Enhanced Nutraceutical bioactivity to fight cancer

1. Introduction
Nutraceuticals are biologically active molecules found in foods that may not be essential for maintaining normal human functions, but may enhance human health and wellbeing by inhibiting certain diseases or improving human performance (Gupta, 2016; Wildman & Kelley, 2007). Numerous different classes of nutraceuticals are found in both natural and processed foods including carotenoids, flavonoids, curcuminoids, phytosterols, and certain fatty acids (Gupta, 2016).

Many of these nutraceuticals have the potential to act as anticancer agents, and may therefore be suitable for incorporation into functional or medical foods as a means of preventing or treating certain types of cancer. Nutraceuticals vary considerably in their chemical structures, physiochemical properties, and biological effects (Bagchi, Preuss, & Sarwoop, 2015; Gupta, 2016).

For example, nutraceuticals vary in their molar mass, structure, polarity, charge and functional groups, which influences their chemical reactivity, physical state, solubility characteristics, and biological fate and functions (McClements, 2015b).

Some nutraceuticals are naturally present in whole foods, such as fruits, vegetables, and cereals, and are therefore often consumed in this form. Conversely, other nutraceuticals are isolated from their natural states and converted into additives that can be incorporated into functional foods, dietary supplements, or pharmaceuticals.

In this article, we will mainly focus on the delivery of nutraceuticals using foods, as it has been proposed that increased consumption of foods rich in nutraceuticals may decrease the risk of certain types of chronic diseases, including cancer. However, if consumers are going to benefit from consuming foods containing nutraceuticals, it is important that they have certain characteristics:

There are a number of factors that currently limit the utilization of many types of
anticancer nutraceuticals in functional foods (Gleeson, Ryan, & Brayden, 2016;
McClements, 2015a; McClements, Li, & Xiao, 2015b). Firstly, many of nutraceuticals
cannot easily be incorporated into foods because they have poor-solubility characteristics,
or they cause undesirable changes in appearance, texture, or flavor of foods. Second,
many nutraceuticals are chemically or biochemically unstable and therefore lose their
bioactivity because they are degraded within food products or the human body. Third,
many nutraceuticals have a low bioavailability and therefore only a small fraction of
them are actually absorbed and utilized by the body. Fourth, for some nutraceuticals the
optimum dose has not been established, and therefore it is unclear how much to deliver in
a bioactive form, e.g., the anticancer efficacy of resveratrol actually decreases as the dose
increases (Cai, Scott, Kholghi, Andreadi, Rufini, Karmokar, et al., 2015)
The purpose of this review article is to highlight how food matrices can be designed
to enhance the biological activity of anticancer nutraceuticals. In particular, we focus on
two different approaches that can be utilized for this purpose (Figure 1): very systems: In this approach, nutraceuticals isolated from their original environment are encapsulated within a delivery system that is specifically designed to enhance their bioavailability (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016; McClements, Decker, & Weiss, 2007; Sagalowicz & Leser, 2010). An example of this approach would be a carotenoid ingredient isolated from carrots that is encapsulated within an oil-in-water emulsion that could then be added to foods such as soft drinks, yogurts, dressings, or sauces (Salvia- Trujillo, Qian, Martin-Belloso, & McClements, 2013a).

In this approach, a nutraceutical-rich food is co-ingested with an excipient food, which is again specially designed to improve the bioavailability of the nutraceuticals (McClements & Xiao, 2014). An example of this approach would be an oil-in-water excipient emulsion consumed at the same time as carotenoid-rich carrots (Zhang, Zhang, Zou, Xiao, Zhang, Decker, et al., 2016b).
nutraceuticals should initially be present in functional foods at a sufficiently high level to have a beneficial physiological effect. The nutraceuticals should remain stable within the functional foods during manufacturing, storage, and utilization, otherwise they may lose their beneficial health effects.
nutraceuticals should not have an adverse effect on the color, taste, or shelf- life of a food product. After ingestion, the nutraceuticals should be released from the functional foods and delivered to the appropriate site-of-action within the human body.

Initially, a brief overview of some of the most important anticancer nutraceuticals that have been identified in foods is given. Some of the major factors limiting the bioavailability of nutraceuticals is then discussed, and potential approaches to overcome these limitations are highlighted. The design of delivery and excipient systems to enhance the bioavailability of anticancer nutraceuticals is then described.

2. Anticancer nutraceuticals
Accumulating evidence suggests that many nutraceuticals, such as curcumin, resveratrol, tea polyphenols, sulforaphane, anthocyanins, genistein, quercetin and
lycopene, exhibit anticancer activities against various forms of cancer (Arvanitoyannis & van Houwelingen-Koukaliaroglou, 2005; Ullah & Khan, 2008).

One of the important advantages of utilizing nutraceuticals to prevent and treat cancer is that they generally exhibit little or no adverse effects frequently associated with pharmaceutical agents after long-term administration. Nutraceuticals have been found to exert a wide range of cellular effects. The possible mechanisms of action of anticancer nutraceuticals include induction of cell cycle arrest and apoptosis in cancerous cells, detoxification of highly reactive molecules, activation of the host immune system, and sensitization of malignant cells to cytotoxic agents (Kotecha, Takami, & Espinoza, 2016; Pan & Ho, 2008). In this section, we focus on several important anticancer nutraceuticals that have beenintensively investigated with particular emphasis on the clinical evidence supporting the safety and efficacy of these compounds in cancer prevention and treatment.

Curcumin: Curcumin is a polyphenol found in turmeric (Curcuma longa), a member of the ginger family (Zingiberaceae) (Joe, Vijaykumar, & Lokesh, 2004). A large number of in vitro and in vivo studies have shown that curcumin inhibits the development of various cancers by inducing cell cycle arrest and cellular apoptosis, through pleiotropic modulation on several key cancer targets such as Wnt/-catenin, nuclear factor kappa B (NF-  B), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF- ), STAT-3 and cyclin D1(Perrone, Ardito, Giannatempo, Dioguardi, Troiano, Lo Russo, et al., 2015). Several phase I and phase II clinical trials have been conducted and demonstrated the safety and anticancer effects of curcumin in patients with different malignancies including myeloma, pancreatic and colorectal cancer (Kotecha, Takami, & Espinoza, 2016). Since curcumin is preferentially distributed in the colonic mucosa, in comparison to other tissues, many clinical trials have been focused on its chemo-preventive efficacy on colorectal cancer (Bar-Sela, Epelbaum, & Schaffer, 2010).

In a phase IIa clinical trial, patients taking 4 grams of curcumin daily were found to have a 40% decrease in the number of aberrant crypt foci (ACF) lesions, which are one of the early histologic signs seen in the colon that may lead to cancer (Carroll, 2012). One of the most important factors limiting the bioefficacy of curcumin is its poor bioavailability.

In animal studies, various edible delivery systems have been developed to improve the bioavailability and bioactivities of curcumin, including liposomes, phospholipid complexes, organogel-based nanoemulsions, chitosan-based nanoparticles and self-emulsifying drug delivery systems (Ting, Jiang, Ho, & Huang, 2014). For example, polylactide co-glycolide nanocapsulated curcumin suppressed cell proliferation, induced cancer cell apoptosis and improved pathological structures in a hepatocellular carcinoma model, whereas an identical concentration of free curcumin was found to be ineffective (Ghosh, Choudhury, Ghosh, Mandal, Sarkar, Ghosh, et al., 2012).

In a recent crossover study, liquid micellar formulations of curcumin showed a 185-fold enhancement in bioavailability within 24 hours without increased toxicity in healthy subjects, compared to powdered curcuminoids (Schiborr, Kocher, Behnam, Jandasek, stede, & Frank, 2014).

Resveratrol is a natural phenol produced by many fruits and plants such as grapes, blueberries, raspberries and peanuts (Bhat, Kosmeder, & Pezzuto, 2001; Kundu & Surh, 2008; Smoliga, Baur, & Hausenblas, 2011a). Animal studies have shown protective effects of resveratrol against several types of cancer, such as breast, skin, gastric, colon, prostate and pancreatic cancers, by interfering with multiple stages of carcinogenesis (Shukla & Singh, 2011).

Recently, clinical trials have established the safety and potential anticancer effects of resveratrol as both a single agent and a constituent of foods (Smoliga, Baur, & Hausenblas, 2011b). A phase I pilot study in colorectal cancer patients determined the anticancer effects of freeze-dried grape powder containing a low dose of resveratrol in combination with other bioactive components. The results suggest that dietary intake of the dry grape powder inhibited the Wnt signaling pathway in the colon, which may have contributed to the observed inhibitory effects on colon carcinogenesis (Nguyen, Martinez, Stamos, Moyer, Planutis, Hope, et al., 2009).

The anticancer effect of resveratrol also has been heavily investigated in breast cancer patients. In a randomized, double-blind placebo-controlled trial, twice daily resveratrol supplement (5 or 50 mg) for 12 weeks decreased the methylation of four cancer-related genes in mammary tissue in women with high risk for developing breast cancer (Zhu, Qin, Zhang, Rottinghaus, Chen, Kliethermes, et al., 2012). Research has been conducted to enhance the bioavailability of resveratrol. Using a prostate cancer mice model, liposome encapsulation of resveratrol and curcumin successfully was shown to increase the oral bioavailablity of both nautraceuticals.

Moreover, encapsulation of both nutraeuticals in the same liposome system resulted in a synergistic reduction of prostate cancer incidence (Narayanan, Nargi, Randolph, & Narayanan, 2009). However, as mentioned earlier, the optimum dose for resveratrol has not been established yet, since some studies have shown that higher levels are less effective at inhibiting cancer than low doses (Cai, et al., 2015).

Sulforaphane: Sulforaphane is an isothiocyanate mainly found in cruciferous vegetables, especially abundant in broccoli and broccoli sprouts (Dinkova- Kostova & Kostov, 2012; Houghton, Fassett, & Coombes, 2013). A large number of cell culture and animal studies have shown that sulforaphane is a potent chemo-preventive agent against various type of cancer.

The molecular targets of sulforaphane vary upon cancer type and stage. The major anticancer mechanism by which sulforaphane protects normal cells from carcinogenesis is through Nrf2- mediated induction of phase II antioxidant and detoxifying enzymes. These enzyme systems enhance cell defense against oxidative damage, and facilitate the removal of carcinogens. Sulforaphane also exerts anticancer activities through various mechanisms of action that are involved in regulating cell proliferation, differentiation, apoptosis, and cell cycle progression (Clarke, Dashwood, & Ho, 2008; Juge, Mithen, & Traka, 2007). To date, only a few clinical trials have been conducted on sulforaphane in cancer patients or high-risk populations. In a phase II study, patients who had recurrent prostate cancer were given 200 moles/day of Sulforaphane-rich extracts for a maximum period of 20 weeks.

Although there was no large decline (by ≥50 %) in prostate-specific antigen (PSA), 7 out of 20 patients experienced moderate PSA declines (by <50 %). Moreover, the on- treatment PSA doubling time (PSADT) was significantly lengthened compared to the pre-treatment PSADT (Alumkal, Slottke, Schwartzman, Cherala, Munar, Graff, et al., 2015). In another clinical trial with Helicobacter pylori-infected humans, daily oral intake of sulforaphane-rich broccoli sprouts (70 gram/day; containing 420 moles of sulforaphane precursor) for 2 months reduced the incidence of infection in humans. This trial suggests that sulforaphane may enhance chemo-protection of the gastric mucosa against H. pylori-induced oxidative stress (Yanaka, Fahey, Fukumoto, Nakayama, Inoue, Zhang, et al., 2009). In a melanoma mouse model, sulforaphane-loaded albumin microspheres demonstrated a significantly stronger suppression on tumor growth as compared to non-encapsulated sulforaphane, and no adverse effects were observed (Do, Pai, Rizvi, & D'Souza, 2010). polyphenols: Tea polyphenols are the major bioactive components in tea leaves, and include epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) (Cabrera, Artacho, & Gimenez, 2006; Crozier, Jaganath, & Clifford, 2009; Nijveldt, van Nood, van Hoorn, Boelens, van Norren, & van Leeuwen, 2001). EGCG and ECG can act as potent antioxidants to prevent DNA damage by scavenging reactive oxygen species (ROS), which in turn prevents carcinogenic mutagenesis in normal cells. In pre-clinical studies, tea polyphenols have been found to regulate multiple key cell signaling pathways, resulting in the suppression of angiogenesis, the modulation of the immune system, and the activation of phase II detoxifying enzymes (Yang, Li, Yang, Guan, Chen, & Ju, 2013). Several clinical trials have suggested that teas and tea polyphenols have the potential to prevent multiple types of cancer, including oral leukoplakia, liver, lung and bladder cancer (Kotecha, Takami, & Espinoza, 2016). In a recent phase II randomized presurgical placebo-controlled trial, bladder cancer patients taking 800 or 1200 mg of polyphenon E (a green tea polyphenol formulation mainly consisting of EGCG) showed a dose-dependent tissue accumulation of EGCG in benign and malignant bladder urothelium, which was associated with reduction of cell proliferation and increase of apoptosis (Gee, Saltzstein, Kim, Kolesar, Huang, Havighurst, et al., 2015). However, many clinical studies showed overall inconsistent results regarding the potential anticancer effects of tea polyphenols in different populations, suggesting different types of teas and variable tea preparations may significantly affect the chemo-preventive properties of teas (Sun, Yuan, Koh, & Yu, 2006). Moreover, the food matrix and gastrointestinal tract are known to impact the bioavailability of tea polyphenols (Manach, Williamson, Morand, Scalbert, & Remesy, 2005). flavonoids: Flavonoids are a large group of polyphenolic secondary metabolites of fruits, vegetables, and other plants with a broad-spectrum of bioactivities, including inhibitory effects on a wide range of human cancers (Tapas, Sakarkar, & Kakde, 2008; Weng & Yen, 2012). Epidemiological
evidence suggests a positive correlation between flavonoids-rich diets and low
risk of colon, breast and prostate cancer. Flavonoids can be categorized into
flavones, flavonols, flavanones, flavanols, anthocyanins and isoflavones based on
their structures. Flavonoids can target multiple signaling pathways during
carcinogenesis to inhibit cancer cell proliferation, suppress tumor angiogenesis,
and induce apoptosis in cancer cells (Batra & Sharma, 2013). Quercetin is one of
the most-studied flavones, and is abundant in onions and apples (Chirumbolo,
2013). Many in vitro and in vivo studies have shown the anticancer potential of
quercetin against a variety of human cancers, such as cervical, breast, colon,
prostate, liver and lung cancer (Yang, Song, Wang, Wang, Xu, & Xing, 2015). A
recent phase I study in patients with chronic Hepatitis C, a major causative factor
of liver cancer, reported that quercetin was safe when consumed at levels up to 5
grams per day, and that there was a modest reduction in viral load, suggesting a
potential for liver cancer prevention (Lu, Crespi, Liu, Vu, Ahmadieh, Wu, et al.,
2016). Quercetin normally exhibits a poor oral bioavailability due to its low
absorption. A microencapsulation approach has been shown to enhance the effects
of quercetin in reducing the oxidative damage and attenuating inflammation in a
mouse colitis model, presumably due to increased absorption (Guazelli, Fattori,
Colombo, Georgetti, Vicentini, Casagrande, et al., 2013). Tangeretin is another
well-studied flavonoid mainly found in citrus fruits (Weng & Yen, 2012). Poor
water-solubility limits the oral bioavailability and efficacy of tangeretin. In a
recent study, an emulsion-based delivery system was shown to significantly
enhance the inhibitory activity of tangeretin on colitis-associated colon
carcinogenesis in mice (Ting, Chiou, Pan, Ho, & Huang, 2015).
3. Factors limiting the bioavailability of anticancer nutraceuticals
The design of functional foods to improve the oral bioavailability of anticancer
nutraceuticals relies on an understanding of their fate within the human gastrointestinal
tract (GIT), as well as of the physicochemical and physiological events that typically
restrict their bioavailability. The factors limiting the oral bioavailability (BA) of
anticancer nutraceuticals can be divided into three main categories, as highlighted
schematically in Figure 2 (Arnott & Planey, 2012; Gleeson, Ryan, & Brayden, 2016;
McClements, 2013; McClements, Li, & Xiao, 2015b; Yao, McClements, & Xiao, 2015):
285 BA = B*  A*  T* (1) 286

