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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

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 (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).

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 (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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1. Introduction
The importance of the mitochondrion in the maintenance and preservation of cellular homeostasis and function is well established and there is a sufficient body of evidence to show that mitochondrial injury or loss of function is deleterious (Durhuus et al., 2015). The mechanisms leading to mitochondrial dysfunction are varied and include genetic changes occurring at the nuclear or the mitochondrial genome, environmental insult or alterations in homeosis. In all cases, the end result of mitochondrial dysfunction is cellular dysfunction that can limit or severely modulate organ function and ultimately increase morbidity and mortality.

In our research, we have focused on the myocardium, a highly aerobic organ in which mitochondria comprise 30% of cellular volume (Faulk et al., 1995; Faulk et al., 1995a; Toyoda et al., 2000; Toyoda et al., 2001; McCully et al., 2003). The mitochondria supply the energy requirements of the myocardium. This energy is derived through oxidative phosphorylation in the myocardium and is dependent upon the coronary circulation. Under equilibrium conditions the mitochondria within the heart extract greater than 79% of arterial oxygen from the coronary arteries (Fillmore et al., 2013). As heart rate increases or if myocardial workload is increased the oxygen demand is increased and is dependent upon increased coronary flow. Thus, any interruption or impedance in coronary blood flow will significantly limit oxygen delivery to the heart and significantly decrease function and hemostasis (Akhmedov et al., 2015; Doenst et al., 2013; Kolwicz et al., 2013). It is generally accepted that the cessation of coronary blood flow, and thus oxygen delivery, is the initial step in the process leading to myocardial ischemic injury. The sequence of events and the mechanisms associated with this injury are many and are reviewed elsewhere (Lesnefsky et al., 2003; Kalogeris et al., 2012; Ong et al., 2015 Kalogeris et al., 2016 ; Lesnefsky et al., 2017). The end result of ischemia is loss of high energy synthesis and the depletion of high energy stores such that the heart is unable to support hemostasis and maintain function (Rosca et al., 2013 ) .

This reduction of high energy synthesis and stores is rapid. 31Pnuclear magnetic resonance studies have shown that following regional or global ischemia wherein the blood flow to the heart is temporarily ceased, high energy phosphate synthesis and stores are rapidly decreased within 6 minutes and that this decrease continues for at least 60 180 minutes after the restoration of blood flow and is associated with significantly decreased myocardial cellular viability and myocardial function (Tsukube et al., 1997).

Mitochondrial modulations induced by ischemia in the myocardium are many. We and others have demonstrated that following ischemia there are changes in mitochondrial morphology and structure (Rousou et al., 2004; Lesnefsky et al., 2004; McCully et al., 2007). Transmission electron microscopy and lightscattering spectrophotometry have shown that ischemia significantly increases mitochondrial matrix and cristae area and mitochondrial matrix volume (McCully et al., 2007). In addition, there is a decrease in mitochondrial complex activity, cytochrome oxidase I Vmax and a decrease in oxygen consumption and an increase in mitochondrial calcium accumulation (Faulk et al.,1995a).

These changes occur in concert with changes in mitochondrial transcriptomics, with downregulation of annotation clusters for mitochondrion function and energy production and the downregulation of cofactor catabolism, generation of precursor metabolites of energy, cellular carbohydrate metabolism, regulation of biosynthesis, regulation of transcription, and mitochondrial structure and function (enrichment score >2.0, P<.05 black et al. masuzawa in addition there are changes evident overall protein synthesis. proteomic analysis has shown that ischemia significantly alters mitochondrial proteins involved fatty acid and glucose metabolism atp biosynthesis oxidoreductase activity change>1.4, P 2. Mitochondrial transplantation
We rationalized that the therapeutic approach to cardioprotection should be comprehensive and rather than involving a single or multiple mechanistic pathways, intervention should be specific. To this end, we speculated that the replacement or augmentation of mitochondria damaged during ischemia and reperfusion should be the target for therapeutic intervention. We hypothesized that viable mitochondria isolated form the patient’s own body, from a nonischemic area, and then delivered by direct injection into the ischemic organ would replace or augment damaged mitochondria; thus, allowing for the rescue of myocardial cells and restoration of myocardial function. We have termed this therapeutic intervention; mitochondrial transplantation (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016).
2.1 Mitochondrial isolation

Mitochondrial transplantation is based on the delivery of isolated, viable mitochondria to the target organ. The isolation of mitochondria can be performed using a variety of techniques and methodologies. In our initial studies, we used a standard procedure requiring consecutive low and high speed centrifugation to isolate purified mitochondria. This procedure required approximately 90120 minutes to complete. The repetitive centrifugation steps increases the time for mitochondrial isolation and ultimately reduce mitochondrial viability (Graham 2001; Frezza et al., 2007; Pallotti and Lenaz 2007; Wieckowski et al., 2009; FernándezVizarra et al., 2010; Schmitt S, et al., 2013).

In cardiac surgery, and in many other surgical interventions, the interventional time is 4560 minutes and therefore mitochondrial isolation times of 90120 minutes are inappropriate for clinical usage. Mitochondrial isolation time must be short and efficient so that the therapeutic use of mitochondrial transplantation would not extend the surgical time and possibly add to patient morbidity or mortality. To meet the demands and requirements for clinical application, we have developed a rapid methodology for the isolation of autologous mitochondria (Preble et al., 2014; Preble et al., 2014a).

Firstly, two small pieces of autologous tissue are obtained from the patient’s own body during the surgical procedure. The tissue is dissected out using a #6 biopsy punch. The amount of tissue is less than 0.1 gram. The source of tissue is dependent upon the surgical entry point and access. The only requirement being that the tissue source must be free from ischemia and be viable.

In our studies, we have used viable, nonischemic skeletal muscle tissue as a source for isolated mitochondria. The muscle tissue was obtained from the pectoralis major or the rectus abdominis based on standardized minithoracotomy or sternotomy, respectively. Other tissue sources can also be used and we have used liver tissue with excellent results. The tissue, once obtained is immediately used for mitochondria isolation.

The methodology for the isolation of mitochondria for use in mitochondrial transplantation is simple and rapid and can be performed in under 30 minutes. The freshly isolated tissue is homogenized using a commercial automated homogenizer. For our uses we have used the Miletenyi gentleMACS Dissociator (Miltenyi Biotec Inc., San Diego, CA).

This homogenizer was chosen over the more standardized PotterElvehjem homogenizer (glass with Teflon pestle) or bead based homogenizers as it provides a sterilized, disposable unit for homogenization with fixed plastic wings for homogenization and an automated programable homogenization protocol allowing for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization methods.

The other systems require cleaning and sterilization for each usage and allow for variance in homogenization based on the operator. Bead homogenization systems were not considered due to the possibility of contamination of the mitochondria by bead fragments. Once the tissue is obtained homogenization can be performed in 90120 seconds.

The homogenized tissue is then subjected to brief digestion (10 minutes on ice) with subtilisin A (Protease from Bacillus licheniformis, Type VIII, Sigma, Aldrich, St. Louis, MO) and the digested homogenate filtered through a series of disposable sterile mesh filters. The filtration can all be performed in 23 minutes.

The mitochondria can then be used for direct application or can be concentrated by centrifugation (9000 rpm at 4 oC for 10 minutes). The mitochondrial yield using this methodology is approximately 1 x 109 to 1 x 1010 mitochondria, using the two biopsy tissue samples (2.2 Mitochondrial delivery
The direct injection of mitochondria is simple and allows for focal concentration of the injected mitochondria. In our studies the number of mitochondria used for direct injection is 13 x 107 mitochondria. The mitochondria are suspended in 1 mL respiration buffer and injected at 810 sites within the area at risk using a 1 mL tuberculin syringe with a 2832 gage needle (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). Mitochondrial concentrations > 2 x 108 are not fully suspended in 1 mL of respiration buffer and are therefore not advised for use for mitochondrial transplantation by direct injection.

Fluorescence microscopy has demonstrated that transplanted mitochondria delivered by direct injection are present and viable for at least 28 days following injection into the myocardium, the end point of our animal experiments (Masuzawa et al., 2013, Kaza et al., 2016).

The transplanted mitochondria are widely distributed from the epicardium to the subendocardium >2–3 mm from the injection site (McCully et al., 2009; Masuzawa et al., 2013). The majority of injected mitochondria are found initially within the interstitial spaces between cardiomyocytes. Within 1 hour postdelivery, the transplanted mitochondria are detectable within cardiomyocytes residing near the sarcolemma between Zlines of the sarcomeres and in clusters around endogenous damaged mitochondria as well as near the nucleus. Enumeration of injected mitochondria has shown that 43.52% ± 4.46 (mean ± SEM) of the injected mitochondria are attached to or found within cardiomyocytes (Cowan et al., 2016).

We have speculated that the mechanisms through which the transplanted mitochondria distribute within the myocardium following direct injection into the myocardium may be associated with alterations in myocardial structure that occur after myocardial ischemia. Our studies have demonstrated that following ischemia there is a significant increase in myocardial interfibrillar space that provides for both longitudinal and transverse myocardial interfibrillar separations (Tansey et al., 2006).

These structural changes are not associated with alterations in conduction velocity anisotropy or in tissue edema but occur coincident with significant decreases in postischemic functional recovery and increased myocardial apoptosis and necrosis. We have hypothesized that these interfibrillar separations allow for the distribution of injected mitochondria within the myocardium.

While the delivery of mitochondria by direct injection is practical for many applications, it does not allow for global distribution of the transplanted mitochondria.

Multiple injections are needed for global distribution within the heart and require organ manipulation to access posterior and lateral aspects. To allow for global distribution of mitochondria we have recently demonstrated that mitochondria can also be delivered to the target organ by vascular infusion (Cowan et al., 2016).

In preliminary investigations, we have found that for optimal distribution 1 x 109 mitochondria in 5 mL respiration buffer is efficacious and multiple injections can be performed. Using this protocol we have demonstrated that vascular delivery of mitochondria through the coronary arteries results in the rapid and widespread distribution of exogenous mitochondria throughout the heart, within 10 minutes and provides for cardioprotection.

