and Jeffery, 2001). Therefore, the preferable conversion of glucoraphanin to sulforaphane is important to optimize health benefits of the extract (Matusheski and Jeffery, 2001). Optimization of the process is depended on the chemical conditions of the conversion. A low pH and ferrous ions favor the formation of nitrile when glucosinolates are broken by myrosinase (Kurata and Arakawa, 1986), whereas neutral pH conditions favor conversion to sulforaphane (Fig. 9.2; Vaughn and Berhow, 2005; Kurata and Arakawa, 1986; Gil and MacLeod, 1980; Matusheski et al., 2004). Besides, it has been reported that nitrile formation is linked to epithiospecifier protein activity. For example, a recombinant protein with epithiospecifier

286

Glucosinolates: Properties, Recovery, and Applications

activity was shown to direct hydrolysis to the formation of nitrile instead of sulforaphane (Matusheski et al., 2006). This fact indicates that a genetic manipulation monitoring the expression of epithiospecifier activity in broccoli could increase the preferable conversion of glucoraphanin to sulforaphane, and this way enhance potential health benefits of the substrate (Matusheski et al., 2006).

9.4 Procedures and analysis data Determination of sulforaphane or sulforaphene has been conducted using different analytical methodologies such as spectrophotometry (Zhang et al., 1992a), gas chromatography (GC), GC/mass spectrometry (GC/MS) (Matusheski and Jeffery, 2001; VanEtten et al., 1976; Cole, 1976; Spencer and Daxenbichler, 1980; Chiang et al., 1998), HPLC (Daxenbichler et al., 1977; Zhang et al., 1992b; Bertelli et al., 1998; Nakagawa et al., 2006; Liang et al., 2006; Sivakumar et al., 2007; Campas-Baypoli et al., 2010), and HPLC/MS (Al Janobi et al., 2006; Egner et al., 2008). Aliphatic isothiocyanates like sulforaphane and sulforaphene are able to react quantitatively with 1, 2-benzenedithiol (when the latest exist in excess), generating 1, 3-benzodithiole-2-thione. The latest compound can be measured using a spectrometer. Therefore, UV spectrometry methods have been developed to determine pure isothiocyanates and isothiocyanates contained in plant extracts. Sulforaphane and sulforaphene are included in this determination. GC that is a more sensitive method has also been applied for the determination of both compounds (VanEtten et al., 1976; Cole, 1976; Spencer and Daxenbichler, 1980; Daxenbichler et al., 1977). However, the temperature of this process is critical because sulforaphane decomposes to 3-butenyl isothiocyanate and other derivatives when high temperature conditions are applied during injection into GC equipment (Chiang et al., 1998). To avoid decomposition, a 4.0 mm i.d. splitless inlet liner and precise control of the carrier gas flow rates have been suggested. However, the reduction of thermal degradation was only 5% using this approach. Thereby, many researchers prefer nondestructive techniques to determine these thermolabile compounds. HPLC coupled with a UV detector has also been proposed by many researchers for the analysis of sulforaphane or sulforaphene (Daxenbichler et al., 1977; Zhang et al., 1992b; Bertelli et al., 1998; Liang et al., 2006; Sivakumar et al., 2007; Campas-Baypoli et al., 2010). However, analytical problems exist also in this method, e.g., there is lack of strong UV chromophores in sulforaphane or sulforaphene. To this line, an evaporative

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

287

light-scattering detector coupled with HPLC has been proposed as a more sensitive approach (detection limit reaching 0.0028 mmol) for the determination of both compounds and other isothiocyanates compared with the aforementioned HPLC-UV methods (Nakagawa et al., 2006). Besides, HPLC/MS methods have been developed, too (Al Janobi et al., 2006; Egner et al., 2008). Table 9.2 summarizes the different methodologies for the determination of sulforaphane and sulforaphene. The HPLC method has been applied for the determination of quantitate sulforaphane in fresh broccoli and cabbage samples. Even at dietary doses, sulforaphane can affect the balance of carcinogen metabolism toward deactivation by altering the xenobiotic-metabolizing enzyme systems (Yoxall et al., 2005). Thus, the determination of sulforaphane’s content in edible broccoli and cabbage may be more important. The analytical procedure includes homogenization of 5 g of freshly harvested broccoli or cabbage for 5 min. The vegetable meal is thereafter left to autolyze for 30 min at room temperature. The meal is then extracted twice with 50 mL methylene chloride and 2.5 g anhydrous sodium sulfate. The methylene chloride fraction is concentrated under vacuum on a rotary evaporator at 30
C. The residue is dissolved in acetonitrile prior filtering through a membrane of 0.22 mm and injection into HPLC. Analysis of samples was conducted using an HPLC apparatus (column oven temperature of 30
C and flow rate of 1 mL/min) equipped with Hitachi model L-7100 pumps, L-7420 tunable absorbance detector, and reversed-phase C18 column (2504.6 mm, 5 mm, Diamodsil). The solvent system initially consisted of 20% acetonitrile in water, prior changing it linearly over 10 min to 60% acetonitrile, and maintaining at 100% acetonitrile for 2 min to purge the column. Sulforaphane was detected by absorbance at 254 nm, and Fig. 9.3 illustrates corresponding HPLC chromatograms of the compound’s elution profiles. The sulforaphane content of 18 different varieties of broccoli and cabbage is presented in Table 9.1. Broccoli contains almost fivefold higher concentration of sulforaphane compared with cabbage. Table 9.2 shows the concentration of sulforaphane in different type tissues of broccoli (Table 9.2). The highest concentration of sulforaphane was found in the edible part of broccoli (florets), whereas the lowest one was found in the leaves. Indeed, the content of sulforaphane in florets was almost 10- and 3fold higher than that in leaves and stalks, respectively. These results indicate the importance of selecting the correct broccoli variety with the highest sulforaphane content.

288

Glucosinolates: Properties, Recovery, and Applications

Figure 9.3 Chromatograms of the HPLC elution profile of sulforaphane from broccoli (A) and cabbage (B) sample (Liang et al., 2006).

9.5 Purification The first recovery step of sulforaphane and sulforaphene from hydrolyzed and defatted seed meal is organic solvent extraction using methylene chloride (Kore et al., 1993; Matusheski and Jeffery, 2001) and ethyl acetate

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

289

Table 9.1 Sulforaphane content (mg/g, FW) in the edible tissues of 18 varieties of broccoli and cabbagea (Liang et al., 2006). Broccoli Cabbage Accession

ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. Mean a

9 16 18 20 26 27 28 145 159 163 166 168 172 175 176 180 187 188

Content (mg/g, FW)

Accession

Content (mg/g, FW)

19.2  1.8 17.6  2.0 14.4  0.6 22.1  2.3 32.9  1.5 6.0  0.8 9.2  1.2 5.4  0.4 20.4  3.2 8.4  1.4 11.5  1.5 12.1  0.9 1.4  0.1 18.6  1.5 23.2  1.6 17.0  2.1 2.3  0.4 20.5  1.8 14.6

CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. CA no. Mean

3.2  1.2 5.3  1.8 1.8  0.2 4.5  0.4 4.7  0.5 4.3  0.8 2.9  0.3 1.2  0.4 4.7  1.2 2.5  0.2 2.1  0.2 2.2  0.5 4.3  0.3 3.3  0.5 0.7  0.0 0.7  0.1 2.5  0.2 2.7  0.1 3.0

6 48 49 100 105 152 163 174 185 194 197 203 205 213 217 227 316 318

Values represent mean  S.D., n ¼ 3.