The parameters B*, A* and T* represent the fractions of the nutraceuticals that are
bioaccessible, absorbed, and biologically active within the GIT, respectively. These
parameters depend on: (i) the molecular and physicochemical characteristics of the
anticancer nutraceuticals; (ii) the composition and structure of the surrounding food
matrices; and (iii) the complex events occurring within the human GIT. Improved
knowledge of the major factors limiting the BA of specific nutraceuticals will lead to a
more rational design of foods with enhanced beneficial health effects.

3.1. Bioaccessibility
The bioaccessibility (B*) of an anticancer nutraceutical represents the fraction of the total
amount of orally consumed nutraceuticals that is in a form that can be readily absorbed
by the GIT (Figure 2):
B*= mB/mI (2) 300
Here, mB is the mass of nutraceutical that is in a form that can be absorbed, and mI is the
total mass of the nutraceutical that is ingested. In the case of lipophilic nutraceuticals, B*
is usually taken to be the fraction of nutraceuticals solubilized in the mixed micelles in
the small intestinal fluids.
The bioaccessibility may be affected by three main factors in the GIT: liberation;
solubility; and interactions (McClements, 2015c; McClements, Li, & Xiao, 2015b).

 3.2. Absorption 

 After an anticancer nutraceutical is released from any structures containing it, and 

 then solubilized within the gastrointestinal fluids, it has to be transported through the GIT 

 contents and then be absorbed by the epithelial cells lining the GIT (Gleeson, Ryan, & 

 Brayden, 2016). The absorption of nutraceuticals is affected by numerous 

 physicochemical and physiological factors

 3.3. Transformation 

 The bioavailability and bioactivity of anticancer nutraceuticals is a result of their 

 precise chemical structures and mole
cular conformation. Changes in the structure or 

 conformation of these nutraceuticals due to chemical or biochemical reactions within the 

 GIT fluids may therefore alter their bio-efficacy. 

 nutraceuticals by using food components that modulate their chemical or 

 biochemical changes within the GIT.

4. Classification of the bioavailability of anticancer nutraceuticals
Foods can be specifically designed to improve the bioavailability profiles of
anticancer nutraceuticals by modulating their bioaccessibility, absorption, and
transformation in the GIT. Nutraceuticals can be classified according to the major factors
that limit their bioavailability, which means that generic food compositions and structures
can often be developed for a broad range of different nutraceuticals. A brief overview of
a recently developed classification schemes for nutraceuticals is given here (McClements,
Li, & Xiao, 2015b). This system is related to the Biopharmaceutical Classification
Scheme (BCS) commonly used to classify the factors limiting the bioavailability of
pharmaceuticals based on their solubility and permeability characteristics (Dahan, Miller,
& Amidon, 2009; Kawabata, Wada, Nakatani, Yamada, & Onoue, 2011; Lennernas &
Abrahamsson, 2005).

The Nutraceutical Bioavailability Classification Scheme (NuBACS) uses a three-
coordinate system (B*A*T*) to classify a nutraceutical according to the primary factors
that limit its bioavailability (McClements, Li, & Xiao, 2015a). Here, B* is the
Bioaccessibility, A* is the Absorption, and T* is the Transformation of the nutraceutical.
Each coordinate is designated “(+)” if it does not limit bioavailability, and “(-)” if it does.
Additional insights into the precise mechanisms associated with the poor bioavailability
are provided using subscripts, such as limited liberation from the food matrix (L), poor
solubility in the GIT fluids (S), and high susceptibility to metabolism (M) or chemical
degradation (C) (Table 1). As an example, curcumin, which is a highly hydrophobic
anticancer nutraceutical whose bioavailability is limited by poor solubility in GIT fluids,
metabolism, and chemical degradation is classified as B*(-)S A*(+) T*(-)M,C.
One of the major advantages of using this classification scheme is that a common
strategy may be developed to increase the bioavailability of a group of nutraceuticals
with similar properties. For example, food matrices (delivery or excipient systems)
containing digestible lipids could be used to increase the bioavailability of many
lipophilic nutraceuticals, i.e., B*(-)S.

5. Boosting the bioavailability of anticancer nutraceuticals using food
matrix design

Conventionally, the composition and structure of foods is usually only optimized to
enhance their quality attributes, such as appearance, texture, mouthfeel, and taste. More
recently, foods have been designed to improve their nutritional profiles by reducing the
levels of macronutrients believed to have adverse health impacts (such as saturated fats,
digestible carbohydrates, and salt) or to enrich them with food components that are
believed to bring beneficial health effects (such as vitamins, minerals, dietary fibers or
nutraceuticals). In this section, we focus on two different food-based approaches that
may be utilized to enhance the bioavailability, and therefore bioactivity, of anticancer
nutraceuticals (Figure 1).

5.1. Delivery Systems
Delivery systems are specifically designed to contain the anticancer nutraceuticals to
be delivered (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016). If the anticancer
nutraceutical is soluble or dispersible in one of the components (e.g., oil, water, or
powder) used to prepare a traditional food then it can often be simply dissolved or
dispersed in this component prior to preparing the final product. Nevertheless, the food
may still have to be carefully formulated to avoid potential adverse effects, such as
undesirable appearance, taste, or aroma caused by the presence of the nutraceutical.
Moreover, the food matrix may have to be carefully designed to avoid the chemical
degradation of the nutraceutical within the product during food manufacturing, storage,
and utilization. In many cases, an anticancer nutraceutical cannot be simply dissolved or
dispersed into a food matrix. Instead, specialized colloidal delivery systems have to be
fabricated that consist of the anticancer nutraceutical encapsulated within a small particle
(Figure 2).

5.1.1. Particle types
Numerous different types of colloidal particles have been developed that could
potentially be used as delivery systems to encapsulate, protect, and deliver anticancer
nutraceuticals (McClements, 2015c; Yao, McClements, & Xiao, 2015) (Figure 3). Some
of the most promising ones for commercial applications are highlighted here.
Microemulsions: Oil-in-water microemulsions contain colloidal particles dispersed in
water that consist of small clusters of surface active molecules (surfactants) that have
their hydrophobic tails located towards the interior and their hydrophilic heads located
towards the exterior (Figure 3A).

Conversely, water-in-oil microemulsions contain
colloidal particles dispersed in oil where the surfactants are organized so that their
hydrophilic heads form the interior and their hydrophobic tails face toward the exterior.
Both types of microemulsions are thermodynamically stable systems that usually contain
relatively small colloidal particles (5 nm < d < 100 nm). O/W microemulsions are most suitable for encapsulating hydrophobic nutraceuticals, whereas W/O microemulsions are most suitable for encapsulating hydrophilic ones (Flanagan & Singh, 2006; Spernath & Aserin, 2006). Because they are thermodynamically favorable, these systems should form spontaneously when the required components are mixed together. Nevertheless, some energy often has to be applied to ensure thorough mixing of the ingredients and to overcome any kinetic energy barriers. Emulsions: Oil-in-water emulsions (d > 200 nm) or nanoemulsions (d < 200 nm) consist of emulsifier-coated oil droplets dispersed within water (Figure 3B), and are therefore most suitable for encapsulating non-polar nutraceuticals inside the hydrophobic interior of the oil droplets (McClements, 2012; McClements, Decker, & Weiss, 2007). Conversely, water-in-oil emulsions or nanoemulsions consist of emulsifier-coated water droplets dispersed in oil, and are therefore more suitable for encapsulating polar nutraceuticals within the hydrophilic interior of the water droplets. Typically, a relatively hydrophilic emulsifier is required to form an O/W emulsion, whereas a relatively hydrophobic one is required to form a W/O emulsion. In practice, the formation, stability, and functional performance of emulsion-based delivery systems is highly dependent on the nature of the emulsifiers used. Emulsions may be made using a variety of high-energy (high pressure homogenization, microfluidization, and sonication) or low- energy (spontaneous emulsification or phase inversion temperature) methods (McClements & Rao, 2011). The nature of the homogenization method utilized to prepare an emulsion depends on many factors, including the type of ingredients used, the required particle size distribution, and the amount of material that should be produced. Solid lipid nanoparticles: Solid lipid nanoparticles (SLN) are similar to oil-in-water emulsions or nanoemulsions, but the oil phase is crystallized (Figure 3C) (Guri, Guelseren, & Corredig, 2013; Mehnert & Mader, 2012). Crystallization of the oil phase may improve the physical stability of the particles, as well as improving the retention and stability of the encapsulated nutraceuticals. SLNs are usually fabricated by forming an O/W emulsion or nanoemulsion using a high melting point lipid, and then cooling the system to promote crystallization of the lipids. Hydrogel beads: Hydrogel beads (microgels) consist of spherical particles, typically in the range of about 1 to 1000 m, which consist of cross-linked biopolymers that trap water (Figure 3D) (Chen, Remondetto, & Subirade, 2006; Joye & McClements, 2014; Shewan & Stokes, 2013; Zhang, Zhang, Chen, Tong, & McClements, 2015). Filled hydrogel beads also contain other types of colloidal particles dispersed within the beads, such as lipid droplets or liposomes. Hydrogel beads can be formed using a wide range of different methods depending on the nature of the biopolymers and cross-linking agents used. Typically, food-grade proteins (such as whey protein, caseinate, or gelatin) or polysaccharides (such as agar, alginate, carrageenan, pectin, or starch) are used as the biopolymers. The gelation mechanism used is largely determined by the nature of the biopolymer utilized and may involve temperature changes (heating or cooling), desolvation (dehydration or solvent removal), or addition of cross-linking agents (such as ions, polyelectrolytes, or enzymes). Certain types of hydrophilic nutraceutical can be directly incorporated into the hydrogel beads interior by mixing them with the biopolymer solution prior to fabrication. Conversely, hydrophobic nutraceuticals may have to be encapsulated into lipid droplets or liposomes prior to being incorporated into the hydrogel beads. The composition, dimensions, pore size and interactions of hydrogel beads can be controlled to create delivery systems with different properties. Liposomes: This type of colloidal particle typically consists of single or multiple lamellar spherical structures fabricated from phospholipids (Desai & Park, 2005; Fathi, Mozafari, & Mohebbi, 2012; Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). The phospholipids are assembled into bilayers with the non-polar tails of each layer being in contact (Figure 3E). Liposomes have a hydrophilic interior that can be used to encapsulate polar nutraceuticals, and a hydrophobic region in the bilayer that can be used to encapsulate non-polar ones. Liposomes can be formed using a number of different methods, including microfluidization and rehydration of surface deposited layers (Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). 5.1.2. Particle properties
The colloidal particles used as delivery systems may vary appreciably in their
properties, which determines their efficacy at encapsulating, stabilizing, and delivering
anticancer nutraceuticals (Figure 4). Some of the most important particle properties that
can be controlled to obtain specific functional attributes in delivery systems are
summarized below (McClements, 2015c):

Composition: Food-grade colloidal particles suitable for encapsulating anticancer
nutraceuticals can be fabricated from lipids, proteins, carbohydrates, minerals, surfactants
and/or water. The nature of the components used to fabricate the particles plays a major
role in determining their ability to encapsulate, stabilize, and release the nutraceuticals.
The amount of an anticancer nutraceutical that can be encapsulated within a colloidal
particle largely depends on its solubility within the particle interior. Hydrophobic
nutraceuticals can be solubilized within particle domains comprised of non-polar
components such as lipids, surfactant tails, phospholipid tails, or hydrophobic
biopolymers (e.g., zein). Conversely, hydrophilic nutraceuticals can be solubilized with
particle domains comprised of polar components such as water, hydrophilic proteins, and
polysaccharides. The chemical stability of certain nutraceuticals can be enhanced by
encapsulating them in colloidal particles that restrict the molecular motion of reactants
(such as solid lipid nanoparticles) or by incorporating components that protect the
nutraceutical from degradation, such as antioxidants. The gastrointestinal fate of
anticancer nutraceuticals can also be controlled by manipulating the composition of the
colloidal particles, i.e., the rate and extent of their digestion in different regions of the
GIT. For example, colloidal particles comprised of starch may be initially digested by
amylases in the mouth, those made by proteins or lipids may be digested by gastric or
pancreatic proteases or lipases in the stomach and small intestine, and those made by
dietary fibers may not be digested until they reach the colon due to the action of colonic
bacteria.

