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(R, R).
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. (R, R). Levels are back to baseline within 72 hours (R, R, R).
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 [7]. 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 (R, R), 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(R, R, R, R).
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 (R, R).
Sulforaphane has also bee shown to reduce Aβ plaque, and lessens cognitive impairment in mouse models of Alzheimer’s disease (R, R, R).
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 (R, R).
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 (R, R, R).
Sulforaphane decreases incidence and severity of the following diabetes complications in mice (R, R)
- 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 (R, R).
Sulforaphane May Combat Depression and Anxiety
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 (R, R).
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 (R, R, R, R).
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 (R, R, 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 (R, R,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 (R, R, R).
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 (R, R).
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 (R, R).
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).
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Sulforaphane activates genes and enzymes that stimulate antioxidant production:
- inhibits Phase I enzymes CYP1A1, CYP1A2, CYP1B1, CYP2B2 and CYP3A4 (R, R).
- activates (R, R). SFN reacts with Keap1, thereby releasing Nrf2 from Keap1 binding (R).
- increases other Phase II enzymes: NQO1, GSTA1, and HO-1 (R, R, R, R).
- blocks SXR (R).
Sulforaphane inhibits inflammation:
- inhibits NfkB (R, R, R).
- inhibits TNF-α (R, R), NLRP3, IL-1β, IL-18 (R), IFN-gamma and IL6 (R, R, R).
- inhibits IL-17 (R, R).
- inhibits TGF-β/Smad (R).
- increases IL-10 (R, R, R), IL-4, Arg1, and YM-1 (R).
- inhibits NO, iNOS and COX-2 (R, R, R).
- silences Th17/Th1 (R, R).
- inhibits IL-23 and IL-12 (R).
- inhibits MMP-9 (R, R).
- 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 (R, R).
- SFN decreases miR-21 and TERT (R).
Sulforaphane induces cell death (apoptosis) in cancer:
- SFN activates caspase-3, caspase-7, caspase-8, caspase-9 (R, R).
- 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.
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