Table 9.2 Sulforaphane content (mg/g, FW) in various tissues of broccolia (Liang et al., 2006). Broccoli(mg/g, FW) Accession

ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. ZhQ no. Mean a

9 26 27 28 145 159 166 172 187 188

Florets

Stalks

Leaves

19.2  1.8 32.9  1.5 6.0  0.8 9.2  1.2 5.4  0.4 20.4  3.2 11.5  1.5 1.4  0.1 2.3  0.4 20.5  1.8 12.9

2.7  0.8 5.7  0.8 5.0  0.2 4.6  0.5 6.4  0.6 6.3  0.3 9.3  0.8 0.8  0.2 1.1  0.4 8.7  0.9 5.1

0.7  0.1 4.3  0.8 3.0  0.9 1.0  0.0 0.7  0.0 3.0  1.0 0.4  0.1 0.8  0.2 0.3  0.0 0.4  0.1 1.5

Values represent mean  S.D., n ¼ 3.

290

Glucosinolates: Properties, Recovery, and Applications

(Liang et al., 2005, 2007, 2008). This step is simple but not selective. More specifically, the recovered extracts contain different kind of glucosinolates as well as pigments and contaminants. Besides, solvent extraction is time-consuming and not environmental friendly (it requires large amounts of solvents).

9.5.1 Macroporous resin Following the above consideration, an isolation method using macroporous resins has been successfully developed and applied with high adsorption and desorption ratios. With this methodology, the concentration of sulforaphane and sulforaphene product reached 85.9%, which was 107-fold higher than that in broccoli seeds (Li et al., 2008). Compared with conventional solvent extraction, adsorption on macroporous resins has numerous advantages, e.g., convenience, lower cost, and higher efficiency that allow the production of sulforaphane and sulforaphene from seed meal in large scale. As an example, macroporous resin-based column chromatography has been used for the isolation of sulforaphene from radish seeds after hydrolysis with myrosinase (185 mg of sinigrin consumed/min/g of radish seeds) (Verkerk and Dekker, 2004). ADS-7, H-2801, HPD-450, AB-8, SP-700, HP-20, HP-2MGL, and D-101 were the macroporous resins used in this study, which were selected and evaluated according to their capacities and ratio of adsorption and desorption. Table 9.3 shows the results of adsorption and desorption experiments. SP-700 and HP-20 macroporous resins showed similar and significantly higher sulforaphene adsorption capacities compared with other assayed resins. SP-700, HP-2MGL, and D-101 macroporous resins showed high desorption ratios, whereas the highest was observed for the first one indicating its superior application for this isolation. The high adsorption capacity and desorption ratio of Sp-700 toward sulforaphene can be attributed to its hydrophobic, nonpolar nature and high specific surface area. Isolation with SP-700 macroporous resin-based column chromatography seems to be cost-effective as it resulted in 117.5 g of sulforaphene-rich extract, whereas 65.8% sulforaphene was obtained from 12.5 kg of radish seeds of 0.7% sulforaphene. Fig. 9.4 illustrates the analytical HPLC chromatograms of the sulforaphene-rich extract and the clarified sample solution.

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

291

Table 9.3 The adsorption and desorption capacities and desorption ratio of sulforaphene on different kinds of macroporous resins at 25
C (Kuang et al., 2013a). Adsorption capacity Desorption capacity Desorption Adsorbent (mg/mL) (mg/mL) ratio (%)

ADS-7 H-2801 HPD-450 AB-8 SP-700 HP-20 HP-2MGL D-101

14.41  0.38 18.97  0.41 16.95  0.48 18.53  0.51 28.81  0.32 26.31  0.37 11.34  0.43 15.23  0.38

9.91  0.47 13.57  0.34 11.01  0.36 14.56  0.45 27.96  0.49 17.50  0.68 9.80  0.54 14.37  0.65

68.8  0.5 71.51  1.42 64.97  0.97 78.55  0.56 97.05  0.52 66.50  0.72 86.40  0.53 94.37  0.79

Figure 9.4 Analytical HPLC chromatograms of the crude plant extract (A) and the purified sulforaphene by HSCCC separation (B). Analytical HPLC column: a reversedphase C18 column (4.6  250 mm, 5 mm, Diamodsil) (Kuang et al., 2013a).

292

Glucosinolates: Properties, Recovery, and Applications

9.5.2 Preparative high-performance liquid chromatography When performing purification on preparative HPLC system, the mobile phase, the flow rate, and the sample loading amount are powerful parameters for enhancing selectivity and capacity. A preparative HPLC purification method has been optimized to purify sulforaphene using a flow rate of 6 mL/min and different methanol solutions (from 20% to 45% methanol in ultrapure water) as the mobile phase. This approach was used to purify up to 300 mg sulforaphene-rich extracts. However, for the purity no less than 96% and the recovery no less than 91%, the maximal sample loading amount should be limited to 250 mg. The preparative HPLC chromatogram of 250 mg of the sulforaphene-rich extract (using 30% methanol in ultrapure water as the mobile phase at the flow rate of 10 mL/ min) is depicted in Fig. 9.5. Further optimization of purification process was conducted to increase purification rate and reduce the solvent consumption, additional optimization was conducted. In particular, once the fraction

Figure 9.5 The preparative HPLC chromatogram of the sulforaphene-rich extract. The preparative HPLC column was a reversed-phase C18 column (19  300 mm, 7 mm, symmetry prep). The mobile phase was 30% methanol in ultrapure water. The flow rate was 10 mL/min. And the detection wavelength was 254 nm. 250 milligrams of the sulforaphene-rich extract separated by resin-based column chromatography were dissolved in 10 mL of the mobile phase, and then filtered with a 0.22 mm membrane filter in preparation for further purification by preparative HPLC (Kuang et al., 2013b).

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

293

of sulforaphene was eluted and manually collected (about 22 min, shown in Fig. 9.5), the methanol concentration of the mobile phase was raised to 100% and maintained for 6 min to achieve a complete elution of nonpolar contaminants. Before the next injection, the preparative HPLC system was held under the initial conditions (using 30% methanol in ultrapure water as the mobile phase at the constant flow rate of 10 mL/min) for 10 min to equilibrate the preparative HPLC column. The results of this optimization study could be used to scale up isolation and purification of sulforaphene from radish seeds to application.