Dimensions: Colloidal particles can be fabricated with a wide range of dimensions,
ranging from around 10 nanometers (surfactant micelles) to a few millimeters (hydrogel
beads). The dimensions of colloidal particles influence many of the properties of
delivery systems, including their chemical stability, release rate, optical properties,
physical stability (to gravitational separation and aggregation), rheology, release rate, and
gastrointestinal fate. Anticancer nutraceuticals that are prone to chemical degradation
when they are dispersed in water (such as curcumin under basic conditions) tend to
breakdown more rapidly when they are in small rather than large lipid particles (Zou,
Zheng, Liu, Liu, Xiao, & McClements, 2015). This effect can be attributed to the fact
that the fraction of nutraceutical molecules in close proximity to the oil-water interface
increases with decreasing particle size. Conversely, the rate of lipid particle digestion
and nutraceutical release tends to increase as the particle size decreases because then
there is more oil-water interface available for digestive enzymes to adsorb to (Salvia-
Trujillo, Qian, Martin-Belloso, & McClements, 2013a). The particle size also affects the
incorporation of colloidal particles into food products. Larger particles tend to cream or
sediment more rapidly than smaller ones because the magnitude of the gravitational
forces is proportional to the diameter squared. Gravitational separation of particles may
be an important consideration in functional foods and beverages that have relatively low
viscosities. The optical properties of foods depend on the particle diameter (d) relative to
the wavelength of light (): colloidal dispersions go from being transparent when d <<  to turbid/opaque when d  , and to visibly distinguishable as separate particles when d >> . The dimensions of the particles will also affect their perception within the
mouth: a colloidal dispersion with particles less than about 50 m in diameter will feel
smooth in the mouth, whereas one with larger particles will feel gritty or lumpy.
Obviously, these factors have to be taken into account when designing delivery systems
intended for application in commercial food products that must be perceived favorably by
consumers.

Interfacial properties: The colloidal particles used in the food industry as delivery
systems may have different interfacial properties, e.g., composition, thickness, charge,
and hydrophobicity (McClements, 2015c). The interfacial properties also have a major
impact on the chemical stability, physical stability, release rate, optical properties,
rheology, and gastrointestinal fate. For example, the magnitude and sign of the electrical
charge on the surfaces of the colloidal particles determines their ability to interact with
other electrical substances, such as other charged particles, surfaces, or polymers. In
addition, the charge may impact the chemical stability of encapsulated nutraceuticals by
influencing the attraction or repulsion of pro-oxidants to the particle surfaces (such as
cationic transition metals). The surface chemistry and digestibility of the particle surface
influences the ability of digestive enzymes (such as lipases, proteins, or amylases) to
adsorb to them and hydrolyze the particles. Colloidal particles can often be coated with a
substance that is resistant to digestion in one region of the GIT, but that is digested in
another region. This phenomenon can be used to control the release of encapsulated
nutraceuticals in different parts of the GIT, e.g., mouth, stomach, small intestine or colon.

Physical state: Colloidal particles may be liquid, semi-solid, or solid depending on the
materials used to assemble them and the environmental conditions (such as temperature,
pH, and ionic composition). The oils and water phase used to prepare oil-in-water or
water-in-oil emulsions are typically liquid. However, high melting oils can be used to
create solid lipid nanoparticles that have a crystalline interior. The biopolymers (proteins
and polysaccharides) used to prepare hydrogel beads usually form semi-solid gel-like
particles. The physical state of a particle may change appreciably when environmental
conditions are changed within a food or as it passes through the GIT.

5.1.3. Mechanisms of Action
Appropriately designed delivery systems may enhance the oral bioavailability and
bioactivity of anticancer nutraceuticals due to numerous mechanisms of action:
Enhance Dispersibility: Most non-polar nutraceuticals are so hydrophobic that they
cannot simply be dispersed into aqueous-based foods (such as beverages, sauces,
dressings, yogurts, and desserts) because of their extremely poor water-solubility (Donsi,
Sessa, Mediouni, Mgaidi, & Ferrari, 2011). However, if these nutraceuticals can be
encapsulated within colloidal particles that have a hydrophobic core and a hydrophilic
shell (as in oil-in-water emulsions, nanoemulsions, or microemulsions), then they can
easily be dispersed into these aqueous-based foods (Flanagan & Singh, 2006;

McClements, 2012). Conversely, many polar nutraceuticals are so hydrophilic that they
cannot easily be dispersed into oil-based foods (such as butter, margarine, and spreads).
In this case colloidal particles that consist of a hydrophilic core and hydrophobic shell (as
in water-in-oil emulsions, nanoemulsions, or microemulsions) can be used to encapsulate
them.

Enhance Chemical or Biochemical Stability: Many nutraceuticals are highly
susceptible to chemical or biochemical degradation within food products or inside the
GIT (Braithwaite, Tyagi, Tomar, Kumar, Choonara, & Pillay, 2014; Lu, Kelly, & Miao,
2016; Yallapu, Nagesh, Jaggi, & Chauhan, 2015). Delivery systems can be specifically
designed to protect the encapsulated nutraceuticals from degradation by providing a
physical barrier or by containing specific components (such as antioxidants, buffers or
UV-visible absorbers).

As an example, some nutraceuticals (such as curcumin) are more
prone to chemical degradation when they are dissolved in water than when they are
dissolved in oil. Consequently, their chemical stability can be improved by encapsulating
them in delivery systems that have lipophilic domains, such as vesicles, nanoemulsions,
protein nanoparticles, or solid lipid nanoparticles (Zou, Liu, Liu, Xiao, & McClements,
2015; Zou, Zheng, Zhang, Zhang, Liu, Liu, et al., 2016a, 2016b).

Including antioxidants or chelating agents within a delivery system can inhibit the oxidation of unsaturated
lipids (Davidov-Pardo & McClements, 2015; Qian, Decker, Xiao, & McClements, 2012;
Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014). Delivery systems can be designed so
that chemically unstable nutraceuticals are trapped within colloidal particles with
coatings that physically separate the nutraceuticals from any other components that may
accelerate their degradation.

Enhance Bioaccessibility: Many nutraceuticals have a low oral bioaccessibility
because of their low solubility in aqueous GIT fluids (Arora & Jaglan, 2016;
McClements, Li, & Xiao, 2015b). Delivery systems can be designed to contain digestible
lipids (such as triglycerides) that form free fatty acids and monoacylglycerols that
enhance the solubilization capacity of the mixed micelles for hydrophobic nutraceuticals
(Ozturk, Argin, Ozilgen, & McClements, 2015; Salvia-Trujillo, Qian, Martin-Belloso, &
McClements, 2013b; Salvia-Trujillo, Sun, Urn, Park, & McClements, 2015; Yao,
McClements, & Xiao, 2015).

The type of lipid used is particularly important since it
determines the rates of lipid digestion and mixed micelle formation, as well as the
solubilization capacity of the mixed micelles. The hydrophobic domains in the mixed
micelles (micelles and vesicles) must be large enough to accommodate the nutraceuticals,
so that they can transport them through the mucus layer to the epithelial cells. There is
therefore considerable interest in designing the lipid phase of foods so that they form
mixed micelles in the small intestine that have an appropriate solubilization capacity for
the nutraceuticals being delivered (Yao, McClements, & Xiao, 2015; Yao, Xiao, &
McClements, 2014).

Enhance Absorption: A delivery system may contain specific components that can
enhance the uptake of the anticancer nutraceuticals, such as components that disrupt the
mucus layer, inhibit efflux inhibitors, stimulate active transporters, or increase the
dimensions of tight junctions (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016;
McClements, Li, & Xiao, 2015b; Yao, McClements, & Xiao, 2015). Gleeson and co-
workers have recently highlighted the major strategies for increasing the absorption of
nutraceuticals (Gleeson, Ryan, & Brayden, 2016).

Certain components found in foods,
such as bromelain (an enzyme from pineapple) and papain (an enzyme from papaya),
have the potential to disrupt the mucus layer that normally coats the epithelial cells,
which may lead to enhanced absorption of nanoparticles coated with these substances (de
Sousa, Cattoz, Wilcox, Griffiths, Dalgliesh, Rogers, et al., 2015). Other components
present in foods are able to increase the permeability of epithelial cells, including
medium chain fatty acids like caprylic acid (Gleeson, Ryan, & Brayden, 2016). Other
food components may also have the ability to act as permeation enhancers, such as
cationic biopolymers (chitosan) chelating agents (EDTA), surfactants (polyol or sugar
esters of fatty acids) and phytochemicals (such as piperine) (McClements, Li, & Xiao,
2015b).

These intestinal permeation enhancers are believed to increase absorption by
increasing the fluidity of the phospholipid membranes, increasing the dimensions of the
tight junctions, stimulating active transporters, and/or blocking efflux transporters.
Nevertheless, it is important to realize that there may be adverse health effects associated
with altering the normal absorption mechanisms of nutraceuticals, which should be
considered when designing functional foods based on this principle (McCartney,
Gleeson, & Brayden, 2016).

5.2. Excipient Systems
Unlike delivery systems, excipient systems do not necessarily contain any
nutraceutical components (McClements & Xiao, 2014; McClements, Zou, Zhang, Salvia-
Trujillo, Kumosani, & Xiao, 2015). Instead they are designed to boost the bioavailability
and bioactivity of the nutraceuticals present in other foods that they are co-ingested with,
such as fruits and vegetables. Alternatively, they can be designed to boost the
bioavailability of nutraceuticals in dietary supplements or drugs (Salvia-Trujillo &
McClements, 2016b). Typically, the composition and structure of the excipient food
matrix is carefully controlled so as to improve the bioaccessibility, absorption, or
transformation profile of a nutraceutical-containing substance that is co-ingested with it.
Many of the approaches developed to improve the bioavailability of nutraceuticals using
delivery systems can also be used with excipient systems.

A considerable research effort has recently been carried out with the objective of
developing excipient systems based on emulsions that are suitable for utilization in the
food, dietary supplement, or pharmaceutical industries (McClements & Xiao, 2014;
McClements, Zou, Zhang, Salvia-Trujillo, Kumosani, & Xiao, 2015). These excipient
emulsions consist of small lipid droplets dispersed in an aqueous medium.
The
composition, size, and interfacial properties of the lipid droplets are optimized based on a
number of factors: (i) the ability to extract and solubilize lipophilic nutraceuticals from
food matrices (such as fruits and vegetables); (ii) the ability to be rapidly digested within
the GIT; (iii) the ability to rapidly form mixed micelles that solubilize the lipophilic
nutraceuticals released from the food matrices; (iv) the ability to protect the
nutraceuticals from chemical or biochemical degradation within the GIT. Additional
components may also be added to the aqueous phase of the excipient emulsions to
improve their efficacy, including antioxidants, chelating agents, buffering agents,
biopolymers, or permeation enhancers.

Recent research has shown that excipient emulsions can be used to increase the
bioavailability of carotenoids in a range of produce, including mangoes (Liu, Bi, Xiao, &
McClements, 2016), carrots (Zhang, Zhang, Zou, Xiao, Zhang, Decker, et al., 2015,
2016a; Zhang, et al., 2016b), yellow peppers (Liu, Bi, Xiao, & McClements, 2015), and
tomatoes (Salvia-Trujillo & McClements, 2016a). The composition and structure of the
excipient emulsions has to be carefully optimized to effectively enhance carotenoid
bioavailability (Figure 5). Studies have shown that excipient emulsions containing long
chain triglycerides (LCT) are more effective than those containing medium chain
triglycerides (MCT) at improving the bioaccessibility of carotenoids (Zhang, et al.,
2015). This effect was attributed to the fact that digestion of LCT by lipase leads to the
generation of long chain fatty acids that enhance the solubilization capacity of the mixed
micelle phase by increasing the dimensions of the hydrophobic domains (Cho, Salvia-
Trujillo, Kim, Park, Xiao, & McClements, 2014; Salvia-Trujillo, Qian, Martin-Belloso, &
McClements, 2013b).

Conversely, the hydrophobic domains formed by the medium
chain fatty acids resulting from MCT digestion are too small to accommodate large
carotenoids. Studies with carrots showed that the bioaccessibility of the carotenoids
increased with increasing fat content in the excipient emulsions co-ingested with them
(Zhang, et al., 2016b). Moreover, decreasing the size of the fat droplets in the excipient
emulsions was found to increase carotenoid bioaccessibility, which was attributed to their
higher surface area and rate of digestion, leading to quicker mixed micelle formation and
nutraceutical solubilization (Zhang, et al., 2016a).