To demonstrate uptake and distribution of the transplanted mitochondria we have labeled isolated mitochondria with 18Frhodamine 6G and magnetic iron oxide nanoparticles. The use of these labels allowed for image analysis using positron emission tomography, computed tomography, and magnetic resonance imaging. Our results show that the transplantation of mitochondria by vascular delivery is rapid and effective Decaycorrected measurements of 18Frhodamine 6G using a dose calibrator revealed most of the 18Frhodamine 6G labeled mitochondria remained contained within the injected hearts throughout reperfusion (77.3% ± 5.5, mean ± SEM).

Positron emission tomography and computed tomography revealed that the transplanted mitochondria were distributed from the heart apex to the base. Quantitative assessment of perfused mitochondrial position in the heart tissue demonstrated that 24.76% ± 2.50 (mean ± SEM) and 23.64% ± 2.42 (mean ± SEM) of the transplanted mitochondria were associated with cardiomyocytes and blood vessels, respectively (Cowan et al., 2016).

To demonstrate the efficacy of vascular delivery of mitochondria to the heart, we delivered unlabeled mitochondria to the regionally ischemic heart. In these experiments we demonstrated that vascular delivery of mitochondria through the coronary arteries following transient ischemia and reperfusion significantly decreased myocardial infarct size and significantly enhanced postischemic functional recovery. The reductions in infarct size and the enhancement of regional functional recovery achieved with vascular delivery of mitochondria are not significantly different from that obtained by direct injection of mitochondria (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016). These data indicate that autologous mitochondrial transplantation is efficacious as a cardioprotective therapy whether these organelles ae delivered by directly injection or delivered by vascular infusion through the coronary arteries.

2.3 Localization to endorgan by vascular delivery
In all our studies, we have noted that the distribution of mitochondria following delivery by direct injection or by vascular infusion remains within the heart and is not detectable in other organs. This finding is important as the delivery of mitochondria by vascular infusion provides for localized therapy without crosscontamination to other endorgans. In the heart we deliver mitochondria by injection into the coronary arteries to avoid systemic distribution; however, we have also delivered mitochondria to the lung by vascular infusion through the pulmonary artery.

In these studies, the mitochondria were labeled with 18Frhodamine 6G as above. Positron emission tomography and computed tomography showed that the mitochondria were localized in the lung and were not detectable in any other areas of the body.

At present, we do not have a mechanism for this “endorgan homing”, where the transplanted mitochondria are retained by the immediate downstream organ, but suggest that this observation may play an important therapeutic role in future studies and applications using mitochondrial transplantation.

2.4 Mechanisms of mitochondrial uptake and internalization
In previous studies, we have investigated a variety of mechanisms that may be associated with mitochondrial uptake and internalization following mitochondrial. These studies were performed using well established pharmacological blockers of clathrin mediated endocytosis, actinmediated endocytosis, macropinocytosis, and tunneling nanotubes (Le et al., 2000; BereiterHahn Jet al., 2008; Lou et al., 2012; Islam et al., 2012; Huang et al., 2013; Kitani et al., 2014). Our studies demonstrated that autologous mitochondria delivered by direct injection are internalized by actindependent endocytosis (Pacak et al., 2015).

Mitochondrial uptake by vascular delivery appears to be more complex. The rapid and widespread uptake of mitochondria when delivered by vascular infusion would suggest that mechanisms allowing for the rapid passage of mitochondria through the vascular wall are involved. Previous studies support the concept that cells can routinely escape from the circulation. It has been previously shown that certain cardiac and mesenchymal stem cells appear to be actively expelled from the vasculature in a process different from diapedesis (Cheng et al., 2012; Allen et al., 2017).

Transmigration of stem cells through the vascular wall requires extensive remodeling of the endothelium (Allen et al., 2016). Cheng et al., 2012 have suggested that this occurs in three stages 1) adhesion of infused cells to the microvessel lining; 2) pocketing of infused cells by endothelial projections; 3) breakdown of the adjacent vascular wall, releasing cells into the interstitium.

The first two steps require integrindependent interactions between transplanted cells and host endothelium, while matrix metalloproteinases mediate the subsequent breakdown of the microvessel wall. These steps are unlikely to occur rapidly or to be associated with the mitochondrion.

Another possible mechanism for mitochondrial uptake may be diapedesislike. Previous studies have shown that some cells routinely escape from the circulation. For example, leukocyte extravasation (i.e. diapedesis) between venous endothelial cells is a wellunderstood process that involves cell adhesion proteins.

It is unlikely, however, that isolated, exogenous mitochondria use a similar mechanism as leukocytes to move through the wall of blood vessels as they do not express the array of proteins involved in diapedesis on their outer membrane (Pagliarini et al., 2008; Calvo et al., 2010).

Whether mitochondria pass between or through endothelial cells and the region of the vasculature at which this process occurs remain to be determined. The size of the isolated mitochondria and the rapidity of their uptake by vascular perfusion would not appear to support any currently defined mechanisms. At present, we are investigating several possible mechanisms of mitochondrial uptake by vascular infusion; but, have yet to conclusively identify a definitive mechanistic route.

3. Mitochondrial transplantation for cardioprotection

3.1 Animal models
In the isolated perfused rabbit heart model and subsequent studies in the in vivo rabbit and pig heart models, we have investigated the use of mitochondrial transplantation as a cardioprotective therapy (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). In these studies, the myocardium was made temporarily ischemic by ligating the left anterior descending artery with a snare. This temporary ligation results in the attenuation or cessation of coronary blood flow such that oxygen delivery to the myocardium is insufficient to meet oxygen demand. The resulting injury is termed ischemia/reperfusion injury and is characterized by the loss of myocardial cell viability and decreased contractile function within the area at risk. In general, we subject 2530% of the left ventricular mass to ischemia, resulting in a loss in cell viability of approximately 30% and a reduction in contractile force of approximately 25% based on regional systolic shortening. These decrements in cell viability and function are reproducible between large animal models and mimic events occurring in human acute myocardial infarction or surgical intervention during cardiopulmonary bypass. Following 30 minutes of temporary ligation the snare is released and coronary blood flow to the region is reestablished. To show the efficacy of mitochondrial transplantation, we typically deliver autologous mitochondria by direct injection or by vascular delivery, just prior to or at the very start of reperfusion. Control hearts receive vehicle alone (respiration buffer with no mitochondria).

3.2 Effects of mitochondrial transplantation on arrhythmogenicity
The transplantation of mitochondria has similarities in methodology with stem cell transplantation. In stem cell transplantation using skeletal muscle myoblasts, it has been reported that skeletal muscle myoblasts, clustering of the cells can occur, resulting in arrhythmia with postoperative episodes of sustained tachycardia due to alterations in electrical coupling (Macia et al., 2009). To demonstrate that mitochondrial transplantation is not proarrhythmic we have used serial 12 lead electrocardiogram (ECG) analysis and optical mapping (Masuzawa et al., 2013). Our results show that there is no proarrhythmogenicity associated with mitochondrial transplantation. We observed no ventricular tachycardia, bradycardia, fibrillation, or conduction system defects or repolarization heterogeneity associated with mitochondrial transplantation. In addition, there were no changes in serial ECG, QRS duration or corrected QT interval. There was no evidence of any changes associated with ventricular wall motion disturbances, left ventricle hypertrophy, valve dysfunction, fibrosis, or pericardial effusion either at the time of injection, or at any time up to 4 weeks following transplantation of autogeneic mitochondria (Masuzawa et al., 2013).
To confirm these findings, we also performed optical mapping (Masuzawa et al., 2013). In these studies, a 400fold increase in mitochondria, 8.4 x 107/gram tissue wet weight as compared to 2 x 105/gram tissue wet weight, was directly injected into the heart. The number of mitochondria injected per site was significantly greater than that used in the in situ heart (4.2 × 108 vs. 1.2 × 106), so that any acute arrhythmogenic responses could be observed. Our results showed that even with this highly increased mitochondrial load, sequential isopotential maps from the left ventricles injected with mitochondria showed no detectable abnormal impulse propagation on the myocardial surface associated with mitochondrial transplantation.

3.3 Effects of mitochondrial transplantation on immune and autoimmune response
In all our studies, we have used autologous mitochondria isolated from the patient’s own body for cardioprotection. The use of autologous tissue was based on the assumption that this approach would not cause an immune response and avoid the need for antirejection therapy required for nonautologous cellbased therapies (Kofidis et al., 2005). To confirm these expectations serial analysis immune and inflammatory markers was performed. The effects of mitochondrial transplantation were also investigated using multiplex (42plex) analysis of cytokines and chemokines (Masuzawa et al., 2013). The results from these studies confirmed that there was no immune or inflammatory response associated with autologous mitochondrial transplantation (Masuzawa et al., 2013). In addition there was no upregulation of cytokines associated with the immune response that is seen in patients with acute heart transplantation rejection (Rose, 2011).
The possibility of autoimmune response due to the presence of increased mitochondrial number in the myocardium was also investigated. The need for these studies was based on previous studies indicating that oxidative modification of E2 subunits of mitochondria pyruvate dehydrogenase, branched chain 2oxoacid dehydrogenase, and 2oxoglutarate dehydrogenase is a critical step leading to the induction of an autoimmune response in the liver as demonstrated by the presence of antimitochondrial antibodies (Leung et al., 2007). Indirect immunofluorescence was used to test for the presence of antimitochondrial antibodies (AMA) in serial blood samples. AMAs were not detected in the serum of any animals treated with autologous mitochondrial transplantation indicating that the transplantation of mitochondria does not induce an autoimmune response (Masuzawa et al., 2013).
3.4 Effects of mitochondrial transplantation on cellular viability and function