9.5.3 High-speed countercurrent chromatography Another modern isolation technique that could be used is HSCCC. The latest is a simple, low pressure, and low-cost method that provides an easy scale-up separation with minimum sample preparation and cleanup procedures, permitting both normal- and reversed-phase operation (Foucault, 1991; Sutherland et al., 1998). It is based on the separation of compounds between two immiscible liquids, providing several advantages, e.g., no adsorption and complete recovery of the chromatographed sample, use of technical grade solvents, etc. (Feger et al., 2006). This chromatographic method has been investigated for the recovery and purification of sulforaphane from broccoli seed meal. At this case, glucosinolates were extracted from seeds using sequential extraction steps with ethyl acetate, prior applying a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v). The solvent mixture was equilibrated in a separation funnel at room temperature, and the two phases (the upper was the stationary and the lower was the aqueous) were separated shortly before usage. The solvent system is the most important factor of HSCCC separation. Indeed, sulforaphane must be stable and soluble, whereas the two phases should be clearly and quickly separated. Besides, the partition coefficient (K) of sulforaphane should be ranged between 0.5 and 2.0 (Ito, 2005). Four different solvent systems (nhexane/ethyl acetate/methanol/water, n-hexane/ethyl acetate/methanol/ water, ethyl acetate/methanol/water, and ethyl acetate/methanol/water) were assayed in this study providing acceptable K values at 0.61, 0.99, 1.34, and 1.59, respectively. As a result of their high K values, the third and fourth solvents systems took much longer to elute sulforaphane. The addition of n-hexane to ethyl acetate/methanol/water mixture could improve system’s phase separation and respective K values, but at the same

294

Glucosinolates: Properties, Recovery, and Applications

time it increases sensitivity and sulforaphane’s distribution in the lower phase. This is happening because sulforaphane is not soluble in n-hexane that is separated in the upper phase. Conclusively, the mixture of n-hexane/ ethyl acetate/methanol/water was selected as the most appropriate one for sulforaphane separation using HSCCC. The retention of the stationary phase is an important parameter for the determination of peak resolution. Therefore, the effects of flow rates and revolution speeds on the retention volume of the stationary phase were studied. An improved peak resolution (Rs) occurs when the retention volume of the stationary phase (Vs) is increased. This could happen using a low flow rate and a high revolution speed help (Ito, 2005; He et al., 2007). For example, when applying a flow rate of 2.0 mL/min HSCCC separation, an important loss of the stationary phase occurred. In addition, higher retention of the stationary phase resulted in better separation of sulforaphane using a revolution speed of 1000 instead of 800 rpm. A flow rate of 1.5 mL/min and a revolution speed of 1000 rpm were therefore used in the present study, providing a stationary phase retention of 52% as illustrated in Fig. 9.6. Fig. 9.7 depicts chromatograms of sulforaphane obtained by crude extraction and HSCCC. Table 9.4 compared the separation and isolation of sulforaphane from broccoli seed meal induced by HSCCC and HPLC (Liang et al., 2007). Both methodologies provided higher than 95% purity of sulforaphane. However, the recovery yield of HSCCC was more than double compared with this obtained with HPLC (186 mg instead of 71.3 mg of sulforaphane, respectively). In addition, HSCCC does not require any sample pretreatment as in the case of HPLC, whereas it required less separation time and solvent volume (240 min and 360 mL instead of 360 min and 2000 mL in preparative HPLC). In studies to date, normal-phase liquid chromatography (NPLC) (Liang et al., 2005) and preparative reverse-phase HPLC (Kore et al., 1993; Matusheski and Jeffery, 2001; Liang et al., 2007) have been used for the purification of high purity sulforaphane. However, an important problem of these techniques is the irreversible adsorption of impurities on normalphase packing material on the chromatography column. This fact affects adversely the separation yield of the column reverse-phase liquid chromatography has the advantage of lower solvent consumption compared with NPLC. Besides, the current preparative reverse-phase HPLC methodologies applied to purify sulforaphane have high cost and thus cannot be applied for industrial productions. To eliminate the impurities’ effect during separation, sample pretreatment like gel filtration (Kore et al., 1993) or

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

295

Figure 9.6 HSCCC chromatogram of the crude extract of broccoli seed meal in the nhexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v) solvent system (Liang et al., 2008).

Figure 9.7 Analytical HPLC chromatograms of sulforaphane obtained by crude extraction (A) and HSCCC (B). The detected absorbance was set at 254 nm. The column was reversed-phase C18 (250 mm  4.6 mm, 5 mm, Diamonsil). The solvent system consisted of 20% acetonitrile in water, then changed linearly over 10 min to 60% acetonitrile, and was subsequently maintained at 100% acetonitrile for 2 min to purge the column. The column oven temperature was set at 30
C. The flow rate was 1 mL/ min (Liang et al., 2008).

296

Glucosinolates: Properties, Recovery, and Applications

Table 9.4 Comparison of HSCCC and HPLC in separation and isolation of sulforaphane from broccoli seed meal (Yang et al., 2016). Item for comparison HSCCC HPLC

Scale of apparatus Separation of column Flow rate Sample quantity Each separation time Total separation time Separation model Yield Recovery of the target Purity of the target Cost

Semipreparative 250 mL 2 mL/min 850 mg crude extract 240 min 240 min One-step separation 186.0 mg 98.50% 97% Lower

Preparative 300  19 mm 12 mL/min 300 mg crude extract 60 min 360 min SPE þ prep-HPLC 71.3 mg 87.40% 95% Higher

solid-phase extraction (Liang et al., 2007) has been employed. Likewise, simultaneous high-purity and high-recovery of sulforaphane cannot be conducted when the sample loading is increased because of the incomplete purification of sulforaphane in the crude preparation. Another innovative HSCCC method has been developed to produce larger amount of pure sulforaphane (Liang et al., 2008). This approach is known to permit both normal- and reversed-phase operation (Sutherland et al., 1998). Besides, HSCCC has been referred to purify sulforaphane up to or greater than 97% from an ethyl acetate extract (recovered by broccoli seed meal) without using any clean-up steps (Liang et al., 2008). At this study, it was confirmed that HSCCC has numerous advantages against HPLC, e.g., higher yield and purity, shorter separation time, and lower solvent consumption. The different purification methods and procedures of sulforaphane from broccoli seed meal and their relationships are figured in Scheme 1.

9.6 Anticarcinogenic activity of sulforaphane and sulforaphene 9.6.1 In vitro inhibition of cancer cells Inhibition of proliferation and induction of apoptosis are usually considered as important mechanisms for the inhibition of carcinogenesis and cancer growth. Sulforaphane and sulforaphene are known to have a direct inhibitory action on cancer cells of prostate (Chiao et al., 2002), leukemia (Fimognari et al., 2002; Misiewicz et al., 2003), colon (Gamet-Payrastre, 2006), bladder (Tang and Zhang, 2004; Shan et al., 2006), breast (Pledgie-