Excipient emulsions have also been used to improve the bioavailability of curcumin
from turmeric powder (Zou, Liu, Liu, Liang, Li, Liu, et al., 2014; Zou, Zheng, Liu, Liu,
Xiao, & McClements, 2015). A higher amount of curcumin reached the simulated small
intestine when the turmeric powder was co-ingested with an excipient emulsion, which
was attributed to a number of effects. First, some of the curcumin was transported into
the hydrophobic interior of the small lipid droplets. Second, the chemical stability of the
curcumin was higher within the hydrophobic interior of the lipid droplets than in the
surrounding aqueous phase. Third, the mixed micelles formed after digestion of the lipid
droplets were able to solubilize the non-polar curcumin molecules in the aqueous GIT
fluids.

The concept of excipient foods is fairly new, and a considerable amount of research
is still required in this area. Nevertheless, it is a promising approach for improving the
bioavailability characteristics of many anticancer nutraceuticals in functional foods,
dietary supplements, and pharmacological drugs.

6. Conclusions
787 There is growing interest in using specific nutraceuticals commonly found in foods
as chemo-preventative agents. Cell culture and animal studies suggest that ingestion of
these nutraceuticals may inhibit certain types of cancers. Nevertheless, many anticancer
nutraceuticals cannot simply be incorporated into foods because of their poor solubility,
stability, and bioavailability characteristics. There is a considerable potential to create
functional food products designed to overcome these challenges, and therefore increase
the potential efficacy of anticancer nutraceuticals. These foods may be designed to
contain the nutraceuticals themselves (delivery systems) or they may be designed to boost
the bioavailability of nutraceuticals in other foods (excipient systems). Despite their
potential, a considerable amount of research is still needed to demonstrate the efficacy of
nutraceuticals, and the ability of delivery or excipient systems to enhance their bio-
efficacy.

Unlike pharmaceuticals, which are typically taken in a well-defined dose at a
particular time, nutraceuticals are obtained from numerous sources at levels that vary
from person to person and from time to time as part of a complex diet. In addition, the
beneficial effects of nutraceuticals may arise from taking relatively low levels over an
extended period.

Consequently, it is often difficult to carry out clinical studies (double
blind randomized feeding trials) using humans to prove their efficacy. Nevertheless, it
may be possible to gain some insights into their efficacy using long-term animal feedings
studies where the diet is carefully controlled. Having said this, there is also considerable
skepticism about the bioactivity demonstrated by certain nutraceuticals established using
in vitro and in vivo assays.

For instance, recent articles have criticized the evidence
suggesting that curcumin has a high bioactivity due to its ability to interfere with many
types of assays used to measure biological activity (Baker, 2017; Nelson, Dahlin, Bisson,
Graham, Pauli, & Walters, 2017). This may at least partially account for the lack of
efficacy that curcumin has demonstrated in many clinical trials.

Sulforaphane a powerful tool to fight cancer, aging, and other inflammatory health issues

Sulforaphane has been shown to be an effective antioxidant, antimicrobial, anticancer, anti-inflammatory, anti-aging, neuroprotective, and anti-diabetic (R). It also protects against cardiovascular and neurodegenerative diseases (R).

Many test-tube and animal studies have found sulforaphane to be particularly helpful for suppressing cancer development by inhibiting enzymes that are involved in cancer and tumor growth (R, R, R).

According to some studies, sulforaphane may also have the potential to stop cancer growth by destroying cells that are already damaged (R, R).

Sulforaphane appears to be most protective against colon and prostate cancer but has also been studied for its effects on many other cancers, such as breast, leukemia, pancreatic and melanoma (R).

Sulforaphane may also help reduce high blood pressure and keep arteries healthy — both major factors in preventing heart disease (R).

Recent research shows that Sulforaphane can help control blood glucose levels in type 2 diabetic patients as effectively as the most commonly used prescription medicine Metformin (R).

Sulforaphane and Broccoli Sprouts

Sulforaphane (SFN) is an isothiocyanate. It is derived from glucoraphanin, found in cruciferous vegetables such as broccoli, cabbage, cauliflower, brussels sprouts and kale (R, R).

Glucoraphanin is stable, but when the vegetables are cut or chewed, it comes in contact with the enzyme myrosinase, that also occurs naturally in these vegetables, and Sulforaphane is formed(RR).

Unlike the glucoraphanin, sulforaphane degrades quickly (R).

The quantity of glucoraphanin varies greatly in different plants. In general, levels of glucoraphanin and sulforaphane are highest in broccoli sprouts (R), but 3 day-old sprouts can contain up to 100 times more glucoraphanin than in mature plants (R).

Sulforaphane  is rapidly absorbed, reaching peak concentration after 1-3 hours. (RR). Levels are back to baseline within 72 hours (RRR).

Daily consumption of cruciferous vegetables can maintain levels of Sulforaphane in the body, if properly prepared (R), however most people find  it is easier to take a daily supplement.

Sulforaphane activation of  AMPK pathway is key to wide range of benefits

There are many hundreds of studies of Sulforaphane’s  effect in fighting various disease and metabolic problems.  Activation or Inhibition of several different genes are described in how it manages to impact such a wide range of health problems.

As with vitamin antioxidants the notion that supplements act as “antioxidants” in human cells is called into question []. Emerging evidence suggests that the most effective supplement exert their intracellular effects not as direct “antioxidants” per se but as modulators of signaling pathways.

Compared with widely used phytochemical-based supplements like curcumin, silymarin, and resveratrol, sulforaphane more potently activates Nrf2 – which researchers call the “Master Regulator” of Cell Defense (R).

A list of genes and enzymes Sulforaphane influences is at the bottom of this page.

Perhaps even more meaningful is that like Metformin and Berberine (R), Sulforaphane strongly activates AMPK, which raises the intracellular NAD+ concentrations and activates SIRT1 which has been shown to have numerous disease fighting and anti-aging potential(R).

It’s possible many of the health benefits are at least partially related to this AMPK/NAD+/SIRT activity.

Here is a  short list of studies showing Sulforaphane activating AMPK to fight Cancer(R),Diabetes(R),Obesity(R),Neurological disease (R),Heart disease (R),HIV (R),Colitis(R).

Sulforaphane helps prevent and can even kill cancer

3-5 servings per week of Cruciferous vegetables decrease the risk of cancer by 30-40% (R).

Even ONE serving of cruciferous vegetables per week significantly reduced the risk of pharynx, colorectal, esophageal, kidney and breast cancer (R).

In vitro, Sulforaphane has been demonstrated to kill breast cancer cells (R),oral squamous cell carcinoma cells (R), colorectal cancer cells (R),  cervical, liver, prostate, and  leukemia cancer cells (RR), while having little to no effect on healthy cells (R)

Sulforaphane combats cancer by multiple mechanisms:

  • Sulforaphane reduces inflammation by inhibiting the NF-κB pathway(R).
  • Sulforaphane induces cancer cell death (R).
  • SFN inhibits Phase I enzymes that enable cancer cell growth (R).
  • SFN induces Phase II enzymes that clear  DNA damaging chemicals (R).
  • Sulforaphane thereby inhibits cancer cell proliferation  (R)

In addition to the numerous cancer fighting mechanism of Sulforaphane, it is also very effective at enhancing commonly used anti-cancer drugs such as    cisplatin, gemcitabine, doxorubicin, and 5-fluorouracil    , allowing for smaller dosages and limiting toxicity to healthy cells (R).

Sulforaphane helps lower Cholesterol


Clinical studies with humans has shown eating broccoli reduces LDL cholesterol.

Twelve healthy subjects that consumed 100 grams per day of broccoli sprouts lowered LDL cholesterol, increased HDL cholesterol, and improved maarkers for oxidative stress  (R).

Sulforaphane May Help Parkinson’s, Alzheimers, Huntingtons

In mouse models of Parkinson’s disease, Sulforaphane increased dopamine levels in the brain to alleviate loss of motor coordination(RRRR).

A buildup of amyloid beta ( Aβ ) peptides are thought to be the cause of Alzheimer’s disease.  Broccoli sprouts were shown to prevent amyloid beta buildup and cell death (RR).

Sulforaphane has also bee shown to reduce Aβ plaque, and lessens cognitive impairment in mouse models of Alzheimer’s disease (RRR).

Sulforaphane activates a protein that slows huntingtins disease in mice (R).

Sulforaphane Prevents and Combats Heart & Cardiovascular Disease


Observational studies in humans has shown those who eat 3-5 servings of cruciferous vegetables a week have a significantly decreased risk of cardiovascular disease (R).

Research with mice shows Sulforaphane decreases blood pressure (RR).

Rats that were given Sulforaphane after heart attack exhibited  reduced heart damage  (R).

Sulforaphane helps prevent atherosclerosis (R) and minimizes inflammation caused by hardening of arteries in mice (R).

Sulforaphane reduces formation of  blood clots and platelet aggregation in humans (R).

Lastly, Sulforaphane has proven beneficial in minimizing damage from strokes, with decreased brain tissue damage (R), and loss of neurological function (R).

Sulforaphane helps control Diabetes and fight  Obesity


In humans, eating Broccoli sprouts  increased  HDL cholesterol, and lowered  triglycerides, insulin, insulin resistance,oxidative stress, and C-Reactive Proteins (RRR).

Sulforaphane decreases incidence and severity of the following diabetes complications in mice (RR)

  • vascular complications .
  • diabetes-induced heart dysfunction
  • heart damage in mice
  • nephropathy
  • tissue damage

Mice fed a high fat diet to induce obesity that were subsequently treated with sulforaphane for 3 weeks had significantly less weight gain, and improved insulin resistance, glucose and cholesterol levels (R,R).

Sulforaphane is Antiviral

Eating Broccoli sprouts increase the bodies natural anti-virus response and reduce influenza (R, R).

In vitro, Sulforaphane  combats  influenza, HIV, Epstein-Barr virus (R) and hepatitis C virus (R).

Sulforaphane Combats Bacterial and Fungal Infections

Human β-defensin-2 (HBD-2) is a key part of our defense against bacterial invasion. Sulforaphane increases HBD-2 in response to  to 23 of 28 bacterial species (R).

Cystic fibrosis patients have increased levels of Mycobacterium abscessus.  Treatment with Sulforphane of such macrophages  significantly decreased bacterial burden (R).

Sulforaphane Combats Inflammation

Nuclear Factor Kappa-B (NF-kB) is a well known driver of inflammation. Sulforaphane greatly decreases NF-kB activity (R).

As previously mention, Sulforaphane very strongly activates Nrf2, which lowers inflammation (RR).

Sulforaphane May Combat Depression and Anxiety


Inflammation has been recognized as one of the causes of depression. By reducing inflammation, sulforaphane can help combat depression.

Repeated SFN administration reverses depression– and anxiety-like behaviors in chronically stressed mice, likely by inhibiting the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory responses to stress (RR).

In another study, it was shown that Nrf2 deficiency in mice results in depressive-like behavior, while the induction of Nrf2 by sulforaphane has antidepressant-like effects (R).

Also, dietary intake of glucoraphanin during the juvenile and adolescent periods in mice prevents the onset of depression-like behaviors at adulthood (R).

Sulforaphane Protects the Brain and Restores Cognitive Function

Sulforaphane increases neuronal BDNF in mice, a factor that supports the survival of existing neurons and encourages the formation of new neurons and synapses (R).

SFN reduces brain inflammation in various animal models of pathogen-induced neuroinflammation and neurodegenerative disease (RRRR).

Sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 state. This reduces brain inflammation and restores spatial learning and coordination in rats (R).

Sulforaphane is beneficial in various pathological conditions:

  • SFN improves cognitive performance and reduces working memory dysfunction in rats after traumatic brain injury (R).
  • SFN attenuates cognitive deficits in mouse models of psychiatric disease. Also, the intake of glucoraphanin during the juvenile and adolescent periods prevents the onset of cognitive deficits at adulthood (R).
  • SFN alleviates brain swelling in rats, by attenuating the blood-brain barrier disruption, decreasing the levels of pro-inflammatory cytokines, and inhibiting NF-κB (R). SFN also increases AQP4 (a water channel protein) levels, thereby reducing brain swelling (R).
  • SFN prevents memory impairment and increases the survival of hippocampal neurons in diabetic rats (R).

Sulforaphane recovers memory in mice and rats with chemically induced memory impairment (RR R).

SFN exerts positive effects against brain damage induced by acute COpoisoning in rats (R).

Sulforaphane protects human neurons against prion-mediated neurotoxicity (R).

Insufficient NRF2 activation in humans has been linked to neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (R). SFN, as a potent Nrf2 activator, may help in the treatment of these diseases.

Sulforaphane Improves Symptoms of  Autism

Sulforaphane activates several genes that lower inflammation and protect cells from oxidative stress and DNA damage, which are much higher in those with Autism (R).

In a clinical trial of 29 young men with  moderate to severe autism (age 13-27), Sulforphane treatment over 13 weeks resulted in a 35% improvement in disruptive behavior(R).

Sulforaphane relieves Gastrointestinal inflammation, colitis, and ulcers

Aspirin and NSAID’s are very effective at relieving pain, but can damage stomach lining and even cause ulcers.  Sulforaphane has been shown to protect agains such damage in mice (RR,R).

Sulforaphane has also been shown to increase Nrf2 and decrease inflammation in mice with colitis (R,R).

Sulforaphane May be Beneficial in Airway Inflammation and Asthma

Sulforaphane received airway inflammation and asthma symptoms in mice  (R)

Broccoli sprout extract  relieved airway inflammation in humans exposed to vehicle exhaust levels similar to those on a Los Angeles freeway (R).