Our results have demonstrated that direct injection or vascular delivery of mitochondria to the heart rescues cell function and myocardial contractile function following ischemia and reperfusion. Creatine kinaseMB isoenzyme (CKMB) and cardiac troponin–I (cTnI) are specific and sensitive markers of myocardial injury, and elevated levels indicate myocardial injury (Pourafkari et al., 2015). Mitochondrial transplantation significantly decreased serum CKMB and cTnI indicating that myocardial injury following 30 minutes of transient ischemia was decreased (Masuzawa et al., 2013; Kaza et al., 2016). These effects were confirmed by infarct analysis using triphenyl tetrazolium chloride analysis to determine necrosis and terminal deoxynucleotidyl transferasemediated dUTP nickend labeling (TUNEL) and caspase activity to determine apoptosis in hearts treated with mitochondrial transplantation and those treated with vehicle alone (Masuzawa et al., 2013). Our results demonstrated that mitochondrial transplantation significantly decreased myocardial injury, including both necrosis and apoptosis, resulting from transient ischemia.
Increased myocardial cellular viability would be expected to correlate with enhanced myocardial function and our studies confirm this assumption. Our results show that 10 minutes following mitochondrial transplantation myocardial function is significantly enhanced as compared to hearts receiving injection of respiration media (vehicle) alone and that this function remains enhanced for at least 28 days – the end point of our studies (Masuzawa et al., 2013; Kaza et al., 2016). This is in contrast to hearts receiving vehicle alone that had persistent left ventricular hypokinesis.
3.5 Mechanisms of mitochondrial transplantation
The mechanisms through which mitochondrial transplantation provides cardioprotection have yet to be fully elucidated. At present, our studies have shown that the transplanted mitochondria act both extraand intracellularly. Once transplanted, the mitochondria increase total tissue ATP content and ATP synthesis. This increase in high energy acts rapidly, as early

as 10 minutes following delivery of the mitochondria to the heart, to enhance cardiac function as determined by echocardiography and pressure volume measurement. The mitochondria then act to upregulate proteomic pathways for the mitochondrion and the generation of precursor metabolites for energy and cellular respiration (P 2.0) (Masuzawa et al., 2013).
At 10 minutes to one hour following transplantation the transplanted mitochondrial are internalized into cardiomyocytes, by actindependent endocytosis (Pacak et al., 2015). Once internalized, the transplanted mitochondria further increase cardiomyocyte ATP content and upregulate cardioprotective cytokines (Masuzawa et al., 2013). These cytokines have been shown to be associated with enhanced cardiac function by stimulating cell growth, proliferation, and migration, enhancing vascularization, providing protection against cardiomyocyte apoptosis and improving functional cardiac recovery and cardiac remodeling independent of cardiac myocyte regeneration (Masuzawa et al., 2013).
We have recently shown that transplanted mitochondria also act at the mitochondrial genomic level. In previous experiments, we have shown that following ischemia there is damage to mitochondrial DNA resulting in the reduction of ATP synthesis (Levitsky et al., 2003). These alterations were associated with poor recovery following cardiac surgery in humans. Our studies demonstrate that mitochondrial transplantation replaces damaged mitochondrial DNA with intact mitochondrial DNA and rescues myocardial cell function (Pacak et al., 2015).
The transplanted mitochondria maintain viability and function for at least 28 days, the limit of our studies (Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). This is in contrast to xenoand allotransplanted cells that are rapidly rejected leading to the loss of transplanted cells, despite the use of antirejection pharmaceuticals (Yau et al., 2003; Hamamoto et al., 2009).

4 Human application
Premised upon these in vivo studies demonstrating the efficacy of mitochondrial transplantation for cardioprotection we have recently performed the first clinical application of mitochondrial transplantation (Emani et al., 2017). The study was performed in pediatric patients who suffered myocardial ischemiareperfusion injury. All procedures were performed under an Innovative Therapies process developed by the Boston Children’s Hospital Institutional Review Board. An individual review of the proposed therapy for each patient was provided by two independent physicians, not involved in the patient’s care. Families were extensively counseled regarding the experimental nature of the procedure and a separate Innovative Therapies consent form was signed.
Five pediatric patients in critical condition who were unable to be weaned off extracorporeal membrane oxygenation (ECMO) support due to myocardial dysfunction related to ischemia and reperfusion were treated with autologous mitochondria. Patient diagnosis included dextrotransposition of the great arteries (4 days and 25 days of age ), hypoplastic left heart syndrome (6 days), left ventricular outflow tract obstruction (6 months of age) and tricuspid atresia 1B (2 years of age) (Emani et al., 2017).
The cause of ischemia was coronary artery obstruction that was relieved in 4 patients, and LV distension with subendocardial ischemia in one patient. The autologous mitochondria were isolated from the patient’s rectus abdominis muscle. The patients received 10 mitochondrial injections, 100 uL each containing 1 x 107 ± 1 x 104 mitochondria. The mitochondria were delivered to the myocardium by direct injection with a 1 mL tuberculin syringe (28gauge needle). All injections were delivered to the area affected by ischemiareperfusion that was identified by epicardial echocardiography as being hypokinetic. Following mitochondrial transplantation, all 5 patients had significant improvement in their myocardial systolic function. Epicardial echocardiography showed moderate to severe systolic function ventricular dysfunction with regional hypokinesis prior to treatment (Emani et al., 2017). Ventricular function was improved to mildmoderate to normal systolic function at 46 days following autologous mitochondrial transplantation and improved to mild dysfunction in one patient and normal systolic function with no regional hypokinesia detected in any patient at 10 days following autologous mitochondrial transplantation (Emani et al., 2017).

All but one patient were successfully weaned off ECMO support by the 2nd day post mitochondrial transplantation. The single patient who was unable to wean off ECMO support suffered irreversible multiorgan failure despite the recovery of myocardial function following mitochondrial transplantation. This patient was on ECMO support for 15 days prior to treatment with mitochondrial transplantation. There were no adverse complications such as arrhythmia, intramyocardial hematoma or scarring with mitochondrial transplantation, in agreement with our animal studies. This case study demonstrates for the first time the potential role mitochondrial transplantation to improve ventricular dysfunction following ischemiareperfusion injury in humans.

While this is the first clinical usage of mitochondrial transplantation in humans, the same protocol can be used in adults and in other settings of ischemiareperfusion injury (Emani et al., 2017). The ability to use mitochondrial transplantation for clinical intervention in situations such as the stunned myocardium is enhanced by the fact that mitochondrial harvest and isolation can be performed within 2030 minutes during the same procedure and involves minimal manipulation of muscle tissue.

5 The timing of mitochondrial transplantation delivery
Studies by us and by others have shown that the mechanisms associated with ischemic myocardial injury converge on the mitochondrion. Rapidly following the onset of ischemia there are alterations in mitochondrial structure, complex activity, oxygen consumption, high energy synthesis and changes in mitochondrial transcriptomics and proteomics.

All these changes occur during ischemia and persist following reestablishment of coronary blood flow (reperfusion). For practical efficacy we therefore deliver the transplanted mitochondria just prior to reperfusion or during early reperfusion in order to limit the effects of ischemia on the transplanted mitochondria. We reasoned that if the mitochondria were transplanted at the start of ischemia they too would be damaged. This approach has been shown to be efficacious in our animal studies. In the human trial described above the delivery of mitochondria was many days after the ischemic insult. In these cases, the patient’s heart was unable to provide sufficient contractile force to be weaned from ECMO (Emani et al., 2017).

We believe that the role of mitochondrial transplantation is the rescue of cells and cellular function. This is premised on our animal studies. The results obtained in the clinical studies in humans are most likely the result of reversal of myocardial stunning. Stunning “describes the mechanical dysfunction that persists after reperfusion despite the absence of myocellular damage and despite the return of normal or nearnormal perfusion (Kloner et al., 1998). Mentzer, 2011, has noted that myocardial stunning, is a frequent consequence after heart surgery and is characterized by a requirement for postoperative inotropic support despite a technically satisfactory heart operation. In the stunned heart the myocardial cells remain alive but there is a prolonged depression of cardiac contractility after reperfusion. This depression in contractility can last days but with extended time results in patient mortality.
In the human cases in which we have used mitochondrial transplantation it is likely that we have rescued the stunned myocardium. The mechanisms most likely involve those we have demonstrated in our animal models, namely, restoration of high energy synthesis and replacement of damaged mitochondrial DNA. The veracity of these mechanisms remains to be elucidated.

6 Mitochondrial transplantation for the rescue of other tissues
While our studies have focussed on the heart and ischemia and reperfusion, other groups have shown that mitochondrial transplantation can be used to enhance drug sensitivity in human breast cancer cells (Elliott et al., 2012); to rescue cell function in cells harboring the mitochondrial DNA mutation (Chang et al., 2013); Parkinson’s disease (Chang et al., 2016); liver ischemia/reperfusion injury (Lin et al., 2013); in cellular studies demonstrating that isolated mitochondria rescue mitochondrial respiratory function and improved the cellular viability in cardiomyocytes (Kitani et al., 2014) and neurorecovery after stroke ( Hayakawa et al., 2016).

These studies demonstrate the potential of mitochondrial transplantation in a variety of diseases. The utility in other organ related diseases and syndromes remains to be investigated. However, diabetes, Alzheimer’s disease and dementia, posttraumatic stress disorder, concussion and others have all been shown to be associated with alterations occurring at or affecting mitochondrial function. Included in these pathological disorders are ischemiareperfusion events affecting pulmonary, renal, hepatic, cerebral, ocular and skeletal muscles. We expect that the application and usage of mitochondrial transplantation will provide for a simple and efficacious therapeutic approach to many of these disease and will significantly ameliorate morbidity and mortality.

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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 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 200 nm) or nanoemulsions (d 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 > . 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.

The post Enhanced Nutraceutical bioactivity to fight cancer appeared first on Alivebynature - Evidence Based Reviews.

Study published here: Glucoraphanin ameliorates obesity and insulin resistance through adipose tissue browning and reduction of metabolic endotoxemia in mice

Low-grade sustained inflammation, triggered by chronically high levels of proinflammatory cytokines and gut microbiota-derived circulatory lipopolysaccharide (LPS), links obesity with comorbidities such as insulin resistance and nonalcoholic fatty liver disease (NAFLD) (1,2).

Although a number of pharmacological treatments for obesity and NAFLD have been tested, few drugs are clinically available owing to the lack of long- term efficacy and safety concerns (3,4). Thus, a novel therapeutic approach that would improve energy metabolism and reduce chronic inflammation in obesity is sorely needed.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a basic leucine zipper transcription factor, is widely expressed in human and mouse tissues, and serves as a defense response against extrinsic and intrinsic stressors (5). Upon exposure to electrophilic and oxidative stress, Nrf2 detaches from its repressor, Kelch-like ECH-associated protein 1-nuclear factor (Keap1), and is translocated from the cytoplasm into the nucleus.