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

297

Tracy et al., 2007), ovary (Chaudhuri et al., 2007), pancreas (Pham et al., 2004), melanoma (Misiewicz et al., 2003), and medulloblastoma (Gingras et al., 2004). In addition, the mediation of these compounds in cell cycle arrest has been reported in several cell lines, whereas alterations in cell cycle progression have been referred in G1 phase (Chiao et al., 2002; Shan et al., 2006; Wang et al., 2004), S phase (Tang and Zhang, 2004), and/or G2/M phase (Tang and Zhang, 2004; Pham et al., 2004; Jackson and Singletary, 2004; Parnaud et al., 2004; Liang et al., 2008a). Following the results of these studies, both compounds are able to mediate apoptosis through activating mitochondria- (Gamet-Payrastre, 2006; Tang and Zhang, 2005) and death receptoremediated apoptosis (Kim et al., 2006; Matsui et al., 2006; Singh et al., 2005). Likewise, many regulators of cell death and cell cycle have been linked to sulforaphane activity, e.g., MAPK’s and caspases’ activation, cyclins’, cdks’, and Bcl-2 family proteins’ modulation, mitochondria’s damage, downregulation of Cdc25C, upregulation of p21 and p27, inhibition of histone deacetylase, and finally tubulin polymerization (Gamet-Payrastre, 2006; Tang and Zhang, 2004, 2005; Jackson and Singletary, 2004; Xu et al., 2006; Cho et al., 2005; Choi and Singh, 2005; Myzak et al., 2004; Singh et al., 2004; Zhang and Tang, 2007; Mi and Chung, 2008; Telang et al., 2009; Hahm and Singh, 2010). Sulforaphane is able to inactivate histone deacetylase 6-mediated HSP90 deacetylation. Subsequently, this ability leads to attenuation of androgen receptor signaling (a mechanism that could help in prostate cancer prevention) (Gibbs et al., 2009). Sulforaphane has also been referred to exert differential effects on histone deacetylases activity and downstream targets in normal and cancerous prostate epithelial cells (Clarke et al., 2010). Autophagy induction comprises a defense mechanism against sulforaphaneinduced apoptosis in human prostate cancer cells (Herman-Antosiewicz et al., 2006). Human colon cancer cells undergo dose-dependent autophagy and autophagy’s inhibition resulted in potentiation of the proapoptotic effect of sulforaphane (Nishikawa et al., 2010). The combination of sulforaphane treatment with autophagy inhibition may also be a promising approach to control breast cancer (Kanematsu et al., 2010). Besides, the combined treatment of sulforaphane with other anticancer agents has attracted attention. For instance, the combination of sulforaphane with doxorubicin allows the latest to be administered at lower doses (Fimognari et al., 2007). Besides, the combination with resveratrol is known to inhibit cell proliferation and migration, induce release of lactate dehydrogenase, reduce cell viability and prosurvival Akt phosphorylation, and accelerate the

298

Glucosinolates: Properties, Recovery, and Applications

activation of caspase-3 (Jiang et al., 2010). The combination with gemcitabine enhances growth inhibition in HeLa cells in comparison with individual activities (Sharma et al., 2011). Likewise, it has been referred that sulforaphane boosts the ability of numerous compounds (e.g., cisplatin, gemcitabine, doxorubicin, 5-flurouracil) to induce apoptosis in cancer stem cells of pancreas and prostate. Indeed, the combined treatment of sulforaphane with these compounds inhibited clonogenicity, spheroid formation, and aldehyde dehydrogenase 1 activity along with Notch-1 and c-Rel expression. This fact indicates the combined treatment tailored action against cancer stem cell (Kallifatidis et al., 2011). Besides, the anticarcinogenic activity of sulforaphane and the involved molecular mechanisms have been discussed by numerous studies (Gamet-Payrastre, 2006; Tang and Zhang, 2005; Myzak et al., 2004; Singh et al., 2004; Zhang and Tang, 2007; Telang et al., 2009; Herman-Antosiewicz et al., 2006; Nishikawa et al., 2010; Kanematsu et al., 2010; Juge et al., 2007). The effectiveness of both sulforaphane and sulforaphene has been investigated and shown in different human lung cancer cell lines (A549, H460, H446, HCC827, H1975, and H1299) and primary lung cancer cells isolated from lung cancer tissues (Fig. 9.8). By increasing sulforaphane concentration from 10 to 40 mmol/L, the anticancer effect was much more pronounced, whereas toxicity to normal human cells was limited. And, our data suggested that SFE might be a more efficient compound inducing (Fig. 9.9) apoptosis of lung cancer cells.

9.6.2 The anticarcinogenic mechanism of sulforaphane and sulforaphene Accumulated evidences indicate that the PI3K-AKT signaling pathway plays an essential role in lung cancer development. To this line, the effect of sulforaphene and sulforaphane on this pathway was investigated. Although AKT protein expression levels did not show any alteration in both A549 and H460 cells after treatment, phosphorylated AKT levels altered after exposure of these two ITCs (Fig. 9.10). After 24 h of sulforaphene treatment, phosphorylation of AKT in both cell lines reduced by half. In addition, sulforaphene 24-h treatment resulted in a five- and threefold increase of PTEN protein expression in A549 and H460 cells, respectively. From these outcomes, it is evident that sulforaphene may upregulate tumor suppressor PTEN, reduce AKT activation, and subsequently inhibit PI3KAKT signaling pathway in lung cancer. Nevertheless, although elevated PTEN expression and depressed AKT phosphorylation were found in

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

299

Figure 9.8 In vitro evaluation of anticancer effects of sulforaphene (SFE) in multiple lung cancer cells as well as toxicity of SFE to human peripheral blood mononucleated cells (PBMC). (AeF) Anticancer effects of SFE in A549, H460, H1299, H446, HCC827, and H1975 lung cancer cell lines. (G) Toxicity of SFE to primary lung cancer. (H) Toxicity of SFE to human PBMC. All results of the mean of triplicate assays with standard deviation are presented. *P < .05, **P < .01 (Yang et al., 2016).

300

Glucosinolates: Properties, Recovery, and Applications

Figure 9.9 SFE inhibits lung cancer cell proliferation via inducing apoptosis. (A) A549 cell apoptosis was examined at 24 and 48 h after 30 mmol/L SFE treatment. (B) H460 cell apoptosis was examined at 24 and 48 h after 30 mmol/L SFE treatment. All results of the mean of triplicate assays with standard deviation of the mean are presented. *P < .05, **P < .01 (Yang et al., 2016).

A549 cells, results were not repeated in H460 cells. A possible explanation is that sulforaphane may inhibit different signaling pathway in various subtype of lung cancer cells. Furthermore, multiple known PTEN-targeting miRNAs (miR-10a, miR-205, miR-221, and miR-222) were detected to further explore how PTEN was upregulated in lung cancer cells. All assayed miRNAs were significantly downregulated after treating lung cancer A549 and H1299 cells with sulforaphene. It was proposed that the upregulation mechanism of PTEN is partially due to suppressed expression of these miRNAs that target and inhibit PTEN expression (Fig. 9.10E).

9.6.3 Acute toxicity testing in mice The up-and-down method was used to quantify the acute toxicity of sulforaphene. Mice were treated with sulforaphene at different doses after fasted overnight. After 14 days of administration, mice treated with 126.6 mg

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

301

Figure 9.10 SFE inhibits the PI3K-AKT cell signaling pathway. (A, B) SFE can significantly depress phosphorylated AKT levels and induce PTEN expression in A549 cells. (C, D) SFE can significantly depress phosphorylated AKT levels and induce PTEN expression in H460 cells. (E, F) SFE can significantly downregulate miR-10a, miR-205, miR-221, and miR222 expression in A549 cells. All results of the mean of triplicate assays with standard deviation of the mean are presented. *P < .05, **P < .01 (Yang et al., 2016).