But in other studies,  broccoli sprouts did not alleviate symptoms of asthma (R),  COPD (R) or ozone-induced airway inflammation (R).

Sulforaphane Can Be Beneficial in Arthritis

We previously pointed out that Sulforaphane strongly activates Nrf2, which relieves inflammation in many conditions.  In additions, sulforaphane was found to inhibit metalloproteinases that cause osteoarthritis and cartilage destruction (R).

Sulforaphane also decreases inflammatory cytokines, reducing symptoms of arthritis in mice (R).

Sulforaphane Protects the Eyes

Sulforaphane protects photoreceptor cells from excessive light exposure damage  (R) and  degeneration (R) in humans, and light-induced retinal damage in mice (R).

Sulforaphane protects human retinal cells and delays onset of cataracts (R), and helps prevent complications after cataract surgery (R).

In mice, sulforaphane helped maintain vitamin A and C levels in retinal cells to prevent damage from oxidative stress (RRR).

Negative Side Effects

Possible liver Toxicity at extreme dosages

There has been a single report of liver toxicity in one individual that consumed over 800 ml per day of broccoli juice for 4 weeks, but function returned to normal wishing 15 days or discontinuing the juice (R).

Note this individual was making juice from mature broccoli plants which have many different active substance and are not recommended for source of glucoraphanin/sulforaphane as the young sprouts have up to 100 times higher glucoraphanin levels.

Maximizing Bioavailability

Glucoraphanin Sources

Broccoli has the highest amounts of glucoraphanin of any vegetable, but it is also found in Brussel sprouts, Kale, Cabbage, Bok Choy, and several others (R).

As previously mentioned, young broccoli sprouts have up to 100 times more glucoraphanin than mature broccoli, making them ideal sources if you want to grow or purchase them (RR).

Myrosinase also required

Remember, Sulforaphane is only formed when it comes in contact with the enzyme myrosinase, by chewing or chopping or other processing.

Myrosinase is a fragile enzyme that is quickly damaged by heating (boiling over 1 minute), or freezing (R).

Many Supplements do not provide active Myrosinase

Broccoli has long been known to provide many health benefits and as such, broccoli sprout supplements are not new to the market.  Unfortunately, many of these were developed before researchers realized the Myrosinase + Glucoraphanain = Sulforaphanin  equation required great care in processing to protect the Myrosinase.

As a result, most broccoli supplements do not provide ANY active myrosinase  (RR).

Sulforaphane was found to be 7 times greater in fresh broccoli sprouts vs supplements with inactive myrosinase  (R).

Glucoraphanin powder with inactive tyrosinase can be combined with a good source of tyrosinase such as broccoli sprouts or mustard seeds to greatly increase Sulforaphane absorption (R).

Mustard seed

The myrosinase found in broccoli is quite fragile and inactivated by freezing or heat. A much more robust form of myrosinase  is found in Mustard seeds. The addition of powdered mustard seed to heat processed broccoli dramatically increases sulforphane (R).

WHAT WE RECOMMEND

We’ve looked at all the leading brands sold on Amazon. There are several that are good, and many that are garbage. The best quality we found is this one by Avmacol.


 

Sulforaphane activates genes and enzymes that stimulate antioxidant production:

  • inhibits Phase I enzymes CYP1A1, CYP1A2, CYP1B1, CYP2B2 and CYP3A4 (RR).
  • activates (RR). SFN reacts with Keap1, thereby releasing Nrf2 from Keap1 binding (R).
  • increases other Phase II enzymes: NQO1, GSTA1, and HO-1 (RRRR).
  • blocks SXR (R).

Sulforaphane inhibits inflammation:

  • inhibits NfkB (RRR).
  • inhibits TNF-α (RR), NLRP3, IL-1β, IL-18 (R), IFN-gamma and IL6 (RRR).
  • inhibits IL-17 (RR).
  • inhibits TGF-β/Smad (R).
  • increases IL-10 (RRR), IL-4, Arg1, and YM-1 (R).
  • inhibits NO, iNOS and COX-2 (RRR).
  • silences Th17/Th1  (RR).
  • inhibits IL-23 and IL-12 (R).
  • inhibits MMP-9 (RR).
  • inhibits LDH and PGE2 (R).

Sulforaphane changes gene expression:

  • Sulforaphane inhibits DNMT1 and DNMT3A (R)
  • SFN is one of the most potent (histone deacetylase) HDAC inhibitors found to date (R).
  • SFN inhibits HDAC1, HDAC2, HDAC3, and HDAC4 (RR).
  • SFN decreases miR-21 and TERT (R).

Sulforaphane induces cell death (apoptosis) in cancer:

  • SFN activates caspase-3, caspase-7, caspase-8, caspase-9 (RR).
  • SFN decreases anti-apoptotic Bcl-2 (R) and Bcl-XL (R).
  • SFN increases pro-apoptotic Bax (R).
  • SFN induces p21 (CDKN1A) (R) and p53 (R).
  • SFN inactivates PARP (R).
  • SFN decreases HIF1A (R).
  • SFN decreases β-catenin (CTNNB1) (R).

More on Sulforaphane and Cancer

The mechanisms of SFN effects on cancer cells have been well studied. It suppresses the proliferation of cancer cells via diverse mechanisms including cell-cycle arrest, apoptosis induction, ROS production, and manipulation of some signaling pathways (166). SFN inhibits proliferation of PC-3 cells in culture in concentrationand time-dependent manner. Singh et al. (167) showed that oral administration of SFN led to >50% reduction in PC-3 xenograft tumor volume in SFN-treated mice in 10 days and more than 70% reduction in 20 days after starting treatment with no effect on body weight.

They also reported that SFN changes the Bax: Bcl-2 ratio, activates caspases 3, 8, and 9, and cleaves and inactivates PARP protein. The authors proposed that SFN induces apoptosis in PC-3 xenograft tumors in a p53-independent manner through cytoplasmic and mitochondrial pathways. Liquid chromatography–mass spectrometry (LC-MS) analyses performed by Rose et al. (17) showed the presence of 7-methylsulphinylheptyl isothiocyanates in watercress (Rorippa nasturtium aquaticum) extract and 4-methylsulfinylbutyl nitrile and 4-methylsulfinylheptyl isothiocyanates in the broccoli extract. Their investigations showed that these compounds contribute to the inhibitory effects of broccoli and watercress extracts on the invasion of MDA-MB-231 cancer cells through suppression of MMP-9 activity.

Treatment of HEK293 cells with different concentrations of SFN with and without TSA, as a HDAC1 inhibitor, leads to the increase in TOPflash reporter activity without affecting b-catenin protein levels. Further studies showed that this increase is due to the decrease in HDAC activity and consequently the increase in histone acetylation following SFN treatment (168).

It has been demonstrated that mamosphere formation in breast cancer cells is dependent on E-cadherin expression (168). It is showed that SFN could target breast cancer stem cells. The mammosphere formation test on two cancer cell lines, MCF7 and SUM195, indicated that SFN could reduce the proportion of cell with stem cell properties, and this was further supported by ALDEFLUOR assay. In vivo examination results of SFN effects on xenograft SUM159 cells in NOD/SCID mice were consistent with the in vitro results. More importantly, cells derived from SFN-treated primary tumors could not produce secondary tumors, while cells derived from the nontreated primary tumors rapidly produced the secondary tumors in the contralateral mammary fat pad of the same mice (168).

Aldehyde dehydrogenase activity is a stem cell marker for enriching tumorigenic stem/progenitor cells (169,170). Five mmol/L of SFN led to >80% reduction of ALDH-positive SUM159 cells in vitro, and daily treatment of xenograft of SUM159 tumors with 50 mg/kg of SFN for 2 weeks led to 50% reduction in tumor size through the reduction in ALDH-positive SUM159 cells by 50%, with no effect on body weight (171). ApcMin/C mice consumed SFN in their diet have fewer tumors with lower sizes in comparison with a control group, albeit, immunohistochemical (IHC) staining revealed that the b-catenin expression was not affected by SFN consumption (172).

Furthermore, the effect of SFN treatment on selfrenewal contributing to signaling pathway, Wnt pathway, was examined by analysis of b-catenin and some other downstream genes at mRNA and/or protein levels (171).
Treatment of T24 bladder cancer cells with SFN results in induction of miR-200c expression (173).

Previous studies demonstrated that miR-200c targets the E-cadherin repressors ZEB1 and ZEB2. Ectopic expression of miR-200c resulted in upregulation of E-cadherin in cancer cells (174). Therefore, treatment of T24 cells with SNF led to E-cadherin induction and EMT suppression (173). However, it seems that these results depend upon cell type and treatment conditions. Although clinical trials seem necessary, there is a large body of investigations about anticancer effects of SFN, and the explicit point is that SFN inclusion into the diet promises a safe and confident strategy.

Another active ingredient of broccoli and other cruciferous vegetables is Indole-3-carbinol (I3C) that has anticancer effects too. Meng et al. (175,176) reported despite a somehow prohibiting effect of I3C on cell attachment in vitro, and I3C could also suppress the invasion and motility of cells. The effect of I3C on cellular metastasis was also evaluated by injecting treated cells into the tail vein of mice and tracing surface metastasis in the lung of the sacrificed animal. Their results indicated that I3C treatment reduced the metastatic capability of the cells.

Bioavailability and new biomarkers of cruciferous sprouts consumption

There are epidemiological evidences of the benefit of consuming cruciferous foods on the reduction of cancer risk (Royston & Tollefsbol, 2015), degenerative diseases (Tarozzi, et al., 2013) and the modulation of obesity-related metabolic disorders (Zhang, et al., 2016), after cruciferous intake. Cruciferous sprouts are especially rich in bioactive compounds compared to the adult plants, due to their young physiological state, being an excellent choice for consuming healthy fresh vegetables (Pérez-Balibrea, Moreno, & García-Viguera, 2011; Vale, Santos, Brito, Fernandes, Rosa, & Oliveira, 2015). The highest benefit of cruciferous foods occurs when are consumed fresh, as young sprouts, avoiding degradation of the enzyme myrosinase by cooking, which is necessary to hydrolyse their characteristic sulphur and nitrogen compounds, the glucosinolates (GLS), to the bioactive isothiocyanates (ITC) and indoles. In case of sprouts, the degradation of GLS after consumption occurs during chewing, in presence of the plant’s enzyme myrosinase, and also is mediated by β-thioglucosidases in the gut microbiota (Angelino & Jeffery, 2014).

There is growing evidences that ITC, such as sulforaphane (SFN) and sulforaphene (SFE), as well as the indole-3-carbinol (I3C), play antioxidant, anti-inflammatory and multi-faceted anticarcinogenic activities in cells (Stefanson & Bakovic, 2014), through the in vivo inhibition of the activation of the central factor of inflammation NF-κB (Egner, et al., 2011), and the induction of the Keap1/Nrf2/ARE pathway related with antioxidant genes and detoxifying enzymes, such as glutathione S-transferases (GST) (Baenas, Silván, Medina, de Pascual-Teresa, García-Viguera, & Moreno, 2015; Myzak, Tong, Dashwood, Dashwood, & Ho, 2007), and also blocking carcinogenic stages in vitro and in vivo by induction of apoptosis, cell cycle arrest and inhibition of histone deacetylases, among others finally, metabolised in the liver with N-acetyl-L-cysteine (-NAC). During the last years, some conjugated ITC, such as SFN-NAC, and other secondary compounds, such as 3,3’diindolylmethane (DIM), which is released by I3C in acid medium (i.e. the stomach), have been used as biomarkers of cruciferous intake (Angelino & Jeffery, 2014; Fujioka, et al., 2016a). However, the bioavailability of the GLS glucoraphenin (GRE) and its isothiocyanate SFE, from radish sprouts, which only differ from SFN in a double bond between the third and fourth carbon (see Figure 2 in section 3), have not been yet investigated.

To our concern, there are no publications studying the bioavailability of radish sprouts compounds, and it is unknown if SFE is metabolised also by the mercapturic acid pathway. Furthermore, there are no commercially available conjugated metabolites of SFE, which would be needed for study its bioavailability by a rapid and sensitive UHPLC-QqQ-MS/MS method, stablishing their appropriate ionization conditions and MRM transitions.

On the other hand, the presence of SFN has been described in radish (Pocasap, Weerapreeyakul, & Barusrux, 2013), maybe by the hydrolysis of GRE, or through a modification of SFE once formed to SFN, suggesting its possible transformation by the mercapturic acid pathway. Therefore, the aim of this study was to evaluate and compare the bioavailability and metabolism of GRA and GRE, from broccoli and radish sprouts, respectively, and the study of possible different biomarker profiles of broccoli and radish consumption for the first time. Also the urine profile evolution of ITC, indoles and conjugated metabolites, after consumption of both broccoli and radish sprouts, were evaluated in a 7 days-cross-over trial with 14 healthy adult women.

2. Material and methods
2.1. Plant material
Broccoli (Brassica oleracea var. italica) and radish (Raphanus sativus cv. Rambo) 8-day-old sprouts were supplied by Aquaporins & Ingredients, S.L. (Murcia, Spain). These sprouts were bioestimulated during production (4 days previous to delivery) with the natural compound methyl jasmonate 250 μM (Baenas, Villaño, García-Viguera, & Moreno, 2016), in order to obtain cruciferous sprouts up to 2-fold richer in bioactive compounds. Three trays of sprouts

(n=3) were collected once a week during the study, then, samples were flash frozen and lyophilised prior analysis of GLS and ITC (see 2.3. section), through an hydro-methanolic (Baenas, Garcia-Viguera, & Moreno, 2014) and aqueous extraction (Cramer & Jeffery, 2011), respectively, as previously described.