This translocation leads to the transcriptional activation of genes encoding phase 2 detoxifying and antioxidant enzymes (6).

In addition to the ubiquitous induction of cytoprotective genes, Nrf2 regulates a large number of genes involved in glucose and lipid metabolism. In the liver, the constitutive activation of Nrf2 via Keap1 knockdown represses the expression of genes involved in gluconeogenesis (7) and lipogenesis (8), thereby alleviating obesity, diabetes, and hepatic steatosis.

Accordingly, synthetic Nrf2 inducers such as synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO)-imidazolide (9), CDDO-methyl ester (known as bardoxolone methyl) (10), and dithiolethione analog, oltipraz (11), have been shown to ameliorate high-fat diet (HFD)-induced obesity and diabetes.

These synthetic Nrf2 inducers also decrease liver and adipose tissue lipogenesis, and enhance glucose uptake in skeletal muscles. However, the mechanisms by which Nrf2 enhances energy metabolism in response to a HFD remain largely unknown.

Although enhanced Nrf2 signaling has shown promising results in several animal studies, the synthetic Nrf2 inducers have caused adverse cardiac events and gastrointestinal toxicities in clinical trials (12,13).

These observations prompted us to explore a safer Nrf2 inducer for the treatment of obesity, insulin resistance, and NAFLD.

Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is one of the most potent naturally occurring Nrf2 inducers; this compound exhibits anticancer activity in cancer cell lines and in carcinogen-induced rodent models (14).

Among the cruciferous vegetables, broccoli sprouts are the best source of glucoraphanin, a stable glucosinolate precursor of sulforaphane (15). In both rodents and humans, glucoraphanin is hydrolyzed by gut microbiota-derived myrosinase into bioactive sulforaphane prior to intestinal absorption (16). A recent clinical study demonstrated the safety of orally administered glucoraphanin (17).

In the present study, we examined the dietary glucoraphanin-mediated modulation of systemic energy balance and the mitigation of chronic inflammation, insulin resistance, and NAFLD in diet-induced obese mice.

Research Design and Methods Glucoraphanin preparation
The sulforaphane precursor, glucoraphanin, was prepared as previously described (17) with minor modifications. Briefly, one day after germination from broccoli seeds (Caudill Seed Company, Louisville, KY), sprouts were boiled in water for 30 min.

The water extract was mixed with dextrinized cornstarch and subsequently spray-dried to yield an extract powder containing 135 mg of glucoraphanin per gram (0.31 mmol/g) (18). The total glucoraphanin titer in the resulting powder was determined by HPLC as previously reported (19).

Mice and diets
Male C57BL/6JSlc mice were purchased from Japan SLC (Hamamatsu, Japan) at 7 weeks of age. Nrf2-knockout (Nrf2-/-) mouse strain (RBRC01390; C57BL/6J background) was provided by RIKEN BRC (Tsukuba, Japan) (6). After a week of acclimation, mice were fed normal chow (NC; containing 2.2% dextrinized cornstarch, 10% kcal from fat, Research Diets, New Brunswick, NJ), NC containing 0.3% glucoraphanin (NC- GR; containing 2.2% extract powder), a high-fat diet (HFD; containing 2.2% dextrinized cornstarch, 60% kcal from fat, #D12492; Research diets), or a HFD containing 0.3% glucoraphanin (HFD-GR; containing 2.2% extract powder) for 14 weeks.

Both the NC and the HFD containing cornstarch or glucoraphanin were prepared by Research Diets. All mice studied were maintained on a 12 h light/dark cycle at 24–26°C with free access to water and food. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kanazawa University, Japan.

Indirect calorimetry
After 3 weeks of feeding, mice were individually housed in an indirect calorimeter chamber at 24–26°C (Oxymax; Columbus Instruments, Columbus, OH). Calorimetry, daily body weight, and daily food intake data were acquired during a 3-day acclimation period, followed by a 2-day experimental period. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in each chamber every 20 minutes. The respiratory exchange ratio (RER = VCO2/VO2) was calculated using Oxymax software. Energy expenditure was calculated as shown below and normalized to the body mass of each subject.
Energy expenditure = VO2 × [3.815 + (1.232 ×RER)]

Metabolic measurements and biochemical analyses
Metabolic parameters, body fat composition, insulin sensitivity, and glucose tolerance were assessed as previously described (20). Plasma LPS levels were analyzed using a Limulus amebocyte lysate assay kit (QCL-1000; Lonza, Allendale, NJ). Plasma LPS binding protein (LBP) levels were determined using an ELISA kit (Enzo Life Sciences, Farmingdale, NY).

Immunoblotting was performed with primary antibodies (Supplementary Table 1) as previously described (20). mRNA expression levels were determined by quantitative real-time PCR using SYBR Green with the primers (Supplementary Table 2) as previously described (20).

Isolation and differentiation of inguinal white adipose tissue-derived primary beige adipocytes
Stromal vascular fractions (SVFs) from inguinal white adipose tissue (WAT) of 7- week-old wild-type and Nrf2-/- mice were prepared as previously reported (21). At confluence, SVF cells were induced for 2 days with differentiation medium containing DMEM/F-12 supplemented with 10% FBS, 20 nM insulin, 1 nM T3, 5 μM dexamethasone, 500 μM isobutylmethylxanthine, 125 μM indomethacin, and 0.5 μM rosiglitazone (all from Sigma-Aldrich, St. Louis, MO).

Induced cells were subsequently cultured in maintenance medium (DMEM/F-12 containing 10% FBS, 20 nM insulin, and 1 nM T3) for 5 days and treated with DMSO or sulforaphane (Toronto Research Chemicals, Toronto, Canada) at the indicated concentrations for 48 h.

Fluorescence-activated cell sorting (FACS)
Cells from the liver and epididymal WAT were prepared as previously described (22). Isolated cells were incubated with Fc-Block (BD Bioscience, San Jose, CA), and subsequently incubated with fluorochrome-conjugated antibodies (Supplementary Table 1).

Flow cytometry was performed using a FACSAria II (BD Bioscience), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Analysis of gut microbiota via pyrosequencing of the 16S rRNA gene
Metagenomic DNA was extracted from mouse cecal content with a QIAamp DNA stool kit (Qiagen, Hilden, Germany). The V1–V2 region of the 16S rRNA gene was amplified using primer sets as previously reported (23).

Mixed samples were prepared by pooling approximately equal amounts of PCR amplicons from each sample and subjected to a GS Junior System (Roche Diagnostics, Basel, Switzerland) for subsequent 454 sequencing.

Pre-processing and taxonomic assignment of sequencing reads were conducted as described previously (23) and separated by unique barcodes. The 16S rRNA sequence database was constructed by retrieving 16S sequences of bacterial isolates (1200–2384 bases in length) from the Ribosomal Database Project Release 10.27.

We used 4000 filter- passed reads of 16S sequences for the operational taxonomic unit (OTU) analysis of each sample. Clustering of 16S sequence reads with identity scores > 96% into OTUs was performed using UCLUST (www.drive5.com).

Representative sequences with identity scores > 96% for each OTU were assigned to bacterial species using BLAST. Principal component analysis using EZR software (https://www.jichi.ac.jp/saitama- sct/SaitamaHP.files/statmedEN.html) was applied for assessment of alterations of cecal bacterial phylum associated with diets.

Statistical analyses
Data were expressed as mean ± SEM. P < 0.05 was considered statistically significant. Statistical differences between pairs of groups were determined by a two-tailed Student’s t-test. An overall difference between more than two groups was determined using a one-way ANOVA.

If one-way ANOVAs were significant, differences between individual groups were estimated using a Bonferroni post hoc test All calculations were performed using SPSS Statistics (v19.0, IBM, Armonk, NY).

Results


Glucoraphanin decreases weight gain and adiposity, and increases energy expenditure in high-fat diet (HFD)-fed mice
To investigate the effects of glucoraphanin on systemic energy balance, we examined the body weight of wild-type mice fed normal chow (NC) or a high-fat diet (HFD), supplemented with glucoraphanin or vehicle (i.e., cornstarch only). Glucoraphanin reduced weight gain only in HFD-fed mice without affecting food intake (Fig. 1A and Supplementary Fig. 1A).

This reduction was not accompanied by evidence of gross toxicity. We determined the plasma concentration of sulforaphane in NC-GR and HFD-GR mice, but not in NC or HFD mice, indicating that glucoraphanin was absorbed as a sulforaphane following food consumption (Supplementary Fig. 1B).

The reduction of weight gain in glucoraphanin-treated HFD-fed mice was largely attributed to the decreased fat mass but not lean mass (Fig. 1B). To assess energy expenditure, we placed the mice in indirect calorimetry cages after 3 weeks of feeding, before an evident change in the body mass of HFD-fed mice was observed (HFD: 29.9 ± 0.5 g vs. HFD-GR: 28.5 ± 0.5 g).

Glucoraphanin-treated mice fed the HFD exhibited consistently higher VO2 and VCO2 than vehicle-treated HFD-fed controls (Fig. 1C and D), leading to increased energy expenditure (Fig. 1E); however, they displayed similar RER (Fig. 1F), suggesting that glucoraphanin supplementation enhanced sugar and fat use under HFD conditions. On NC-fed mice, glucoraphanin did not affect these parameters of energy balance (Fig. 1B–F).

Consistent with increased energy expenditure, glucoraphanin increased the core body temperature of HFD-fed mice by approximately 0.5°C (Fig. 1G).

Glucoraphanin improves diet-induced insulin resistance and glucose tolerance
After 14 weeks of feeding, glucoraphanin supplementation did not affect plasma triglycerides, total cholesterol, and free fatty acid (FFA) levels in either NC- or HFD-fed mice (Table 1). On NC, blood glucose levels were not altered by glucoraphanin, but on the HFD, glucoraphanin-treated mice exhibited significantly lower fasted blood glucose compared with vehicle-treated controls (Table 1).

Additionally, glucoraphanin significantly decreased plasma insulin concentrations in HFD-fed mice under both fasted and fed conditions, resulting in lower homeostatic model assessment-insulin resistance (HOMA-IR; Table 1).