302

Glucosinolates: Properties, Recovery, and Applications

Figure 9.11 Acute toxicity testing of SFE in mice. Forty-eight mice were treated with SFE at five different doses of vehicle control or 400, 300, 225, 168.8, and 126.6 mg/kg via lavage administration (n ¼ 8 for each group). Weight changes of mice were showed (Yang et al., 2016).

sulforaphene/kg survived. In addition, no physical or abnormal changes were observed in sleep patterns, behavior patterns, fur, skin, eyes, mucus membranes, tremors, or salivation. The LD50 value of sulforaphene was found to be up to 202.7 mg/kg, whereas no differences between mice treated with 126.6 mg sulforaphene/kg or sterile H2O were observed (Fig. 9.11).

9.6.4 Pharmacokinetics in mice Pharmacokinetics analysis of sulforaphene has also been investigated. For this purpose, half female ICR mice were treated with 10 mg sulforaphene/ kg via intravenous injection and other half mice were treated with 0 mg sulforaphene/kg via oral gavage. Plasma was obtained at specified time intervals, and concentrations of sulforaphene were determined using LC-MS/MS equipment. Fig. 9.12 shows how sulforaphene was absorbed rapidly, reaching after 30 min maximum plasma concentrations of 6.75 and 8.25 mg/mL after intravenous injection and oral administration, respectively. Elimination half-lives were 1.05 and 1.14 h for intravenous injection and oral administration, respectively.

9.6.5 In vivo anticancer effects of sulforaphene The in vivo anticancer properties of sulforaphene were estimated treating nude Balb/C mice with A549 xenograft (Fig. 9.13). At this case, mice

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

303

Figure 9.12 In vivo pharmacokinetics assays. (A, B) The pharmacokinetic properties of SFE were evaluated in ICR mice after intravenous injection. (C) The pharmacokinetic properties of SFE were evaluated in ICR mice after oral gavage. Three mice were measured at each data point and all results of the mean with standard deviation are presented (Yang et al., 2016).

304

Glucosinolates: Properties, Recovery, and Applications

Figure 9.13 In vivo characterization of the anticancer potential SFE on human xenograft lung cancer tumors. (AeD) Nude Balb/C mice with A549 xenograft treated with SFE via injection administration. (EeH) Nude Balb/C mice with A549 xenograft treated with SFE via oral administration. (AeC, EeG) The growth of tumors from SFE treatment was inhibited significantly compared with that of tumors treated with vehicle control. (D, H) There were no significant differences of mice weight between SFE or vehicle control treated groups. NS, not significant (Yang et al., 2016).

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

305

received treatment in a dose of 50 and 100 mg/kg via injection and oral gavage, respectively. After 29 days of treatment with injection, the average tumor volume of the sulforaphene group was 48% of that observed for the solvent control group. Similarly, the average tumor weight of sulforaphenetreated mice was 51.4% compared with the control group. Nevertheless, no significant mice body weight changes were observed between two groups. Treatment with sulforaphene resulted also in a decrease of the mice’s tumor volume and weight compared with the control group. However, significant higher inhibition efficiency in mice treated with sulforaphene via injection was observed compared with the oral treatment, indicating that sulforaphene intake has in vivo inhibitory effects on lung cancer tumor growth. In conclusion, sulforaphene exhibited antiproliferative effects against multiple kinds of lung cancer cells ex vivo and in vivo while having little adverse impacts. These outcomes are consistent to the role of sulforaphene in different cancers as referred by other studies (Shapiro et al., 2006; Perocco et al., 2006; Daxenbichler et al., 1991).

9.7 Conclusions Sulforaphane and sulforaphene have been investigated as potential chemopreventive agent over the last two decades. Since their discovery, new analytical and purification methods of these compounds are introduced. These innovations contribute to improved understanding of different properties of these compounds, e.g., the formation, conversion, bioactivity, metabolism, and stability. This chapter discussed the applicability of these methodologies to establish a better chemopreventive research of sulforaphane and sulforaphene. Besides, these chemopreventive properties have been shown by numerous in vitro and in vivo studies, reflecting modulation of critical metabolism enzymes through antioxidant and detoxification functions, inhibition of cancer cell proliferation, and induction of apoptosis, carcinogen-induced tumors prevention, and finally antiinflammation properties. Based on research results summarized herein, opportunities for future research are proposed. For instance, further investigation is needed to show the efficacy of natural pure sulforaphane and sulforaphene treatments in animal models and human populations. Human clinical studies are still rather limited. These studies can elucidate the chemopreventive ability and toxicity of pure sulforaphane and sulforaphene, instead of the activity found in vegetable extracts. Exploration of combined chemopreventive effects with other compounds is also very important and challenging to develop

306

Glucosinolates: Properties, Recovery, and Applications

novel formulations to improve or strengthen the stability, absorption, and bioavailability of sulforaphane and sulforaphene. Last but not the least, it is important to investigate large-scale production of natural sulforaphane and sulforaphene with high purity to support research on animal experiments and human trials.

References Al Janobi, A.A., Mithen, R.F., Gasper, A.V., Shaw, P.N., Middleton, R.J., Ortori, C.A., Barrett, D.A., 2006. Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 844, 223e234. Bertelli, D., Plessi, M., Braghiroli, D., Monzani, A., 1998. Separation by solid phase extraction and quantification by reverse phase HPLC of sulforaphane in broccoli. Food Chemistry 63, 417e421. Bianchini, F., Vainio, H., 2004. Isothiocyanates in cancer prevention. Drug Metabolism Reviews 36, 655e667. Campas-Baypoli, O.N., Sánchez-Machado, D.I., Bueno-Solano, C., Ramírez-Wong, B., López-Cervantes, J., 2010. HPLC method validation for measurement of sulforaphane level in broccoli by-products. Biomedical Chromatography 24, 387e392. Chaudhuri, D., Orsulic, S., Ashok, B.T., 2007. Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells. Molecular Cancer Therapeutics 6, 334e345. Chiang, W.C., Pusateri, D.J., Leitz, R.E., 1998. Gas chromatography/mass spectrometry method for the determination of sulforaphane and sulforaphane nitrile in broccoli. Journal of Agricultural and Food Chemistry 46, 1018e1021. Chiao, J.W., Chung, F.L., Kancherla, R., Ahmed, T., Mittelman, A., Conaway, C.C., 2002. Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. International Journal of Oncology 20, 631e636. Cho, S.D., Li, G., Hu, H., Jiang, C., Kang, K.S., Lee, Y.S., Kim, S.H., Lu, J., 2005. Involvement of c-Jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by sulforaphane in DU145 prostate cancer cells. Nutrition and Cancer 52, 213e224. Choi, S., Singh, S.V., 2005. Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent. Cancer Research 65, 2035e2043. Ciska, E., Martyniak-Przybyszewska, B., Kozlowska, H., 2000. Content of glucosinolates in cruciferous vegetables grown at the same site for two years under different climatic conditions. Journal of Agricultural and Food Chemistry 48, 2862e2867. Clarke, J., Yu, Z., Ho, E., 2010. Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal and prostate cancer cells. The FASEB Journal 107, 5. Cole, R.A., 1976. Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in cruciferae. Phytochemistry 15, 759e762. Daxenbichler, M.E., Van Etten, C.H., Spencer, G.F., 1977. Glucosinolates and derived products in cruciferous vegetables. Identification of organic nitriles from cabbage. Journal of Agricultural and Food Chemistry 25, 121e124. Daxenbichler, M., Spencer, G., Carlson, D., Rose, G., Brinker, A., Powell, R., 1991. Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry 30, 2623e2638.