2.2. Human subjects and study design. 

A total of 14 women, aged 27-36 years, non-smokers with stable food habits and not receiving medication, during the experimental procedure, were selected to participate in the study. Due to the sex-related disparities in pharmacokinetics and bioavailability results reported by different authors (Soldin, Chung, & Mattison, 2011; Soldin & Mattison, 2009), the choice of a single genus was chosen to avoid a high dispersion in the data, and the availability of healthy young adult female volunteers for the study. Written informed consent was obtained from all subjects. The present study was conducted according to guidelines and procedures approved by the CSIC Committee of Bioethics for the AGL-2013-46247-P project. Subjects were randomly assigned to a seven-by-seven cross-over design (Figure 1), one group receiving broccoli sprouts and the second receiving radish sprouts. Nobody dropped out of the study. A list of foods containing glucosinolates was given to all the participants in order to avoid consumption during the study. Experimental doses (7 trays of broccoli or radish sprouts of 20 grams each) were given at once, on Friday. Subjects were instructed to ingest 1 tray per day, at 10 a.m., according to the cross- over design and to keep trays refrigerated (4 °C) at home. The first day of the study, the urine samples were collected from 0 to 12 h, and from 12 to 24 h after ingestion. From day 2 to 7, the urine samples were collected in 24 h periods. All urine samples were kept refrigerated during collection and were frozen upon reception in the laboratory.

2.3. Metabolites analysis 

The quantitative analysis of GLS in sprouts was carried out by HPLC-DAD 1260 Infinity Series (Agilent Technologies, Waldbronn, Germany), according to UV spectra, and order of elution already described for similar acquisition conditions (Baenas, et al., 2014). For samples preparation, briefly, freeze-dried sprouts (50 mg) were extracted with 1mL of methanol 70% V/V in a US bath for 10 min, then heated at 70°C for 30 min in a heating bath and centrifuged (17500 xg, 15min, 4°C). Supernatants were collected, and methanol was completely removed using a rotary evaporator. The dry material obtained was dissolved in 1 mL of ultrapure water and filtered through a 0.22 μm Millex-HV13 filter (Millipore, Billerica, MA, USA). Measurement of metabolites in sprouts and urine (GRA, SFN, SFN-GSH, SFN-CYS, SFN- NAC) was performed following their MRM transition by a rapid, sensitive and high throughput UHPLC-QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany) method, with modifications of the protocol of Dominguez-Perles et al. (2014), for the optimization of new compounds: GRE, SFE, glucobrassicin (GB), I3C, DIM; assigning their retention times, MS fragmentation energy parameters and preferential transitions (Supplemental File 1). The urine samples were centrifuged (11,000g, 5 min) and the supernatants (400 μL) were extracted using SPE Strata-X cartridges (33u Polymeric Strong Cation), following the manufacturer’s instructions (Phenomenex, Inc., Madrid, Spain), and the slight modifications of Dominguez- Perles et al., (2014). Briefly, the cartridges were conditioned and then aspirated until dryness. The target analytes were eluted with 1 mL of MeOH/formic acid (98:2, v/v) and dried completely using a SpeedVac concentrator (Savant SPD121P; Thermo Scientific, Waltham, MA). The extracts were reconstituted with 200 μL of mobile phase solvent A/B (90:10, v/v) previously to their UHPLC/MS/MS analyses. GRA and GRE were obtained from Phytoplan (Diehm & Neuberger GmbH, Heidelberg, Germany) and ITC and indoles were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). No commercially available conjugated metabolites of SFE from radish were found.

2.4. Statistical analysis 

All assays were conducted in triplicate. Data were processed using SPSS 15.0 software package (LEAD Technologies Inc., Chicago, IL, USA). First of all, data were tested by Shapiro-wilk normality test, as these values do not follow a normal distribution (non-parametric data), statistical differences were determined by the Wilcoxon signed-rank test when comparing two samples and by the Friedman test when comparing multiple samples. Values of P<0.05 were considered significant.

3. Results and discussion
3.1. Bioactive compounds present in cruciferous sprouts
Broccoli and radish sprouts from each week of the study were characterised in GLS and ITC (Table 1). Results are presented as commercial serving dose (20 grams of fresh weight (F.W.)). The amount of cruciferous sprouts consumed daily by the participants was considered a normal serving according to the EFSA (2009), which consider that there is no a perfect way of measuring habitual intake, however, the portion size should be convenient for the use in the context of regular dietary habits.
In broccoli sprouts, glucoraphanin (GRA), in the hydromethanolic extract, and its hydrolysis compound sulforaphane (SFN), in the aqueous extract were the predominant compounds, according to previous findings (Angelino & Jeffery, 2014; Cramer & Jeffery, 2011).
Results showed that radish sprouts presented glucoraphenin (GRE) and glucoraphasatin (GPH) as predominant GLS (Table 1), which were hydrolysed to sulforaphene (SFE) and raphasatin (RPS), respectively. Only SFE was detected in the aqueous extract, as RPS is very unstable and rapidly degraded to less bioactive compounds in aqueous media, such as raphanusanins, E- and Z-3-methylsulfanylmethylene-2-pyrrolidinethiones and E-4-methylsulfanyl-3- butenyldithiocarbamate (Kim, Kim, & Lim, 2015; Montaut, Barillari, Iori, & Rollin, 2010). GRA was not detected in radish sprouts, but the hydrolysis product SFN was present in the aqueous extract (Pocasap, et al., 2013). This could be due to the formation of SFN derived from SFE, losing its double bond, or directly hydrolysed from GRE (Figure 2). Forthcoming evaluations of GRE hydrolysis under different conditions would provide more information about its possible transformations.

3.2. Bioavailability and metabolism of GLS/ITC 

After ingestion of a serving portion of broccoli sprouts (20 g F.W.), GRA (64 μmol) was hydrolysed, absorbed and metabolised, through the mercapturic acid pathway, by a 12 % on average. SFN and its conjugated metabolites, with glutathione (-GSH), cysteine (-CYS) and N- acetyl-L-cysteine (-NAC) (Angelino & Jeffery, 2014), were found in urine (~7.6 μmol/ 24 h as the sum of SFN, SFN-GSH, SFN-CYS and SFN-NAC) (Figure 3A), considered markers of bioavailability.

In the case of radish sprouts, the metabolism of SFE, the predominant ITC, has not been described to the present date, and there are not any conjugated SFE metabolites commercially available to evaluate its bioavailability, by optimization of MRM-transitions in UHPLC-QqQ- MS/MS system. Therefore, it was hypothesised that conjugated SFN metabolites could be found also in urine after radish sprouts consumption, being also possible biomarkers of intake.

Results showed that GRE (61 μmol in 20 g F.W.) was metabolised in SFE, SFN and SFN metabolites (SFN-NAC, SFN-CYS, SFN-GSH), corresponding to 8% on average (~4.9 μmol/24 h) of the GRE consumed (Figure 3B). Therefore, SFN metabolites (SFN-NAC, SFN- CYS, SFN-GSH) could act also as biomarkers of radish consumption. In this sense, this analysis method allowed us to evaluate radish compounds bioavailability, in addition to differentiate it from the bioactives in broccoli, as well as other cruciferous foods, finding intact SFE metabolite as characteristic biomarker of radish consumption.

The values of bioavailability ranged from 9 to 100 % according to different GLS/ITC profiles of the cruciferous vegetables administered, and the consumption as raw or cooked foods and the influence of the microbiota (Shapiro, Fahey, Wade, Stephenson, & Talalay, 1998; Vermeulen, Klopping-Ketelaars, van den Berg, & V aes, 2008). In our case, after broccoli sprouts consumption, the SFN-NAC was the predominant metabolite found in urine (~80 %), followed by SFN-CYS (~11 %), SFN (7.5 %), and SFN-GSH (~0.9 %) (Figure 3A), as previously found (Clarke, et al., 2011; Dominguez-Perles, et al., 2014). In the case of radish sprouts, the SFE was excreted in higher amounts (~65 %) (Figure 3B), followed by the conjugated metabolites: SFN- NAC (~19 %), SFN-CYS (~4%), SFN (~1.1%) and SFN-GSH (~0.7%). SFN and SFE present the isothiocyanate group (−N=C=S), which central carbon is highly electrophilic and actively interacts with cellular nucleophilic targets; such as, the GSH and/or cysteine residues (Kim, Kim, & Lim, 2010). Little information is available about SFE bioactivity and, only recently, Byun, et al., (2016) showed that SFE reduced the cellular GSH levels in vitro, which could indicate its conjugation with GSH. Thus, the synthesis of SFE conjugates with GSH, CYS or NAC could help to generate knowledge on the metabolism of this compound.

On the other hand, the higher excretion of pure SFE after radish consumption compared to pure SFN after broccoli ingestion, may suggest that this compound could follow a different transformation pathway. Contrary to SFN, SFE contains a double bond between the third and fourth carbon, which could result in a decrease in the electrophilicity of the –N=C=S group (Kim, et al., 2010), and different excretion kinetics and transformation.

According to Holst & Williamson (2004), human studies about Phase I metabolism may contribute considerably to understand the biotransformation of ITC and, consequently, the limit of their bioavailability and health-promoting effects. Therefore, further studies about SFE metabolism and bioactivity are needed to support the health-promoting activities of SFE, now insufficiently studied. For instance, Myzak, et al, (2006) showed differences in the bioactivity of SFN conjugated metabolites, as being SFN-CYS and SFN-NAC, significantly active as HDAC inhibitors, with cancer therapeutic potential in vitro and in vivo, but not for SFN and SFN-GSH (Myzak, Ho, & Dashwood, 2006). On the other hand, pure SFN and SFE have shown bioactivities, inhibiting growth of several colon cancer cells (Byun, et al., 2016). Therefore, whether SFE is metabolised by the mercapturic acid pathway or acting in the cell without transformation, this ITC may provide health-promoting effects through induction of detoxification enzymes and antioxidant activity, which did not appear to be affected by their hydrophilicity or other structural factors (La Marca, et al., 2012).

3.3. Urine profile evolution of SFN, SFE and their metabolites. 

The values of individual metabolites excreted, analysed in urine samples, were represented according to two different criteria: 1) the levels of excretion during 24 h were normalised, first from 0 to 12 h after consumption and then from 12 to 24 h, using creatinine as an index to which refers the results, since the creatinine excretion is a relatively constant value between subjects; 2) when comparing the daily excretion during 7 days, the data were represented in volumes collected every 24 h.

The excretion of SFN and its conjugated metabolites, as well as SFE after radish sprouts ingestion was higher during the first 12 h after consumption of sprouts (Figure 4). It has been described that urinary excretion of SFN metabolites after consumption of fresh broccoli reaches peak concentration 3-6 h after consumption, but it could be delayed until 6-12 h (Vermeulen, et al., 2008). After broccoli sprouts ingestion, non-significant differences were found (Figure 4A) between the two periods. In contrast, significant differences were shown in the values of excretion after radish sprouts consumption in all metabolites except for SFN-GSH and SFN- CYS (Figure 4B). The delayed excretion of metabolites, after broccoli sprouts consumption, could be related to a saturation of the membrane transporters (such as P-glycoprotein) in the cells, as SFN is conjugated with GSH and cysteinylglycine in the cells and exported after protein binding, being available for metabolism and excretion (Hanlon, Coldham, Gielbert, Sauer, & Ioannides, 2009). SFE is mostly excreted during the first 12 h after ingestion of radish sprouts (Figure 4B), suggesting that it might be not subjected to the mercapturic acid metabolism. Nevertheless, the biological activity of the pure ITC has shown to be similar than the N -acetylcysteine conjugates (Tang, Li, Song, & Zhang, 2006), being of great interest either if are metabolised or not.

The levels of excretion after 7 days of ingestion (Supplemental files 2 and 3) were also studied and no differences were found in the median daily excretion of SFE, SFN and its metabolites in both broccoli and radish studies. This suggests that repetitive dosing of sprouts should not produce accumulation of any metabolite in the body, as any factor that increases the metabolites amount in the body will cause a decrease in its excretion (Hanlon, et al., 2009). Also, there is a high interindividual variation in excretion values related to human bioavailability studies, which could be explained by different factors, such as the intensity of chewing, where myrosinase enzymes come into contact with intact GLS, gastric pH, intestinal transporters and the activity of the microbiota, where one subject could metabolize three times more GLS into ITC than another, and also the polymorphisms of GST enzymes may affect ITC metabolism (Clarke, et al., 2011; Egner, et al., 2011; Fujioka, Fritz, Upadhyaya, Kassie, & Hecht, 2016b). Low amounts of intact GRA and GRE, on average 0.011 and 0.04 μmol/24 h, respectively, were also recovered in the urine.

One of several challenges in the design of clinical trials is the selection of the appropriate dosage. In this work, commercial trays of sprouts were used, facilitated and quality certified by the company. The average amount of sprouts (~ 20 g per tray) was chosen as one serving, representing a realistic dietary supply. Different specific dietary intervention studies have estimated that the consumption of 3-5 servings per week of cruciferous foods (broccoli, red cabbage, Brussels sprouts, among others) may produce upregulation of detoxification enzymes, responsible for clearance of chemical carcinogens and ROS (Jeffery & Araya, 2009). Therefore, the daily consumption of cruciferous sprouts may result in potential effects decreasing the risk for cancer, even though further epidemiological trials and in vivo studies testing broccoli and radish sprouts are necessary to further understand these effects.