During the insulin tolerance test (ITT), glucoraphanin significantly enhanced the reduction in blood glucose levels in HFD-fed mice, but not in NC-fed mice compared with the vehicle-treated controls (Fig. 2A). Glucoraphanin improved glucose tolerance in HFD- fed mice during the glucose tolerance test (GTT), but had no effect on NC-fed mice (Fig. 2B).

Insulin secretion during the GTT was not affected by glucoraphanin (data not shown). In line with increased insulin sensitivity, insulin-stimulated Akt phosphorylation on Ser473 was enhanced by glucoraphanin in the liver, muscle, and epididymal WAT of mice fed the HFD (Fig. 2C).

Glucoraphanin does not exert anti-obesity and insulin-sensitizing effects in Nrf2-/- mice
Although the Keap1-Nrf2 pathway is a well-known target of sulforaphane, this isothiocyanate has also been reported to modulate different biological pathways independent of the Keap1-Nrf2 pathway (24,25).

To determine whether the anti-obesity and insulin-sensitizing effects of glucoraphanin are mediated through Nrf2, the effects of glucoraphanin on energy balance and glucose metabolism were assessed in NC- and HFD- fed Nrf2-/- mice.

Although food intake and plasma concentration of sulforaphane in NC-GR or HFD-GR diet-fed Nrf2-/- nice were comparable that detected in wild-type mice fed NC- GR or HFD-GR diet (Supplementary Fig. 1C and D), the effects of glucoraphanin following HFD feeding on weight gain (Fig. 3A), VO2 (Fig. 3B), VCO2 (Fig. 3C), energy expenditure (Fig. 3D), RER (Fig. 3E), rectal temperature (Fig. 3F), insulin sensitivity (Fig. 3G), and glucose tolerance (Fig. 3H) were abolished by the Nrf2 deficiency.

These data are consistent with comparable plasma metabolic parameters between glucoraphanin-treated and vehicle-treated mice on the HFD. These metabolic parameters include lipids, blood glucose, insulin, HOMA-IR, and liver enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) (Supplementary Table 3).

Glucoraphanin blocks HFD-induced reduction of Ucp1 expression in WAT of wild- type mice but not in Nrf2-/- mice

The increased energy expenditure and body temperature of glucoraphanin-treated HFD-fed mice suggest an increase in adaptive thermogenesis. However, glucoraphanin supplementation had little effect on the size and number of lipid droplets in the intrascapular brown adipose tissue (BAT) of HFD-fed wild-type mice (Supplementary Fig. 2A).

In addition, the mRNA expression of uncoupling proteins (Ucps), PGC-1α, and deiodinase 2 in BAT and of Ucps in skeletal muscle was not altered by glucoraphanin supplementation in both NC- and HFD-fed wild-type mice (Supplementary Fig. 2B and C). In BAT, HFD increased Ucp1 protein expression, but glucoraphanin did not alter the expression in both wild-type and Nrf2-/- mice (Fig. 4A).

Brown-like adipocytes expressing Ucp1, also known as beige cells, exist in various WAT depots and can contribute to thermogenesis (26). Compared with NC, HFD significantly decreased Ucp1 protein levels in epididymal and inguinal WAT of both wild-type and Nrf2-/- mice (Fig. 4A).

Glucoraphanin supplementation restored HFD-induced reduction in Ucp1 protein levels in epididymal and inguinal WAT of wild-type mice but not those in Nrf2-/- mice.

To examine whether the effects of glucoraphanin were fat cell-autonomous and Nrf2-mediated, we tested the effects of sulforaphane, an active metabolite of glucoraphanin, on the expression of brown fat-selective genes in primary beige adipocytes obtained from inguinal WAT of wild-type and Nrf2-/- mice.

In beige adipocytes derived from wild-type mice, treatment with sulforaphane induced the Nrf2 target gene, NAD(P)H:quinone oxidoreductase 1 (Nqo1; Fig. 4B) and antioxidant genes (Supplementary Fig. 3A).

Concurrently, sulforaphane significantly increased the mRNA expression of brown-fat selective genes, including Ucp1, Prdm16, Cidea, and Elovl3 (Fig. 4B).

In contrast, in Nrf2-deficient beige adipocytes, sulforaphane failed to activate Nrf2 as judged by unaltered mRNA expression of the target genes and to promote the expression of brown-fat selective genes (Supplementary Fig. 3B and Fig. 4C).

Importantly, Nrf2-deficient beige adipocytes exhibited less differentiation levels associated with attenuated lipid accumulation (Supplementary Fig. 3C) and lower mRNA expression of fatty acid binding protein 4 (Supplementary Fig. 3C) and brown-fat selective genes compared with wild-type beige adipocytes (Fig. 4C).

Glucoraphanin reduces hepatic steatosis and oxidative stress in HFD-fed mice
The HFD causes hepatic steatosis and inflammation, eventually leading to steatohepatitis. As shown in Fig. 5A, the increase in liver weight caused by the 14-week- HFD was alleviated by glucoraphanin supplementation.

Glucoraphanin also attenuated HFD-induced hepatic steatosis (Fig. 5B). Additionally, compared with the HFD group, the lower levels of plasma ALT, plasma AST, liver triglycerides, and liver FFAs in the HFD- GR group indicate that glucoraphanin alleviated HFD-induced liver damage (Fig. 5C and D).

The reduction in hepatic steatosis was accompanied by the decreased expression of the following lipogenic genes: sterol regulatory element binding protein-1c (Srebf1), fatty acid synthase (Fasn), and peroxisome proliferator-activated receptor gamma (Pparγ) (Fig. 5E).

Additionally, hepatic levels of malondialdehyde, a marker of lipid peroxidation, were increased by the HFD. Glucoraphanin attenuated lipid peroxidation (Fig. 5F) and decreased gene expression of the NADPH oxidase subunits gp91phox, p22phox, p47phox, and p67phox (Fig. 5E).

The HFD led to a compensatory increase in the expression of genes involved in fatty acid β-oxidation (Ppara and Cpt1a) and anti-oxidative stress (Cat, Gpx1, and Sod1) in the liver. However, glucoraphanin did not increase the expression of these genes in the liver of HFD-fed mice further (Fig. 5E).

Glucoraphanin suppresses HFD-induced proinflammatory activation of macrophages in liver and adipose tissue
In response to the HFD, liver-resident macrophages (Kupffer cells) increase the production of proinflammatory cytokines that promote insulin resistance and NAFLD in mice (27). In particular, chemokine (C-C motif) ligand 2 (Ccl2) promotes the recruitment of chemokine (C-C motif) receptor 2 (Ccr2)-positive monocytic lineages of myeloid cells into the liver (28). These recruited cells produce a large amount of proinflammatory mediators and activate a lipogenic program (28).

Here, we found a prominent induction of tumor necrosis factor-α (Tnf-α), Ccl2, and Ccr2 in the liver of HFD-fed mice, which was markedly reduced in glucoraphanin-treated mice (Fig. 6A).

Glucoraphanin significantly suppressed HFD-induced inflammatory pathways such as c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk) (Fig. 6B).

Glucoraphanin tended to decrease levels of p-NF-κB p65 (Ser536) in HFD-fed mice, although this decrease was not statistically significant (Fig. 6B). Of note, in the liver of Nrf2-/- mice, glucoraphanin failed to suppress HFD-induced inflammatory signal pathways (Supplementary Fig. 4).

In addition, glucoraphanin significantly decreased the HFD-induced hepatic expression of macrophage markers, including F4/80, Cd11b, and Cd68 (Fig. 6C). Tissue macrophages are phenotypically heterogeneous and have been characterized according to their activation/polarization state as M1-like proinflammatory macrophages or M2-like anti- inflammatory macrophages (29).

Consistent with the decreased expression of macrophage markers, glucoraphanin prevented macrophage (F4/80+CD11b+ cell) accumulation in the liver of HFD-fed mice (Fig. 6D).

Additionally, glucoraphanin decreased the number of M1- like liver macrophages expressing surface markers (F4/80+CD11b+CD11c+CD206−) (Fig. 6E).

In contrast, glucoraphanin increased the number of M2-like liver macrophages (F4/80+CD11b+CD11c−CD206+), resulting in a predominantly M2-like macrophage population (Fig. 6E).

Moreover, glucoraphanin decreased the mRNA expression of Tnf-α and NADPH oxidase in the epididymal WAT of HFD-fed mice (Supplementary Fig. 5A).

Although the HFD-induced expression of macrophage markers and macrophage accumulation in epididymal WAT were not altered by glucoraphanin (Supplementary Fig. 5B and C), the number of M1-like macrophages was significantly decreased in the epididymal WAT of glucoraphanin-treated HFD-fed mice (Supplementary Fig. 5D).

Glucoraphanin decreases circulating LPS and the relative abundance of Proteobacteria in the gut microbiomes of HFD-fed mice

Gut microbiota-derived LPS induces chronic inflammation that eventually leads to insulin resistance in obesity, termed metabolic endotoxemia (1,2).

Based on our observation that glucoraphanin alleviates inflammation in the liver and epididymal WAT of HFD-fed mice, we subsequently investigated the effects of glucoraphanin on metabolic endotoxemia and gut microbiota.

In accordance with previous studies (1,2), the HFD induced a 2-fold increase in circulatory LPS levels, which was reduced by glucoraphanin supplementation (Fig. 7A). Furthermore, plasma and hepatic levels of the LPS marker, LBP, were significantly elevated by the HFD and were reduced by glucoraphanin supplementation (Fig. 7B).

A principal component analysis distinguished cecal microbial communities based on diet and treatment, revealing that the metagenomes of HFD-fed mice formed a cluster distinct from the cluster formed by NC-fed mice (Fig. 7C). However, samples from glucoraphanin-treated HFD-fed mice formed a cluster that was indistinguishable from that of vehicle- or glucoraphanin-treated NC-fed mice (Fig. 7C).

Importantly, consistent with previous reports (30,31), further analysis at the phylum level demonstrated that the proportion of Gram-negative Proteobacteria was significantly elevated in the gut microbiomes of HFD-fed mice, which was suppressed by glucoraphanin supplementation (Fig. 7D and E).