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

307

Drewnowski, A., Gomez-Carneros, C., 2000. Bitter taste, phytonutrients,and the consumer: a review. American Journal of Clinical Nutrition 72, 1424e1435. Egner, P.A., Kensler, T.W., Chen, J.G., Gange, S.J., Groopman, J.D., Friesen, M.D., 2008. Quantification of sulforaphane mercapturic acid pathway conjugates in human urine by high-performance liquid chromatography and isotope-dilution tandem mass spectrometry. Chemical Research in Toxicology 21, 1991e1996. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5e51. Fahey, J.W., Wade, K.L., Stephenson, K.K., Chou, F.E., 2003. Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography. Journal of Chromatography A 996, 85e93. Falk, K.L., Vogel, C., Textor, S., Bartram, S., Hick, A., Pickett, J.A., Gershenzon, J., 2004. Glucosinolate biosynthesis: demonstration and characterization of the condensing enzyme of the chain elongation cycle in Eruca sativa. Phytochemistry 65, 1073e1084. Faulkner, K., Mithen, R., Williamson, G., 1998. Selective increase of the potential anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli. Carcinogenesis 19, 605e609. Feger, W., Brandauer, H., Gabris, P., Ziegler, H., 2006. Nonvolatiles of commercial lime and grapefruit oils separated by high-speed countercurrent chromatography. Journal of Agricultural and Food Chemistry 54, 224e252. Fimognari, C., Nüsse, M., Cesari, R., Iori, R., Cantelli-Forti, G., Hrelia, P., 2002. Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane. Carcinogenesis 23, 581e586. Fimognari, C., Lenzi, M., Sciuscio, D., Cantelli-Forti, G., Hrelia, P., 2007. Combination of doxorubicin and sulforaphane for reversing doxorubicin-resistant phenotype in mouse fibroblasts with p53Ser220 mutation. Annals of the New York Academy of Sciences 1095, 62e69. Foucault, A.P., 1991. Countercurrent chromatography. Analytical Chemistry 63, 569e579. Gamet-Payrastre, L., 2006. Signaling pathways and intracellular targets of sulforaphane mediating cell cycle arrest and apoptosis. Current Cancer Drug Targets 6, 135e145. Gibbs, A., Schwartzman, J., Deng, V., Alumkal, J., 2009. Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6. Proceedings of the National Academy of Sciences of the United States of America 106, 16663e16668. Gil, V., MacLeod, A.J., 1980. The effects of pH on glucosinolate degradation by a thioglucoside glucohydrolase preparation. Phytochemistry 19, 2547e2551. Gingras, D., Gendron, M., Boivin, D., Moghrabi, A., Théorêt, Y., Béliveau, R., 2004. Induction of medulloblastoma cell apoptosis by sulforaphane, a dietary anticarcinogen from Brassica vegetables. Cancer Letters 203, 35e43. Graser, G., Oldham, N.J., Brown, P.D., Temp, U., Gershenzon, J., 2001. The biosynthesis of benzoic acid glucosinolate esters in Arabidopsis thaliana. Phytochemistry 57, 23e32. Hahm, E.R., Singh, S.V., 2010. Sulforaphane inhibits constitutive and interleukin-6induced activation of signal transducer and activator of transcription 3 in prostate cancer cells. Cancer Prevention Research 3, 484e494. Halkier, B.A., Du, L., 1997. The biosynthesis of glucosinolates. Trends in Plant Science 2, 425e427. He, J., Yang, R., Zhou, T., Tsao, R., Young, J.C., Zhu, H., Li, X., Boland, G.J., 2007. Purification of deoxynivalenol from Fusarium graminearum rice culture and mouldy corn by high-speed countercurrent chromatography. Journal of Chromatography A 1151, 187e192.

308

Glucosinolates: Properties, Recovery, and Applications

Heiss, E., Herhaus, C., Klimo, K., Bartsch, H., Gerhser, C., 2001. Nuclear factor k B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. Journal of Biological Chemistry 276, 32008e32015. Herman-Antosiewicz, A., Johnson, D.E., Singh, S.V., 2006. Sulforaphane causes autophagy to inhibit release of cytochrome C and apoptosis in human prostate cancer cells. Cancer Research 66, 5828e5835. Higgins, L.G., Kelleher, M.O., Eggleston, I.M., Itoh, K., Yamamoto, M., Hayes, J.D., 2009. Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents. Toxicology and Applied Pharmacology 237, 267e280. Holst, B., Williamson, G., 2004. A critical review of the bioavailability of glucosinolates and related compounds. Natural Product Reports 21, 425e447. Hong, F., Freeman, M.L., Liebler, D.C., 2005. Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane. Chemical Research in Toxicology 18, 1917e1926. Ito, Y., 2005. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A 1065, 145e168. Jackson, S.J., Singletary, K.W., 2004. Sulforaphane inhibits human MCF-7 mammary cancer cell mitotic progression and tubulin polymerization. Journal of Nutrition 134, 2229e2236. Jiang, H., Shang, X., Wu, H., Huang, G., Wang, Y., Al-Holou, S., Gautam, S.C., Chopp, M., 2010. Combination treatment with resveratrol and sulforaphane induces apoptosis in human U251 glioma cells. Neurochemical Research 35, 152e161. Juge, N., Mithen, R.F., Traka, M., 2007. Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cellular and Molecular Life Sciences 64, 1105e1127. Kallifatidis, G., Labsch, S., Rausch, V., Mattern, J., Gladkich, J., Moldenhauer, G., Büchler, M.W., Salnikov, A.V., Herr, I., 2011. Sulforaphane increases drug-mediated cytotoxicity toward cancer stem-like cells of pancreas and prostate. Molecular Therapy 19, 188e195. Kanematsu, S., Uehara, N., Miki, H., Yoshizawa, K., Kawanaka, A., Yuri, T., Tsubura, A., 2010. Autophagy inhibition enhances sulforaphaneinduced apoptosis in human breast cancer cells. Anticancer Research 30, 3381e3390. Keum, Y.S., Jeong, W.S., Kong, A.N., 2005. Chemopreventive functions of isothiocyanates. Drug News and Perspectives 18, 445e451. Kim, H., Kim, E.H., Eom, Y.W., Kim, W.H., Kwon, T.K., Lee, S.J., Choi, K.S., 2006. Sulforaphane sensitizes tumor necrosis factor-related apoptosisinducing ligand (TRAIL)resistant hepatoma cells to TRAILinduced apoptosis through reactive oxygen speciesmediated up-regulation of DR5. Cancer Research 66, 1740e1750. Kore, A.M., Spencer, G.F., Wallig, M.A., 1993. Purification of theu-(methylsulfinyl) alkyl glucosinolate hydrolysis products: 1-isothiocyanato-3-(methylsulfinyl) propane, 1isothiocyanato-4-(methylsulfinyl) butane, 4-(methylsulfinyl) butanenitrile, and5(methylsulfinyl) pentanenitrile from broccoli and Lesquerella fendleri. Journal of Agricultural and Food Chemistry 41, 89e95. Kristal, A.R., Lampe, J.W., 2002. Brassica vegetables and prostate cancer risk: a review of the epidemiological evidence. Nutrition and Cancer 42, 1e9. Kuang, P., Song, D., Yuan, Q., et al., 2013. Preparative separation and purification of sulforaphene from radish seeds by high-speed countercurrent chromatography. Food Chemistry 136 (2), 309e315. Kuang, P., Song, D., Yuan, Q., et al., 2013. Separation and purification of sulforaphene from radish seeds using macroporous resin and preparative high-performance liquid chromatography. Food Chemistry 136 (2), 342e347.