3.4. Bioavailability, metabolism and urine profile evolution of GLS/indoles 

Glucobrassicin (GB) present in broccoli and radish sprouts is an indole GLS derived from tryptophan and releases bioactive indole-3-carbinol (I3C) upon hydrolysis. This bioactive compound requires acid modification in the stomach to form 3,3’-diindolylmethane (DIM) and other condensates to optimize activity, increasing levels of Phase II enzymes, related to detoxification against lung, colon and prostate cancers (Egner, et al., 2011), and the antiproliferative effects on estrogenic-sensitive tumours (Fujioka, et al., 2016a). In particular, DIM has been associated with the suppression of epigenetic alterations related to carcinogenesis, by suppression of DNA methylation and aberrant histone modifications (Fujioka, et al., 2016b).

Additionally, the induction of Phase I enzymes, including the CYP 1 family, catalysing the oxidation of xenobiotics may also be responsible of the action (Ebert, Seidel, & Lampen, 2005; Watson, Beaver, Williams, Dashwood, & Emily, 2013).
Because of the rapid hydrolysis of I3C to DIM in vivo, high stability of DIM, and the strong correlation between GB intake and the amount of DIM excreted, this compound has been described as a biomarker of cruciferous vegetables consumption (Fujioka, et al., 2014). Other condensated compounds such as indol-[3,2-b]-carbazole and related oligomers, were in non- quantifiable concentrations in other studies (Reed, et al., 2006).

Little is known about the bioavailability of other indole GLS present in broccoli and radish sprouts, such as hydroxyglucobrassicin (HGB), methoxyglucobrassicin (METGB) and neoglucobrassicin (NEOGB), which might be also hydrolysed leading indolyl-3-methyl isothiocyanates, unstable and hydrolysed to their corresponding carbinols (Agerbirk, De Vos, Kim, & Jander, 2008; Hanley & Parsley, 1990).

Additional studies are required to confirm if these indole GLS could be hydrolysed also in I3C and DIM, as well as to evaluate the possible health-promoting effects of their hydrolysis and condensate metabolites.
Results demonstrated that broccoli and radish sprouts content in GB were ~11.4 and ~7.7 μmol/20 g F.W, respectively. After ingestion of broccoli sprouts, 49 % of GB was suitably metabolised and excreted as hydrolysis metabolites, calculated as the sum of I3C and DIM (~5.57 μmol /24 h). Following radish ingestion, the percentage of GB hydrolysed and absorbed was 38 % (~2.92 μmol /24 h). It is remarkable that the DIM excreted correspond to over 99 % of these total metabolites. These results of bioavailability contrast with the extremely low percentage (< 1 %) of GB excreted as DIM after consumption of Brussels sprouts and cabbage in a previous study (Fujioka, et al., 2014). Nevertheless, results show relevant bioavailability of GB and the successful use of DIM as biomarker of cruciferous intake. Further studies about conversion of other indole GLS to I3C and DIM are needed to know more about bioavailability of these compounds, as there is no information in literature.

When urine samples were collected in two periods after the ingestion of the sprouts, higher values of excretion of I3C from 12 to 24 h than from the first period were detected (Figure 5), although non-statistically different. Regarding excretion values of DIM, no differences among results were found from 0 to 12 h and from 12 to 24 h (Figure 5). Even if a previous study in humans has shown that the majority of DIM was excreted in the first 12 h (Fujioka, et al., 2016b), other authors have detected DIM in plasma at 12 and 24 h post ingestion (Reed, et al., 2006). Therefore, the excretion of this compound might be longer than for the ITC, which were almost totally excreted during the first 12 h. According to these results, in vivo evidences show that I3C condensation products were absorbed, preferentially targeting the liver, and were detected within the first hour in urine. However, the amount increased significantly between 12 and 72 h, implying effects on the xenobiotic metabolism (WHO, 2004).

No statistical differences were found in the 24 h urine excretion values of I3C and DIM after 7- days of consumption of sprouts (Supplemental File 3). The high variability between subjects has been described before within a low dose level of GB consumed (50 μmol), which was considerably higher than in this study (Fujioka, et al., 2016a).

Furthermore, the results showed no accumulation of metabolites after 7 days of intervention, an important result for safe consumption, also proven with hyper-doses of GB (400-500 μmol) in humans (Fujioka, et al., 2016a).
Low amounts of intact GB (~0.011 and ~0.04 μmol/24 h, after broccoli and radish ingestion, respectively) were also recovered in the urine, but the biological activities of I3C, administered orally to humans, cannot be attributed to the parental compound but rather to DIM and other oligomeric derivatives (Reed, et al., 2006). Therefore, the evaluation of DIM in urine after broccoli and radish consumption provided a susceptible tool to design future clinical trials.

4. Conclusions 

The measurement of ITC, indoles and conjugated metabolites are useful biomarkers of dietary exposure to cruciferous foods. The SFN-NAC is not the only metabolite present in urine and, as along with DIM, could be used as biomarker of the consumption of cruciferous vegetables.

After ingestion of radish sprouts, SFE, together with SFN-NAC and DIM, could be considered as biomarkers, however, metabolites of SFE are not commercially available yet and, consequently, understudied. Repeated dosing of sprouts does not lead to accumulation or higher urine levels of metabolites over time. Furthermore, human short-term pharmacokinetics (e.g. 48 – 72 h) as well as long-term intervention studies (e.g. 10 – 12- weeks) using different doses, and collecting urine and plasma (several time points), as well as faecal samples (microbial metabolites and potential beneficial changes in the gut/colonic microbiota), are strongly recommended for future research in this area.

Cruciferous Vegetable Intake Is Inversely Associated with Lung Cancer Risk among Current Nonsmoking Men in the Japan Public Health Center Study

Abstract

Background: Cruciferous vegetables, a rich source of isothiocyanates, have been reported to lower the risk of several types of cancer, including lung cancer. However, evidence from prospective observations of populations with a relatively high intake of cruciferous vegetables is sparse.
Objective: We investigated the association between cruciferous vegetable intake and lung cancer risk in a large-scale population-based prospective study in Japan.

Methods: We studied 82,330 participants (38,663 men; 43,667 women) aged 45–74 y without a past history of cancer. Participants were asked to respond to a validated questionnaire that included 138 food items. The association between cruciferous vegetable intake and lung cancer incidence was assessed with the use of Cox proportional hazard regression analysis to estimate HRs and 95% CIs (with adjustments for potential confounding factors).

Results: After 14.9 y of follow-up, a total of 1499 participants (1087 men; 412 women) were diagnosed with lung cancer. After deleting early-diagnosed cancer and adjusting for confounding factors, we observed a nonsignificant inverse trend between cruciferous vegetable intake and lung cancer risk in men in the highest compared with the lowest quartiles (multivariate HR: 0.85; 95% CI: 0.69, 1.06; P-trend = 0.13). Stratified analysis by smoking status revealed a significant inverse association between cruciferous vegetable intake and lung cancer risk among those who were never smokers and those who were past smokers after deleting lung cancer cases in the first 3 y of follow-up [multivariate HR for never smokers: 0.49 (95% CI: 0.27, 0.87; P-trend = 0.04); multivariate HR for past smokers: 0.59 (95% CI: 0.35, 0.99; P-trend = 0.10)]. No association was noted in men who were current smokers and women who were never smokers. Conclusion: This study suggests that cruciferous vegetable intake may be associated with a reduction in lung cancer risk among men who are currently nonsmokers. J Nutr doi: 10.3945/jn.117.247494.

Introduction

Cruciferous vegetables, including broccoli, cabbage, and radish, are a rich source of isothiocyanates, which are known for their protective effect against cancer (1). Although the mechanisms underlying their anticancer properties are not clear, they may enhance the excretion of

1 Supported by the National Cancer Research and Development Fund, Ministry of Health, Labor and Welfare, and Ministry of Agriculture, Forestry and Fisheries. 2 Author disclosures: M Inoue is a beneficiary of a financial contribution from the AXA Research Fund. N Mori, T Shimazu, S Sasazuki, M Nozue, M Mutoh, N Sawada, M Iwasaki, T Yamaji, R Takachi, A Sunami, J Ishihara, T Sobue, and S Tsugane, no conflicts of interest.

carcinogens before they damage DNA (2, 3). Cruciferous vegetable intake is associated with lowering the risk of several types of cancer, such as bladder (4–6) and colorectal cancer (7, 8).

Several prospective epidemiologic studies have considered the association between cruciferous vegetables intake and lung cancer risk (9–17). As for all cruciferous vegetables, 2 studies to our knowledge reported a significant inverse association (12, 17), but most studies reported a nonsignificant inverse asso- ciation (9, 13–16). Among all prospective studies (9–17), 2 found a significant inverse association between individual cruciferous spices, namely Chinese greens (15) and cauliflower (13), and lung cancer risk. Both cruciferous vegetables represented the highest mean intake among the cruciferous vegetables considered in the study (13, 15).

The reason for the weak inverse association may be partly explained by the little variation in the amount of cruciferous vegetables consumed. Seven of the 9 studies were conducted in Western countries (9–14, 17), where the intake of cruciferous vegetable is lower than in Asian countries. The daily intake of total cruciferous vegetables was reported as 0.2 servings (~22.6 g/d) in the United States (18); 59.8 g cabbage, Chinese leaves, and Chinese radish intake per day in Japan (19); and 121.0 g leafy, stalk, or shoot vegetables and the brassica subgroup per day in Hong Kong (20). Therefore, a stronger association may exist in Asian populations with a relatively high intake of cruciferous vegetables. However, few such prospective investigations have been conducted in Asian populations (15, 16).

Furthermore, cigarette smoking is an established risk factor of lung cancer (21), and reports from prospective studies of the association between cruciferous vegetable intake and lung cancer stratified by smoking status are limited and inconsistent (9, 15, 17).

This study was a large-scale population-based prospective study among Japanese residents with a relatively high intake of cruciferous vegetables. We aimed to clarify the association between cruciferous vegetables intake and lung cancer risk while taking smoking status into account.

Methods

Study population. The Japan Public Health Center (JPHC)10 prospec- tive study was launched between 1990 and 1993. The details of the study design have been described previously (22). The study protocol was approved by the institutional review board of the National Cancer Center, Tokyo, Japan.

The study population was defined as registered residents aged 40– 69 y of 11 public health center (PHC) areas of Japan. Ineligible subjects were first excluded because of non-Japanese nationality (n = 51), late report of emigration before the follow-up (n = 207), incorrect birthdate (n = 7), and duplicate registration (n = 10). We then excluded 1 PHC (Tokyo area) because it lacked information on cancer incidence (n = 7078). Furthermore, we excluded 2459 subjects who had died, moved out of the study area, or with whom we lost contact before the 5-y follow-up questionnaire survey.

We used the 5-y follow-up survey as the starting point because the questionnaire used in this survey provided more comprehensive infor- mation on dietary intake than the baseline survey. We included subjects who responded to the questionnaire that contained demographic data, medical history, information on smoking, and dietary intake through an FFQ. Of the remaining 130,608 participants, 98,552 responded to the questionnaire (response rate: 75.5%).

Among the 98,552 subjects, we excluded those who reported or were diagnosed with cancer before the 5-y follow-up questionnaire survey, those who left the frequency of all cruciferous vegetable intake unanswered (n = 2271), those who reported daily energy intake at the upper or lower 2.5% ends of the range (1015 and 4204 kcal for men and 865 and 3689 kcal for women, respectively; n = 4812), and those with incomplete information on smoking status (n = 5725). Finally, 82,330 participants (38,663 men; 43,667 women) were included in the analysis.

FFQ. The FFQ used in the 5-y follow-up questionnaire survey was designed to estimate the habitual dietary intake of 138 food items (including 6 cruciferous vegetables and 3 pickled cruciferous vegetables) during the previous year in standard portion sizes and 9 frequency categories. Standard portion sizes for each food item were small (50% less than the standard serving size), medium (proportional to standard serving size), and large (50% larger than the standard serving size). The 9 frequency categories for each food items were never, 1–3 times/mo, 1–2 times/wk, 3–4 times/wk, 5–6 times/wk, 1 time/d, 2–3 times/d, 4–6 times/d, and $7 times/d. Cabbage, Chinese radish, broccoli, komatsuna, Chinese leaves, pak choi, leaf mustard, and Swiss chard were categorized as cruciferous vegetables according to the grouping rules adopted in the IARC Handbooks of Cancer Prevention (19). The 3 pickled cruciferous vegetables were pickled Chinese radish, rape and leaf mustard, and Chinese leaves. Total cruciferous vegetables did not include pickled cruciferous vegetables in this study in order to make valid comparisons with previous studies (15, 16). Isoflavone intake was also estimated with the use of the FFQ to be included as a covariate because we have previously found an inverse association between isoflavone intake and lung cancer risk (23, 24). The dietary intake of each food item was calculated by multiplying frequency by standard portion and the relative portion sizes for each food item. The food intake was log-transformed and adjusted for total energy intake with the use of the residual method (25). We examined the validity of the FFQ for assessing cruciferous vegetable intake and the reproducibility of the FFQ administered at a 1-y interval (Supplemental Tables 1 and 2).

Follow-up and identification of lung cancer incidence. We followed up participants until 31 December 2012. Participants who died or relocated to other municipalities were identified annually through residential registers in each PHC area. The cause of death was confirmed with the use of mortality data from the Ministry of Health, Labor and Welfare. Among study participants, 12,345 (15.0%) died, 2992 (3.6%) moved away, and 144 (0.2%) were lost to follow-up during the study period.

Data on lung cancer incidence were obtained from local major hospitals in the respective PHC areas and data linkage with population-based cancer registries with permission from the local governments. We also used death certificate information as supplementary data. In our cancer registry system, 6.4% of lung cancer cases were obtained through death certificates only during the study period. During the 1,195,175 person-years of follow-up (median follow-up period: 14.9 y), a total of 1499 participants (1087 men; 412 women) were diagnosed with lung cancer.