The increase in the relative abundance of Proteobacteria in HFD-fed mice is mostly explained by an increase in the relative abundance of bacteria from the family Desulfovibrionaceae (Fig. 7F), key producers of endotoxins in animal models of obesity (30).

In fact, the relative abundance of Desulfovibrionaceae was positively correlated with plasma LPS levels (Fig. 7G). Furthermore, plasma LPS levels were significantly and positively correlated with the hepatic mRNA levels of Tnf-α, gp91phox, and F4/80 (Fig. 7H).

Similarly, the liver expression of other marker genes was both significantly and positively correlated with plasma LPS levels and with one another (Supplementary Table 4).

Discussion
In the present study, we demonstrated that glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, mitigated HFD-induced weight gain, insulin resistance, hepatic steatosis, oxidative stress, and chronic inflammation in mice.

The weight-reducing and insulin-sensitizing effects of glucoraphanin were abolished in Nrf2-/- mice. Additionally, glucoraphanin lowered plasma LPS levels in HFD-fed mice, and decreased the relative abundance of Desulfovibrionaceae.

At the molecular level, glucoraphanin increased Ucp1 protein expression in WAT depots, while suppressing the hepatic mRNA expression of genes involved in lipogenesis, NADPH oxidase, and inflammatory cytokines.

Our data suggest that in diet-induced obese mice, glucoraphanin restores energy expenditure and limits gut-derived metabolic endotoxemia, thereby preventing hepatic steatosis, insulin resistance, and chronic inflammation.

Consistent with previous reports demonstrating the anti-obesity effects of synthetic Nrf2 inducers (9–11), we show that the oral administration of glucoraphanin mitigates HFD-induced weight gain (Fig. 1A). The dose of glucoraphanin used in the current study (about 12 μmol/mouse/day) is similar to that used in other experiments investigating its antitumor effects in mice (14,32,33).

Here, we show that the effect of glucoraphanin on whole-body energy expenditure and the protein expression of Ucp1 in WAT were abolished in Nrf2-/- mice (Fig. 3D and 4A).

A recent study using adipocyte-specific PRDM16- deficient mice indicated that adaptive thermogenesis in beige fat also contributes to systemic energy expenditure (26). The mutant mice in the aforementioned study, which exhibited markedly reduced Ucp1 mRNA expression in inguinal WAT and minimal effects on BAT, developed obesity and insulin resistance in response to a HFD.

Thus, we believe that the increased energy expenditure in glucoraphanin-treated HFD-fed mice stems from, at least in part, an increase in beige fat, even though the expression of Ucps in BAT and skeletal muscle is not altered. Further analysis using Ucp1-knockout mice will elucidate the relative contribution of beige fat to the Nrf2-mediated metabolic effects elicited by glucoraphanin.

Several studies indicated that Nrf2-/- mice are partially protected from HFD-induced obesity and associated with milder insulin resistance compared with wild-type counterparts (9,34,35). Recently, Schneider et al. demonstrated that mitigation of HFD-induced obesity in Nrf2-/- mice, which was 25% less body weight than that of wild-type mice after 6 weeks of feeding (35).

They also found that HFD-fed Nrf2-/- mice exhibit a 20–30% increase in energy expenditure that is associated with an approximately 3-fold up-regulation of Ucp1 protein expression in abdominal WAT (35). In the present study, Nrf2-/- mice gained less weight after 6 weeks of HFD feeding compared with HFD-fed wild-type mice (Figure 1A and 3A; Nrf2-/-: 35.9 ± 0.9 g vs. wild-type: 39.0 ± 0.8 g, P < 0.05).

The lower body mass of Nrf2-/- mice raises the possibility that anti-obesity effect of glucoraphanin was completely phenocopied by Nrf2 gene deficiency. However, several observations suggest that Nrf2-/- mice only partially phenocopy the effect of glucoraphanin on weight gain reduction. 1)

In the present study, the weight difference between Nrf2-/- and wild-type mice was only 8%, which is much less than that in the previous study (35). 2) In Nrf2-/- mice, compared with NC, HFD induced significant weight gain (Fig. 3A), glucose intolerance (Fig. 3H), and insulin resistance as judged by increased HOMA-IR (Supplementary Table 3). 3)

Metabolic rate and energy expenditure of Nrf2-/- mice were comparable with those in wild-type mice (Fig. 1C–E and 3B–D). 4) Ucp1 protein levels in both epididymal WAT and inguinal WAT of HFD-fed Nrf2-/- mice were lower than those in HFD-GR-fed wild-type mice (Fig. 4A).

Taken together, these findings suggest that Nrf2 gene deficiency is not sufficient to block HFD-induced obesity by increasing energy expenditure and Ucp1 expression in WAT depots, and to mask the effect of glucoraphanin.

However, we cannot fully exclude the possibility that the effects of glucoraphanin are mediated by Nrf2 independent-mechanisms. Possible reasons for the discordance in metabolic phenotypes of Nrf2-/- between the previous study (35) and ours may be due to differences in knockout mouse lines and experimental conditions (e.g. age of mice at beginning of HFD feeding, composition of HFD, and temperature in the metabolic chamber).

Our in vitro study of primary beige adipocytes revealed that sulforaphane promotes the expression of brown-fat selective genes (Fig. 4B). Importantly, the concentration of sulforaphane used in cell culture (0.2–5 μM) is comparable with that detected in mice fed NC-GR and HFD-GR diet (Supplementary Fig. 1B). Moreover, we determined that Nrf2 acts as a positive regulator of beige adipocyte differentiation (Fig. 4C and Supplementary Fig. 3C).

The less differentiation levels in Nrf2-deficient beige adipocytes are in agreement with previous reports demonstrating Nrf2 induces white adipocyte differentiation through increasing the gene expression of Pparγ (34) and Cebpβ (36), common transcription factors regulating the differentiation of brown, beige, and white adipocytes.

Furthermore, it is noteworthy that Nrf2 has been reported to bind NF-E2-binding sites in the 5′-flanking region of the human and rodent Ucp1 genes (37).

However, we cannot exclude the possibilities that glucoraphanin affects sympathetic nervous activity or that hormonal factors are regulating fat browning (38). In addition, mitochondrial reactive oxidative species facilitate Ucp1-dependent respiration in BAT and whole-body energy expenditure by promoting the sulfenylation of a specific cysteine residue (Cys253) in Ucp1 (39).

The molecular mechanism by which Nrf2 regulates the expression and thermogenic activity of Ucp1 in beige adipocytes requires further investigation.

Glucoraphanin supplementation improved the systemic glucose tolerance and insulin sensitivity of HFD-fed mice. Although the molecular mechanism by which synthetic Nrf2 inducers enhance glucose uptake is unclear, AMP-activated protein kinase (AMPK) activation may mediate this enhancement in mouse skeletal muscle and adipose tissue (9– 11).

The phosphorylation levels of AMPK (Thr172) and acetyl-CoA carboxylase (Ser79) in peripheral insulin target tissues were comparable between glucoraphanin-treated NC- and HFD-fed mice and vehicle-treated controls (Supplementary Fig. 6).

These data suggest that AMPK activation is not necessary for glucoraphanin to exert its insulin-sensitizing effect on HFD-fed mice. Additional studies using the hyperinsulinemic-euglycemic clamp technique are needed to determine which tissues contribute to the insulin-sensitizing effects of glucoraphanin.

The beneficial effects of glucoraphanin on hepatic lipid metabolism were not accompanied by AMPK activation or the increased expression of fatty acid β-oxidation genes (Fig. 5E).

Instead, glucoraphanin mitigated HFD-induced oxidative stress and inflammation in the liver. In obesity, hepatic inflammation mediated by macrophage/monocyte-derived proinflammatory cytokines promotes lipogenesis through the inhibition of insulin signaling and SREBP activation (40,41). In fact, the depletion of Kupffer cells by clodronate liposomes ameliorates hepatic steatosis and insulin sensitivity in HFD-fed mice (27).

Furthermore, Ccl2- or Ccr2-deficient mice are protected from diet- induced hepatic steatosis even though they still become obese (42,43). Moreover, the specific ablation of M1-like macrophages restores insulin sensitivity in diet-induced obese mice (44), while the deletion of Pparδ, which promotes M2 activation, predisposes lean mice to develop insulin resistance (45).

Therefore, decreased hepatic macrophage accumulation and M2-dominant polarization of hepatic and adipose macrophages account, at least in part, for the protection from hepatic steatosis and insulin resistance in glucoraphanin-treated HFD-fed mice.

One of the most important findings of this study is that glucoraphanin decreases the relative abundance of Gram-negative Proteobacteria, particularly family Desulfovibrionaceae, while reducing circulatory LPS levels (Fig. 7).

Recent studies demonstrated that a significant increase in Desulfovibrionaceae, potential endotoxin producers, in the gut microbiomes of both HFD-induced obese mice and obese human subjects compared to lean individuals (30,31,46). We cannot exclude the possibility that other microbiota-derived products, such as bile acids and short chain fatty acids, also mediate the metabolic action of glucoraphanin.

Whether the interaction between sulforaphane and gut microbiota is affected directly or indirectly by altered host physiology remains to be determined.

However, several studies have suggested that sulforaphane can alter the gut microbiota directly, as isothiocyanates (including sulforaphane) have been shown to exhibit antibacterial activity against Proteobacteria (47,48).

This activity may proceed via redox disruption and enzyme denaturation reactions involving the isothiocyanate reactive group, -N=C=S, the thiol group (-SH) of glutathione and proteobacterial proteins (47,48). Additionally, sulforaphane exhibits antibacterial activity against Helicobacter pylori, a member of the phylum Proteobacteria (49).

We are unaware of any previous reports demonstrating that isothiocyanates inhibit the proliferation of Desulfovibrionaceae. The mechanistic underpinnings of this antibacterial activity require elucidation.

In conclusion, the results of the present study indicate that glucoraphanin may be effective in preventing obesity and related metabolic disorders such as NAFLD and type 2 diabetes.

A recent clinical study demonstrated that supplementation with a dietary dose of glucoraphanin (69 μmol/day) for two months significantly decreased the plasma liver enzymes, ALT and AST, although body mass did not change (18).

Long-term treatment with a higher dose of glucoraphanin (800 μmol/day), which can be safely administered without any harmful side effects (17), may be required to achieve an anti-obesity effect in humans.