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

309

Kurata, U.Y., Arakawa, T.N., 1986. Effects of pH and ferrous ion on the degradation of glucosinolates by myrosinase. Agricultural & Biological Chemistry 50, 2735e2740. Kwak, M.K., Wakabayashi, N., Kensler, T.W., 2004. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers. Mutation Research 555, 133e148. Lampe, J.W., Peterson, S., 2002. Brassica, biotransformation and cancer risk: genetic polymorphisms alter the preventive effects of cruciferous vegetables. Journal of Nutrition 132, 2991e2994. Li, C., Liang, H., Yuan, Q., Hou, X., 2008. Optimization of sulforaphane separation from broccoli seeds by macroporous resins. Separation Science and Technology 43, 609e623. Liang, H., Yuan, Q., 2012. Natural sulforaphane as a functional chemopreventive agent: including a review of isolation, purification and analysis methods. Critical Reviews in Biotechnology 32 (3), 218e234. Liang, H., Yuan, Q., Xiao, Q., 2005. Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 828, 91e96. Liang, H., Yuan, Q.P., Dong, H.R., Liu, Y.M., 2006. Determination of sulforaphane in broccoli and cabbage by high-performance liquid chromatography. Journal of Food Composition and Analysis 19 (5), 473e476. Liang, H., Li, C., Yuan, Q., Vriesekoop, F., 2007. Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography. Journal of Agricultural and Food Chemistry 55, 8047e8053. Liang, H., Li, C., Yuan, Q., Vriesekoop, F., 2008. Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal. Journal of Agricultural and Food Chemistry 56 (17), 7746e7749. Liang, H., Lai, B., Yuan, Q., 2008a. Sulforaphane induces cell-cycle arrest and apoptosis in cultured human lung adenocarcinoma LTEP-A2 cells and retards growth of LTEP-A2 xenografts in vivo. Journal of Natural Products 71, 1911e1914. Matsui, T.A., Sowa, Y., Yoshida, T., Murata, H., Horinaka, M., Wakada, M., Nakanishi, R., Sakabe, T., Kubo, T., Sakai, T., 2006. Sulforaphane enhances TRAILinduced apoptosis through the induction of DR5 expression in human osteosarcoma cells. Carcinogenesis 27, 1768e1777. Matusheski, N.V., Jeffery, E.H., 2001. Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile. Journal of Agricultural and Food Chemistry 49, 5743e5749. Matusheski, N.V., Juvik, J.A., Jeffery, E.H., 2004. Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli. Phytochemistry 65, 1273e1281. Matusheski, N.V., Swarup, R., Juvik, J.A., Mithen, R., Bennett, M., Jeffery, E.H., 2006. Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane. Journal of Agricultural and Food Chemistry 54, 2069e2076. McInnis, M.L., Larson, L.L., Miller, R.F., 1993. Nutrient composition of whitetop. Journal of Range Management 46, 227e231. Mi, L., Chung, F.L., 2008. Binding to protein by isothiocyanates: a potential mechanism for apoptosis induction in human non small lung cancer cells. Nutrition and Cancer 60 (Suppl. 1), 12e20. Misiewicz, I., Skupinska, K., Kasprzycka-Guttman, T., 2003. Sulforaphane and 2-oxohexyl isothiocyanate induce cell growth arrest and apoptosis in L-1210 leukemia and ME-18 melanoma cells. Oncology Reports 10, 2045e2050.

310

Glucosinolates: Properties, Recovery, and Applications

Myzak, M.C., Karplus, P.A., Chung, F.L., Dashwood, R.H., 2004. A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase. Cancer Research 64, 5767e5774. Nakagawa, K., Umeda, T., Higuchi, O., Tsuzuki, T., Suzuki, T., Miyazawa, T., 2006. Evaporative light-scattering analysis of sulforaphane in broccoli samples: quality of broccoli products regarding sulforaphane contents. Journal of Agricultural and Food Chemistry 54, 2479e2483. Nishikawa, T., Tsuno, N.H., Okaji, Y., Shuno, Y., Sasaki, K., Hongo, K., Sunami, E., Kitayama, J., Takahashi, K., Nagawa, H., 2010. Inhibition of autophagy potentiates sulforaphane-induced apoptosis in human colon cancer cells. Annals of Surgical Oncology 17, 592e602. O’Shaughnessy, J.A., Kelloff, G.J., Gordon, G.B., Dannenberg, A.J., Hong, W.K., Fabian, C.J., Sigman, C.C., Bertagnolli, M.M., Stratton, S.P., Lam, S., Nelson, W.G., Meyskens, F.L., Alberts, D.S., Follen, M., Rustgi, A.K., Papadimitrakopoulou, V., Scardino, P.T., Gazdar, A.F., Wattenberg, L.W., Sporn, M.B., Sakr, W.A., Lippman, S.M., Von Hoff, D.D., 2002. Treatment and prevention of intraepithelial neoplasia: an important target for accelerated new agent development. Clinical Cancer Research 8, 314e346. Parnaud, G., Li, P., Cassar, G., Rouimi, P., Tulliez, J., Combaret, L., Gamet- Payrastre, L., 2004. Mechanism of sulforaphane-induced cell cycle arrest and apoptosis in human colon cancer cells. Nutrition and Cancer 48, 198e206. Pereira, F.M., Rosa, E., Fahey, J.W., Stephenson, K.K., Carvalho, R., Aires, A., 2002. Influence of temperature and ontogeny on the levels of glucosinolates in broccoli (Brassica oleracea var. italica) sprouts and their effect on the induction of mammalian phase 2 enzymes. Journal of Agricultural and Food Chemistry 50, 6239e6244. Perocco, P., Bronzetti, G., Canistro, D., Valgimigli, L., Sapone, A., Affatato, A., Pedulli, G.F., Pozzetti, L., Broccoli, M., Iori, R., Barillari, J., Sblendorio, V., Legator, M.S., Paolini, M., Abdel-Rahman, S.Z., 2006. Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver. Mutation Research 595, 125e136. Pham, N.A., Jacobberger, J.W., Schimmer, A.D., Cao, P., Gronda, M., Hedley, D.W., 2004. The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. Molecular Cancer Therapeutics 3, 1239e1248. Pledgie-Tracy, A., Sobolewski, M.D., Davidson, N.E., 2007. Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Molecular Cancer Therapeutics 6, 1013e1021. Prestera, T., Fahey, J.W., Holtzclaw, W.D., Abeygunawardana, C., Kachinski, J.L., Talalay, P., 1996. Comprehensive chromatographic and spectroscopic methods for the separation and identification of intact glucosinolates. Analytical Biochemistry 239, 168e179. Procháska, Z., 1959. Isolation of sulforaphane from hoary cress (Lepidium draba L.). Collection of Czechoslovak Chemical Communications 24, 2429e2430. Procháska, Z., Komersová, I., 1959. Isolation of sulforaphane from Cardaria draba and its antimicrobial effect. Ceskoslovenska Farmacie 8, 373e376. Prochaska, H.J., Santamaria, A.B., Talalay, P., 1992. Rapid detection of inducers of enzymes that protect against carcinogens. Proceedings of the National Academy of Sciences of the United States of America 89, 2394e2398. Rochfort, S., Caridi, D., Stinton, M., Trenerry, V.C., Jones, R., 2006. The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and