The histologic types of lung cancer were coded based on the International Classification of Diseases for Oncology, 3rd edition (C34.0–C34.9) (26). The diagnosis of lung cancer was confirmed with the use of a histo- or cytologic examination in 79% of cases (n = 1187) and was based on the clinical findings or unspecified evidence in the remaining 21% of cases. Histologic type was classified as adenocarci- noma (n = 630; 42%), squamous cell carcinoma (n = 292; 19%), small cell carcinoma (n = 146; 10%), and other histologic types according to the WHO!s histologic classification of lung tumors (27).

Statistical analysis. We prospectively counted the number of person- years of follow-up for each participant from the date of completion of the 5-y follow-up questionnaire until the date of diagnosis of lung cancer, date of death, moving out of the study area, or end of follow-up (31 December 2012), whichever came first.

Cox proportional hazards regression analysis was used to calculate the sex-specific HRs and 95% CIs of lung cancer incidence according to quartile of cruciferous vegetable intake and adjusted for potential covariates. The lowest quartile of cruciferous vegetable intake was used as a reference. P values for linear trends were calculated by assigning ordinal variables for quartiles of cruciferous vegetable intake and entering the number as a continuous variable into the model. Because cigarette smoking is the most important risk factor for lung cancer, we performed a subgroup analysis by smoking status on the association between cruciferous vegetable intake and lung cancer risk. Cox proportional hazards regression analysis was repeated to calculate the HRs and 95% CIs for each smoking status. We then repeated the same analysis after excluding 189 lung cancer cases diagnosed in the first 3 y of follow-up to diminish the potential influence of participants with subclinical cancer who had modified their dietary habits. The multivar- iate model was adjusted for age, PHC area, smoking status, fruit intake, noncruciferous vegetable intake, isoflavone intake, and menopausal status in women.

Because pickled cruciferous vegetables accounted for 26% of the total cruciferous vegetable intake in our subjects, we conducted a subanalysis by including pickled cruciferous vegetable intake in total cruciferous vegetable intake as a main exposure. Further analysis was conducted on the association between the intake of specific cruciferous vegetables and lung cancer risk by smoking status in men. Multivariate HRs and 95% CIs before and after the removal of lung cancer cases diagnosed in the first 3 y of follow-up were calculated. For those cruciferous vegetables with a Spearman!s correlation coefficient of <0.1, additional analyses were performed to investigate the association between the frequency of intake and lung cancer risk. We then calculated P values with the addition of an interaction term in the multivariate model by smoking status. The reported P values are 2-sided, and P < 0.05 was defined as significant. All statistical analyses were performed with the use of SAS version 9.3 (SAS Institute).

Results

The baseline characteristics of participants! cruciferous vegeta- ble intake are shown in Table 1. Those with a higher intake of cruciferous vegetables were less likely to be current smokers, to have a higher BMI, and to have had a chest X-ray or sputum screening. A dietary intake of vegetables, fruit, and isoflavones was significantly higher in the highest quartile of cruciferous vegetable intake in both men and women.

Table 2 shows the association between cruciferous vegetable intake stratified by smoking status and lung cancer risk. Because of the small number of lung cancer cases among women who were past and current smokers, only the results for never smokers are shown. Although we observed trends of decreased cancer risk in the highest quartile among men who were never and past smokers, the association was not significant among never smokers (multivariate HR: 0.66; 95% CI: 0.39, 1.13; P-trend = 0.17) or past smokers (multivariate HR: 0.74; 95% CI: 0.49, 1.09; P-trend = 0.18). After excluding the lung cancer cases diagnosed in the first 3 y of follow-up, multivariate HRs of the highest quartile of cruciferous vegetable intake were signifi- cantly inversely associated with lung cancer risk among both never and past smokers: 0.49 (95% CI: 0.27, 0.87; P-trend = 0.04) and 0.59 (95% CI: 0.35, 0.99; P-trend = 0.10), respectively. When we combined never and past smokers, the association was still significant (multivariate HR: 0.54; 95% CI: 0.37, 0.79; P-trend = 0.007). However, no such association was observed in men who were current smokers (P-trend = 0.92) or in women who were never smokers (P-trend = 0.86). Associ- ations were virtually unchanged when pickled cruciferous vegetable intake was included in the total cruciferous vegetable intake (data not shown).

We also conducted an analysis of specific cruciferous vege- table intake and lung cancer risk by smoking status in men. To investigate the true effect of cigarette smoking, results of never and current smokers were compared (Table 3). After deleting lung cancer cases diagnosed in the first 3 y of follow-up, a significant inverse association was observed in the highest quartile of cabbage intake and lung cancer risk (multivariate HR: 0.57; 95% CI: 0.34, 0.97; P-trend = 0.04) among never smokers. No significant association was noted between other types of cruciferous vegetable intake and lung cancer risk in never or current smokers. Results were unchanged when we conducted additional analyses between the frequency of Chinese leaves, leaf mustard, and Swiss chard and lung cancer risk (data not shown).

We also conducted a likelihood ratio test to calculate P values for interactions by adding the potential confounding factors to the multivariate model. First, we tested the association between cruciferous vegetable intake and lung cancer risk among men only by smoking status (P-interaction = 0.65) and both sexes combined (P-interaction = 0.73). Second, we tested the associ- ation by sex (P-interaction = 0.13).

We further analyzed the association between cruciferous vegetable intake and lung cancer risk stratified by detection types to try to explain the observed null association in women who were never smokers. We found no association between cruciferous vegetable intake and symptom-detected lung cancer (multivariate HR: 0.97; 95% CI: 0.49, 1.89; P-trend = 0.84), whereas we observed a nonsignificant but increased risk of screening-detected lung cancer (multivariate HR: 1.21; 95% CI: 0.68, 2.14; P-trend = 0.36). In men, however, we observed a significant decreased risk of symptom-detected lung cancer (multivariate HR: 0.45; 95% CI: 0.23, 0.85; P-trend = 0.03).

Further investigation was conducted on the association between cruciferous vegetable intake and the risk of lung cancer by histologic type among women who were never smokers. Because of the small number of lung cancer cases (in men: adenocarcinoma, n = 65, 43%; squamous cell carcinoma, n = 24, 16%; and small cell carcinoma, n = 9, 6%; in women: adenocarcinoma, n = 272, 76%; squamous cell carcinoma, n = 12, 3%), only adenocarcinoma among women who were never smokers was analyzed. The multivariate HR among the highest quartile in reference to the lowest quartile was 0.98 (95% CI: 0.67, 1.44; P-trend = 0.85).

Discussion

This prospective cohort study investigated the association between cruciferous vegetable intake and lung cancer risk in a population with a relatively high intake of cruciferous vegeta- bles. We found a significant inverse association between crucif- erous vegetable intake and lung cancer risk among men who were current nonsmokers, although no significant association was observed among men who were current smokers and women who were never smokers. Similarly, an analysis of individual cruciferous vegetable intake revealed a significant association between cabbage intake and lung cancer risk among men who were never smokers. Although our study group previously found no association between total fruit and vegeta- ble intake and lung cancer risk, the association between cruciferous vegetable intake and lung cancer risk was not considered (28).

In addition to containing several bioactive components such as folate, vitamin C, tocopherols, and carotenoids (29), crucif- erous vegetables are a rich source of glucosinolates, which are catalyzed into isothiocyanates by myrosinase. Although the mechanisms underlying the anticancer properties of isothiocy- anates are not fully understood, isothiocyanates have been reported to inhibit the development of various cancers, including lung cancer in an animal model (30).

Asians, including the Japanese, have higher cruciferous vegetable intake than non-Asian populations (19, 20). Whereas

cruciferous vegetable intake is reported to be 0.2 servings/d (~22.6 g/d) in the US population (18), the intake reported in the Japanese population is much higher (59.8 g/d) (19). However, these data only include cabbage, Chinese leaves, and Chinese radish, and total cruciferous vegetable intake is expected to be higher among the Japanese. In fact, our dietary record collected from subsamples of the JPHC study indicated a mean crucifer- ous vegetable intake of 110 g/d for men and 102 g/d for women (Supplemental Table 1).

After removing subjects with early-diagnosed cancer and adjusting for confounding factors, we observed a nonsignifi- cant inverse trend between cruciferous vegetable intake and lung cancer risk in men in the highest compared with the lowest quartiles. The prevalence of smoking reported in 2000 was 51.3% in Japanese men and 27.7% in US men (31). It is likely that because of the much higher prevalence of smoking among Japanese men, we failed to observe a significant inverse association between cruciferous vegetable intake and lung cancer risk in men.

In this study, no significant association was found between cruciferous vegetable intake and lung cancer risk among men who were current smokers. Two prospective studies investigated the association between cruciferous vegetable intake and lung cancer risk among current smokers and reported inconsistent results. Whereas Lam et al. (17) reported a significant inverse association between cruciferous vegetable intake and lung cancer risk among current smokers, Wright et al. (9) found no protective association in both sexes.

 

Circulating antioxidants such as ascorbic acid, a-carotene, b-carotene, and cryptoxanthin are depleted in current smokers (32, 33), which may explain our result. Likewise, Steinmetz et al. (34) found a stronger inverse association between fruit and vegetable intake and lung cancer risk among past smokers than in current smokers. They noted that the continued presence of carcinogens derived from tobacco smoke may have overwhelmed the anticarcinogenic capacity of vegetables. In addition, the residual confounding factor of smoking might have influenced the results among current smokers.

Unlike in men, we found no significant association between cruciferous vegetable intake and lung cancer risk among women who were never smokers. One possible reason for this differ- ence could be a lack of information on secondhand smoking in women who were never smokers from the 5-y follow-up questionnaire, which may have masked the association. Addition- ally, a previous study has noted that the epidermal growth factor receptor (EGFR) gene mutation is associated with adenocarcinoma histology, never smokers, East Asian ethnicity, and women (35). Matsuo et al. (36) found that the protective effect of vegetables against non-small cell lung cancer were only present for wild-type EGFRs. The EGFR gene mutation status may explain the differ- ences found in our study because no association was found between cruciferous vegetable intake and lung cancer risk in women.

In addition, we also investigated the influence of a detection bias with regard to this difference in sex. As expected from the association between healthy behavior, including screen- ing experience and cruciferous vegetable intake, we observed an increased point estimate of screening-detected lung cancer risk in the highest quartile of cruciferous vegetable intake among women who were never smokers.

No association was found in symptom-detected lung cancer risk among women. In contrast, we did not observe an increased risk of screen- or symptom- detected lung cancer in men. These results suggest that women with higher cruciferous vegetable intake were likely to be diagnosed with lung cancer via screening. However, it is not clear why the detection bias arose only among women.

The effect of cooking (37) is important because it has been reported that raw cruciferous vegetables have a higher bioavail- ability of isothiocyanates (38, 39). We identified the amount of raw intake of the 3 most frequently consumed cruciferous vegetables, namely Chinese radish, cabbage, and Chinese leaves, with the use of 28-d dietary records of 289 subjects from the JPHC validation study (40) based on the list of menus provided by the subjects. Within that period, the proportional intake of raw cabbage, Chinese radish, and Chinese leaves (grams per day) were 26.7%, 38.4%, and 0.7%, respectively, without including pickled vegetables (N Mori, T Shimazu, S Sasazuki, M Nozue, M Mutoh, N Sawada, M Iwasaki, T Yamaji, M Inoue, R Takachi, A Sunami, J Ishihara, T Sobue, S Tsugane, unpublished data). In addition, different cruciferous species have different precursors of glucosi- nolate, which can be converted into isothiocyanates with different anticarcinogenic effects (41).

We found a significant inverse association between cabbage intake and lung cancer risk among men who were never smokers. A previous case-control study in Japan found a similar inverse association of raw cabbage intake (42). Taken together, these findings suggest that raw cabbage intake has a preventive effect on lung cancer. Future prospective research is needed to assess the effect of cooking methods or cruciferous species on lung cancer.

The strengths of this study include its prospective study design, recruitment of the general Japanese population with a high response rate to the questionnaire, and low proportion of follow-up loss. However, several limitations should be noted. First, our estimation of cruciferous vegetable intake did not cover the entire intake because our FFQ only covered 6 fresh and 3 pickled cruciferous vegetables.

We examined whether the cruciferous vegetable intake estimated from the FFQ was satisfactory with the use of dietary records. Estimation from the FFQ was deemed to be satisfactory because 67% (in men) and 80% (in women) of total cruciferous vegetable was covered.

Second, dietary intake obtained with the use of an FFQ may be misclassified. Even if misclassification of cruciferous vegetable intake occurred, it may have been nondifferential and would have tended to result in an underestimation of the impact of cruciferous vegetable intake on lung cancer risk. Nevertheless, we revealed a significant inverse association between total cruciferous vegetable and cabbage intake and lung cancer risk among men who were never smokers.

Third, cruciferous vegetable intake may represent healthy eating behavior and may have cofounded the association between cruciferous vegetable intake and lung cancer risk. We therefore added fruit and other vegetable intake into the multi- variate model to investigate the independent association between cruciferous vegetable intake and lung cancer risk. We also tested whether the trend changed by adding the lung cancer screening experience into the multivariate model, but the results were virtually unchanged.

Finally, in our study there may have been a residual confounding effect of smoking (e.g., years since quitting smoking) among past smokers, as well as other remaining confounding factors such as exposure to chemical pollutants.

Further epidemiologic studies with the use of urinary isothiocyanates may be necessary because the bioavailability of isothiocyanates derived from cruciferous vegetables depend on the cooking method (38, 39). Furthermore, a prospective cohort study on the cruciferous vegetable intake and glutathione S-transferase genotype interaction may be helpful to understand the detoxification mechanism. In conclusion, this prospective cohort study conducted in Japan demonstrated a protective association between cruciferous vegetable intake and lung cancer risk among men who were current nonsmokers.