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Image credit: Sven Bulterijs

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs

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The post A NAD+/PARP1/SIRT1 axis in Aging appeared first on Alivebynature - Evidence Based Reviews.

Previous research has demonstrated that overexpressing a protein known as SIRT6 increases lifespan of mice and offers protection against obesity-induced metabolic abnormalities. In contrast mice that lack SIRT6 suffer from various aging-related problems and die before the age of 4 weeks. SIRT6 is part of a class of proteins known as the sirtuins that have been well-studied for their anti-aging effects. Earlier this year we reported on a small molecule drug that could selectively activate SIRT6.

Now, two papers by the Cohen lab further investigate the effect of SIRT6 on life- and healthspan.

In the first paper the researchers developed a SIRT6 transgenic mouse that has extra copies of the SIRT6 gene (termed MOSES mice). Old MOSES mice had improved insulin sensitivity, reduced fasting glucose levels, increased physical activity, reduced inflammation of their fat tissue, and improved hair growth.

What about SIRT6 knockout?

In the second paper the authors investigate the effect of a complete lack (a so called knockout) of SIRT6. Such work had been done before but all mice died before four weeks of age. The authors wanted to investigate the effect of SIRT6 on health parameters later in the mouse lifespan. By changing the genetic background of the mice that carry the SIRT6 knockout the researchers were able to generate SIRT6 knockout mice that reach adulthood. Average lifespan of males was just 140 days while 80% of female mice were still alive after 200 days. Hence the effect of SIRT6 on lifespan is gender-specific. The authors succeeded in investigating the effect of certain health parameters up to 10 months of age.

By 5-6 months of age SIRT6 knockout mice showed severe corneal injury. The cornea is the transparent outer layer of the eye allowing light to pass into the eye. The age-related thinning of the retina, the eye layer where light is sensed, was accelerated in SIRT6 knockout mice. Furthermore, SIRT6 knockout mice also show degradation of retinal function.

Surprisingly the SIRT6 knockout mice had higher glucose uptake in skeletal muscle compared to normal mice. The authors showed that this glucose uptake was insulin-independent. The interpretation of this observation is unknown at this moment.

A link to neurodegeneration

Finally, in a third paper by another lab the effect of brain-specific SIRT6 knockout was investigated. Mice that lacked SIRT6 in the brain showed learning impairment at 4 months of age. The authors further discovered that such mice had higher levels of phosphorylated tau in their brains, a characteristic of several neurodegenerative diseases including Alzheimer’s disease. Hyperphosphorylated tau is believed to be neurotoxic and indeed mice that lacked SIRT6 had higher levels of cell death and DNA damage in their brains. SIRT6 levels in the brain decrease with age. Finally, the authors showed that Alzheimer’s disease patients had lower levels of SIRT6 protein in their brains.

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DNA sequences between mitochondria inside a single cell are vastly different, reported scientists in the Perelman School of Medicine at the University of Pennsylvania. This discovery will help to illuminate the underlying mechanisms of diseases that start with mutations in mitochondrial DNA and provide clues about how patients might respond to specific treatments. The researchers published their findings in the journal Cell Reports this week.

Mutant Mitochondrial DNA

Mitochondria are the tiny organelles that produce energy inside our cells. We have two types of DNA in our bodies, the DNA inside the nucleus of our cells, called nuclear DNA – which stores the vast majority of our genetic code – and a separate DNA inside the mitochondria called mitochondrial DNA (mtDNA).

Mitochondria play a crucial role in human health. Mitochondrial dysfunction is one of the hallmarks of aging, and other researchers have linked mitochondria to an array of metabolic and age-related diseases, including type 2 diabetes, Parkinson’s, Alzheimer’s,  cardiovascular disease, and the slowdown in metabolism that occurs as we age.

Unlike nuclear DNA which you inherit from both parents, you inherit mtDNA, only from your mother. Mitochondrial DNA is small and circular and has about 16,500 base pairs that encode proteins specific to the duties of the mitochondria. Because the primary job of the mitochondrion is to produce energy, most of its genes are involved in pathways in the energy production process.

Although it has a small genetic code, mtDNA contains the blueprints for building many of the enzymes and proteins that are needed to function in those pathways.

While each of the cells in our bodies carries only a single copy of our genome, a single mitochondrion can contain 10 or more different genetic codes, each arranged in a small circular chromosome inside the mitochondrion. Each cell in our body has hundreds to thousands of mitochondria.

A mutation in mtDNA leads to mitochondrial disease, which involves an energy deficit in organs such as the kidney, muscle, and brain. Moreover, researchers have shown that mitochondria play a significant role in cancer, and have found mtDNA mutations in various tumors, including those of the pancreas, lung, colorectal, ovaries, bladder, kidney, and breast.

UPenn Research Findings

The UPenn researchers isolated a single mitochondrion and then extracted its DNA. They compared mutations in a single mitochondrion in individual mouse and human neurons and found that mouse cells accumulated more mutations compared to human cells. The scientists concluded that mice accumulate mutations at a different rate than humans.

Senior author of the study James Eberwine, Ph.D., a professor of Systems Pharmacology and Translational Therapeutics, said that a significant take away of the study is to ensure that mitochondrial diseases or potential therapeutics in cells are examined in animal models where the mutations parallel those that occur in humans.

Over a lifetime, mutations in mtDNA accrue differently in each person. The study addressed similarities and differences in discrete mitochondrial DNA in the same cell and also between cell types such as astrocytes and neurons in the brain. As Professor Eberwine says

“By being able to look at a single mitochondrion and compare mutational dynamics between mitochondria, we will be able to gauge the risk for reaching a threshold for diseases associated with increasing numbers of mitochondrial mutations.”

For example, the findings of the study may improve diagnosis of neurological diseases, such as Alzheimer’s, potentially allowing doctors to detect cells that could become diseased or pinpointing patients who may develop certain conditions. This is particularly likely for diseases that commonly strike the elderly in which mutations in mtDNA accumulate with age.

In the future, the scientists plan to use this knowledge to find ways to slow the rate of accumulation of mutations in mtDNA in hopes of halting the progression of diseases. Professor Eberwine adds

“This roadmap of the location and number of mutations within the DNA of a mitochondrion and across all of a cell’s mitochondria is where we need to start.”

References

University Of Pennsylvania School Of Medicine, “First DNA sequence from a single mitochondria.” [Press Release]. Via EurekAlert. Dec 5, 2017. Link.

Morris, Jacqueline, et al. ‘Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing” Cell Reports, Volume 21, Issue 10, p2706–2713, 5 December 2017, DOI:https://dx.doi.org/10.1016/j.celrep.2017.11.031. Alternate Link.

Disclaimer

Diagnosis, Treatment, and Advice:  This article is intended for educational and informational purposes only and is not a substitute for qualified, professional medical advice.  The information and opinions provided herein should not be used during any medical emergency or for the diagnosis or treatment of any medical condition. Experimental therapies carry a much higher risk than FDA-approved ones.  Consult a licensed and qualified physician for the diagnosis and treatment of any and all medical conditions. Call 911, or an equivalent emergency hotline number, for all medical emergencies. As well, consult a licensed, qualified physician before changing your diet, supplement or exercise programs. Photos, Endorsements, & External Links:  This article is not intended to endorse organizations, companies, or their products. Links to external websites, mention or depiction of company names or brands, are intended for illustration only and do not constitute endorsements.

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Senescent cells are defective older cells that are no longer functioning as they should. Most are cleared out and recycled by our immune systems, but as we age, more and more of these defective cells build up.

Sometimes called “zombie cells”, they cause significant harm by secreting molecules that cause inflammation to surrounding cells, leading to more impairment.

Increased systemic inflammation is now recognized as a leading contributor to all age related disease and aging itself. Clearing out of senescent cells can lower systemic inflammation and turn back aging to some degree.

https://www.ebiomedicine.com/article/S2352-3964(18)30373-6/fulltext

This new study found that Fisetin is more effective than any currently know senolytic compound, destroying 25-50% of senescent cells. The dose used equates to 500 mg per day for five days for a 60kg human.

A Phase 2 human trial is underway now, using 20 mg/kg a day for 2 days.

This current research with Fisetin was exciting because it is:

• very effective at clearing senescent cells
• a well studied natural product with very good safety profile
• has many well documented health benefits
• not required to use daily
• has already passed phase 1 safety tests in humans

Killing Zombie cells is attracting big investors

Therapies to kill these zombie cells is attracting big money investors. San Francisco based Unity biotech received $385m from a group including Jeff Bezos and Peter Thiel, and just went public in May for $700m.

New startups like eattle based Oisin Biotech raised $4m, while Netherlands based Cleara Biotech was formed in June with $3m funding.

They are all focusing on drug combinations that can be patented, which clearly makes business sense as demonstrated by the flow of dollars into this burgeoning field.

Senescent cell clearance and NAD+ boosting therapies

Supplements to restore NAD+ levels are generating a lot of excitement among researchers working to extend Healthspan.  Researchers looking to uncover WHY NAD+ levels drop have found increased systemic inflammation consumes more and more NAD+ as we age.

Increasing NAD+ levels thru supplementation is being shown to have many health benefits, but likely has limits as the increased inflammation will eventually overwhelm any supplementation therapy.

Senescent cells are like little factories, spewing poisons that greatly increase inflammation in surrounding cells. Minimizing systemic inflammation by killing these zombie cells will make it easier to restore NAD+ levels.

How to try Fisetin for killing senescent cells

Fisetin has been widely used for it’s cancer fighting and numerous other health benefits (below), normally at 100-200mg per day.

This new research has spurred much self experimentation among the “bio-hackers”  at Longecity.org, so you might want to read there if you are interested in trying this yourself.

Most users on that forum are trying  1,000 to 2,000 mg per day, for 2-5 days.  The Phase 2 human study with Fisetin used 20 mg per kg of body weight for 2 days.

Quotes from this study:

 the first to document extension of both health span and lifespan by a senolytic with few side effects, even though administration was started late in life.

Taken together, these data establish the natural product fisetin as a potent senotherapeutic

demonstrate that reducing the senescent cell burden in mice even late in life is sufficient to have a significant health impact.