Sulforaphane and sulforaphene: two potential anticancer compounds from glucosinolates

311

preparative high performance liquid chromatography. Journal of Chromatography A 1120, 205e210. Rosa, E.A.S., Heaney, R.K., Fenwick, G.R., Portas, C.A.M., 1997. Glucosinolates in crop plants. Horticultural Reviews 19, 99e215. Shan, Y., Sun, C., Zhao, X., Wu, K., Cassidy, A., Bao, Y., 2006. Effect of sulforaphane on cell growth, G(0)/G(1) phase cell progression and apoptosis in human bladder cancer T24 cells. International Journal of Oncology 29, 883e888. Shapiro, T.A., Fahey, J.W., Dinkova-Kostova, A.T., Holtzclaw, W.D., Stephenson, K.K., Wade, K.L., Ye, L., Talalay, P., 2006. Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study. Nutrition and Cancer 55, 53e62. Sharma, C., Sadrieh, L., Priyani, A., Ahmed, M., Hassan, A.H., Hussain, A., 2011. Anticarcinogenic effects of sulforaphane in association with its apoptosis-inducing and anti-inflammatory properties in human cervical cancer cells. Cancer Epidemiol 35, 272e278. Singh, S.V., Herman-Antosiewicz, A., Singh, A.V., Lew, K.L., Srivastava, S.K., Kamath, R., Brown, K.D., Zhang, L., Baskaran, R., 2004. Sulforaphaneinduced G2/M phase cell cycle arrest involves checkpoint kinase2-mediated phosphorylation of cell division cycle 25C. Journal of Biological Chemistry 279, 25813e25822. Singh, S.V., Srivastava, S.K., Choi, S., Lew, K.L., Antosiewicz, J., Xiao, D., Zeng, Y., Watkins, S.C., Johnson, C.S., Trump, D.L., Lee, Y.J., Xiao, H., HermanAntosiewicz, A., 2005. Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species. Journal of Biological Chemistry 280, 19911e19924. Sivakumar, G., Aliboni, A., Bacchetta, L., 2007. HPLC screening of anticancer sulforaphane from important European Brassica species. Food Chemistry 104, 1761e1764. Smith, J.J., Tully, P., Padberg, R.M., 2005. Chemoprevention: a primary cancer prevention strategy. Seminars in Oncology Nursing 21, 243e251. Spencer, G.F., Daxenbichler, E., 1980. Gas chromatography-mass spectrometry of nitriles, isothiocyanates and oxazolidinethiones derived from cruciferous glucosinolates. Journal of the Science of Food and Agriculture 31, 359e367. Sutherland, A., Brown, L., Forbes, S., Games, G., Hawes, D., Hostettmann, K., McKerrel, E.H., Marston, A., Wheatley, D., Wood, P., 1998. Countercurrent chromatography (CCC) and its versatile application as an industrial purification & production process. Journal of Liquid Chromatography & Related Technologies 21, 279e298. Talalay, P., Dinkova-Kostova, A.T., Holtzclaw, W.D., 2003. Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis. Advances in Enzyme Regulation 43, 121e134. Tang, L., Zhang, Y., 2004. Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells. Journal of Nutrition 134, 2004e2010. Tang, L., Zhang, Y., 2005. Mitochondria are the primary target in isothiocyanate-induced apoptosis in human bladder cancer cells. Molecular Cancer Therapeutics 4, 1250e1259. Telang, U., Brazeau, D.A., Morris, M.E., 2009. Comparison of the effects of phenethyl isothiocyanate and sulforaphane on gene expression in breast cancer and normal mammary epithelial cells. Experimental Biology and Medicine 234, 287e295. VanEtten, C.H., Daxenbichler, M.E., Williams, P.H., Kwolek, W.F., 1976. Glucosinolates and derived products in cruciferous vegetables. Analysis of the edible part from twentytwo varieties of cabbage. Journal of Agricultural and Food Chemistry 24, 452e455. Vaughn, S.F., Berhow, M.A., 2005. Glucosinolate hydrolysis products from various plant sources: pH effects, isolation, and purification. Industrial Crops and Products 21, 193e202.

312

Glucosinolates: Properties, Recovery, and Applications

Verkerk, R., Dekker, M., 2004. Glucosinolates and myrosinase activity in red cabbage (Brassica oleracea L. var. Capitata f. rubra DC.) after various microwave treatments. Journal of Agricultural and Food Chemistry 52, 7318e7323. Wang, L., Liu, D., Ahmed, T., Chung, F.L., Conaway, C., Chiao, J.W., 2004. Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention. International Journal of Oncology 24, 187e192. West, L.G., Meyer, K.A., Balch, B.A., Rossi, F.J., Schultz, M.R., Haas, G.W., 2004. Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, brussels sprouts, kale, and cabbage. Journal of Agricultural and Food Chemistry 52, 916e926. Xu, C., Shen, G., Yuan, X., Kim, J.H., Gopalkrishnan, A., Keum, Y.S., Nair, S., Kong, A.N., 2006. ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells. Carcinogenesis 27, 437e445. Yang, M., Wang, H., Zhou, M., et al., 2016. The natural compound sulforaphene, as a novel anticancer reagent, targeting PI3K-AKT signaling pathway in lung cancer. Oncotarget 7 (47), 76656. Yoxall, V., Kentish, P., Coldham, N., Kuhnert, N., Sauer, M.J., Ioannides, C., 2005. Modulation of hepatic cytochromes P450 and phase II enzymes by dietary doses of sulforaphane in rats: implications for its chemopreventive activity. International Journal of Cancer 117, 356e362. Zhang, Y., Tang, L., 2007. Discovery and development of sulforaphane as a cancer chemopreventive phytochemical. Acta Pharmacologica Sinica 28, 1343e1354. Zhang, Y., Cho, C.G., Posner, G.H., Talalay, P., 1992a. Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Analytical Biochemistry 205, 100e107. Zhang, Y., Talalay, P., Cho, C.G., Posner, G.H., 1992b. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proceedings of the National Academy of Sciences of the United States of America 89, 2399e2403.