Taken together, these studies demonstrate that senolytic compounds can have significant effects on chronic degenerative diseases and age-related pathology.

Thus, new and improved senotherapeutic drugs and combinatorial approaches are needed to eliminate senescent cells safely from multiple organs or even within a single tissue [28–30,42].

Fisetin was most effective at reducing senescent markers.

Fisetin treatment extended the health and lifespan in WT mice even when treatment was initiated in aged animals.

Importantly, no adverse effects of fisetin have been reported, even when given at high doses.

 22–24-month-old mice were treated with 100 mg/kg fisetin for 5 consecutive days by oral gavage, or vehicle only.

The short-course treatment with fisetin resulted in a significant reduction in the fraction of senescent cells in each of these populations (Fig. 4C).

This resulted in an extension of median as well as maximal lifespan (Fig. 5A-B).

improved pancreatic and liver homeostasis (Fig. 5C).

Brain, kidney, liver, lung, and forepaw tissue sections … had reduced age-related pathology in the fisetin diet group compared to the control diet (Fig. 5D).

4. Discussion
Aging is a complex process involving numerous pathways and both genetic and environmental components [64–69]. The biological processes that drive the aging process contribute to the etiology of most chronic diseases including: 1) chronic, “sterile” inflammation; 2) macromolecular changes in proteins, carbohydrates, lipids, mitochondria, and DNA; 3) stem cell and progenitor dysfunction; and 4) increased cellular senescence [5,70]. These processes are linked in that interventions that target one appear to attenuate others. For example, senescent cells accumulate with age and at sites of pathogenesis in chronic diseases [5,70]. Reducing senescent cell burden can lead to reduced inflammation, decreased macromolecular dysfunction, and enhanced function of stem/progenitor cells [1,3,19]. Adult stem cells also become dysfunctional with age, displaying evidence of senescence [71].

Here, we demonstrate that when tested against a panel of other flavonoids, fisetin had the most potent senotherapeutic effects in several cell types in vitro and showed strong anti-geronic effects in vivo. We demonstrated that acute (oral) or chronic (dietary) treatment of progeroid and WT mice with fisetin reduces markers of senescence and senescence-associated secretory phenotype in multiple tissues (Fig. 3A-D, 4A, 5F-I).

Our findings reveal that fisetin targets multiple, but not all types of senescent cells in vivo. Furthermore, by reducing the percent of senescent cells, fisetin reduces expression of senescence markers in multiple organs as measured by qPCR. This results in improved tissue homeostasis and reduction in multiple age-related pathologies, consistent with effects on a fundamental aging process.

The fact that fisetin reduced the fraction of senescent T and NK cells could help amplify the beneficial effects of fisetin, since healthy immune cells are important for clearing senescent cells [72,73]. Similarly, fisetin reduces markers of inflammation and oxidative stress (Figs. 3F-G and 5L-M), consistent with prior literature [45]. This too could contribute to the reduction in senescence markers. The decrease in these markers was observed in tissues harvested several days after completion of fisetin administration.

Since the rapid and terminal half-lives of fisetin are 0.09 and 3.1 h respectively [74], these improvements did not depend on continued presence of circulating fisetin. This is more consistent with fisetin causing removal of senescent cells

Chronic exposure to fisetin improves healthspan and extends the median and maximum lifespan of mice. Importantly, in our study fisetin supplementation was initiated in mice N20 months old.

Fisetin has anti-cancer activity and appears to block the PI3K/AKT/mTOR pathway [80]. We previously found that transiently disrupting the PI3K/AKT pathway by RNA interference leads to death of senescent cells [30], as with other SCAPs that defend senescent cells from their own proapoptotic SASP [28,29]. Fisetin, like some other flavonoids, is a topoisomerase inhibitor, which may also contribute to its anti-cancer activity [81]. It increases the catalytic activity of hSIRT1 at least in vitro [76].

Other biological activities include anti-hyperlipidemic [84–86], anti-inflammatory [85], and neurotrophic [87] effects, some of which could be mediated through a reduction in the senescent cell burden, particularly since when administered intermittently, fisetin alleviated dysfunction despite its short elimination half-life, more consistent with elimination of senescent cells than action on a receptor or enzyme requiring continuous drug presence.

Given that fisetin is a natural product found in common foods and available as an oral dietary supplement and has no reported adverse side effects [45], our pre-clinical data suggest that fisetin should be imminently translatable and could have a significant benefit to the health of elderly patients.

Fisetin Snapshot

Fisetin is a flavonol, a chemical that belongs to the flavonoid group of polyphenols. Fisetin is a type of plant pigment that gives color to many fruits and vegetables (R, R1).

• Great for reducing inflammation
• Improves brain function and memory
• Great antioxidant
• Natural pain killer
• Increases the master antioxidant – Glutathione
• Maintains stable blood sugar
• Good for a diverse array of conditions

Previously Documented Health Benefits of Fisetin

Note that there are very few trials of fisetin with humans and nearly all of the studies listed here are done on animals.

1) Fisetin is Good For Your Brain

Fisetin Encourages New Brain Growth

Out of a number of studied flavanoids, fisetin was found to be the most effective at causing new brain growth (R).

One of the reasons fisetin possesses so many brain-boosting effects is because it is able to cross the blood brain barrier (R).

Fisetin Improves Memory

Fisetin supplementation increases the strength of long term memory pathways (by modifying ERK signaling and increasing BDNF), so it might be useful for memory disorders like Alzheimer’s (R, R1).

Fisetin Protects Against Brain Degeneration

Fisetin has been shown to regulate a number of pathways (e.g. antioxidant & mitochondrial function) that are implicated in age-related decline in brain health (R).

One mechanism by which fisetin might protect against degenerative brain conditions is by activating certain transcription factors such as Nrf2that increase the cellular levels of glutathione – a key cellular antioxidant that has protective effects on nerve cells (R, R1).

Fisetin protects nerve cells from oxidative stress. Oxidative death is one of the many factors associated with cognitive decline, stroke, Alzheimer’s, and Parkinson’s disease (R).

Moreover, Fisetin limits the accumulation of harmful compounds (phosphorylated tau) that accumulate in the brain and cause Alzheimer’s disease (R).

Fisetin is a potentially useful supplement for decreasing inflammation in microglia – immune cells that exert neurotoxic effects and are often activated in neurodegenerative conditions (R).

2) Fisetin Has Anti-Inflammatory Properties

Fisetin inhibits the activity of lipoxygenases (enzymes that act on fatty acids), thereby reducing the production of pro-inflammatory compounds(eicosanoids) and their by-products (R, R).

High blood sugar in diabetics can cause inflammation of the blood vessels and lead to serious damage. These inflammatory processes are inhibited by treatment with fisetin (possibly via inhibition of the HMGB1 signaling pathway) (R, R1).

Fisetin can relieve allergic reactions by inhibiting inflammatory cytokine production (IL-4 and IL-13) by Basophils (R).

Fisetin was found to be an effective treatment for Eczema in animals as it reduced the presence of inflammatory cytokines, eosinophils, mast cells and T-cells (CD4+ & CD8+) that are typically found in atopic dermatitis skin lesions (R).

3) Fisetin May Prevent and Treat Cancer

Fisetin induces cell death and inhibits the growth of various types of cancers(e.g. melanoma, pancreatic cancer, colon cancer, lung cancer and prostate cancer) (R).

Fisetin inhibits the pathways responsible for cell growth and survival that are overactive in cancer patients (R).

Fisetin inhibits the ability of cancer cells to invade healthy tissues (R).

Fisetin inhibits cell proliferation and causes programmed death of chronic myeloid leukemia (white blood cell cancer) cells and possibly other leukemias(R, R1).

Treatment with fisetin was able to kill acute monocytic leukemia cells by increasing levels of nitric oxide and Ca(2+) and activating pathways of apoptosis (cell death) (R).

Fisetin might exert anti-cancer effects by inhibiting the mTOR pathway (R).

Fisetin is such a promising anti-cancer agent because it appears to inhibit and kill cancers without severely affecting the surrounding normal cells – probably because it is able to identify the unique cell-signaling pathways used by cancer cells (R, R1,R2).

4) Fisetin Improves Blood Flow & Lowers Blood Pressure

Fisetin lowers the clumping together (coagulation) of blood cells in mice and, thus, lowers the chance of blockages (R)

By inhibiting Ca(2+) signaling, Fisetin helps reverse vasoconstriction caused by the release serotonin and phenylephrine (R).

Fisetin was able to reverse bad circulation in mice caused by a high fat diet (R).

A rodent study found that Fisetin relaxes drug-induced contraction of blood vessels and, thus, lowers blood pressure. This action occurs regardless of blood vessel function (R).

5) Fisetin May Help Treat Diabetes

Fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and, therefore, may have potential therapeutic use for the treatment of diabetic complications (R).

Diabetic mice fed a diet high in Fisetin stayed diabetic, but the kidney abnormalities normalized (R).

Fisetin administration normalized the increased levels of lipid content in blood, liver and kidney in drug-induced diabetic rats (R).

In diabetic mice, Fisetin reduced the expected formation of cataracts (R).

Fisetin increases HDL and lowered LDL in diabetic rats (R).

6) Fisetin May Extend Lifespan

A number of studies have found that fisetin activates SIRT-1, a gene that has been repeatedly associated with long lifespan; in fact, it is often referred to as the longevity gene (R, R1, R2).

Dietary supplementation with a strawberry extract (which is said to have the highest levels of fisetin) improved the performance of rats in a rodent model of accelerated aging (R).

7) Fisetin May Lower Body Weight

In mice, fisetin supplementation reversed the weight gain caused by a high fat diet (R).

Mice that underwent fisetin treatment had less weight increase than mice that did not undergo treatment (R).

Fisetin prevents diet-induced obesity by regulating cell growth (R).

8) Fisetin Protects Skin From Sun Damage

Fisetin lowers the expression of various genes associated with skin aging (COX-2, MMP-1, MMP-3, MMp-9) that are usually induced by UVB exposure (R).

Fisetin inhibits UVB-induced collagen degradation – a key factor in skin aging (R).

Fisetin reduced  the cellular levels of UV-induced reactive oxygen species, prostaglandin E2, and nitric oxide generation (R).

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