Sulforaphane

Sulforaphane exists in food in its foodbound form known as Glucoraphanin, a glycoside (bound to a sugar) or sulforaphane that is commonly seen as a prodrug or storage form of Sulforaphane.^[1][2] Glucoraphanin is one of a few molecules known as isothiocyanates, existing mostly in cruciferous vegetables alongside Sinigrin (metabolized into Allyl Isothiocyanate), Glucotropaeolin (metabolized into Benzyl Isothiocyanate), Gluconasturtiin (metabolized into Phenethylisothiocyanate) and Glucobrassicin (metabolized into Diindoylmethane).

Sources of sulforaphane and/or glucoraphanin include:

• Broccoli (44-171mg/100g dry weight^[3])
• Broccoli sprouts (1153mg/100g dry weight^[3])

Sulforaphane is an active isothiocyanate, found in food via its storage form of Glucoraphanin

As sulforaphane tends to exist in foods as Glucoraphanin (4-methylsulphinylbutyl glucosinolate) which has its glucose moiety removed by Myrosinase (Thioglucoside glucohydrolase), an enzyme occurring in the broccoli family of plants^[4] that also mediates the conversion of other glucosinolates such as glucobrassicin to Indole-3-Carbinol (which proceeds to turn into Diindoylmethane).

When Myrosinase acts on Glucoraphanin, it produces an unstable intermediate; if epithiospecifier protein (ESP) is active it can convert this intermediate to a sulforaphane nitrile (5-methylsulfinylpentane nitrile) that has no anti-cancer activity.^[5][6][7] If ESP is low-active, then the only other alternate pathway for this unstable intermediate to progress to is towards production of Sulforaphane

Heating for a short period of time denatures ESP and can enhance sulforaphane production in broccoli due to reducing the alternate metabolic fate of Glucoraphanin^[8] while excessive heat application appears to denature Myrosinase itself and abolish all sulforaphane content; as inactivation of Myrosinase during food preparation by excessive heat would mean a lack of sulforaphane absorption (except for perhaps a small content from the colon^[9]) preparing broccoli via heat treatment for the purpose of Sulforaphane preservation appears to be a balancing act. This balancing act exists since ESP is more sensitive to heat than Myrosinase.^[8]

The application of heat can enhance the absorption of Sulforaphane, but excessive application of heat can prevent most sulforaphane from being absorbed; a balancing act exists

With microwaving, 3 minutes in an 800W microwave (with 150mL water) depletes sulforaphane with peak sulforaphane content at 1 minute.^[10] A 900W microwave between 0.5-0.75 minutes appears to maximize Sulforaphane while minimizing nitrile formation, with 1 minute highly depleting nitrile formation but also sulforaphane starting to be affected.^[11]

Boiling broccoli in 1.5L water appears to inactive myrosinase after 1 minute, with the authors of this study concluding that without dipping broccoli into ice water after 30s of heating (to reduce heat application after being removed from liquid) that boiling is unlikely to be an effective method of preparation.^[11]

With steaming sulforaphane peaks at 1-3 minutes while being depleted at 5, but nitrile formation does not appear to exist to any sufficient level with steaming.^[11]

Steaming appears to be the best preservation method, with microwaving coming in second place; boiling broccoli appears to be a highly ineffective way to preserve sulforaphane content

When comparing the bioavailability of Sulforaphane from broccoli that has been purchased frozen against fresh, it appears that the fresh broccoli has 10-fold the Sulphoraphane content; this was credited to blanching processes pre-freezing destroying Myrosinase.^[9]

Hydrogen sulfide is one of three major gasotransmitters in the human body (transmitting molecules which happen to be gasses) alongside nitric oxide and carbon monoxide, and is the major underlying factor in garlic supplementation. Cruciferous vegetables are known to confer a smell of H₂S when cooked, and since garlic has its cardioprotective effects mediated by sulfur^[12] and sulforaphane has similar protective effects^[13] it was investigated if there was a connection and it was found that, in prostatic cancer cells and mouse liver homogenate at 10µM, sulforaphane releases H₂S.^[14] It is thought that any molecule with an isothiocyanate group (-N=C=S) can act as a sulfur donating molecule for the production of H₂S.

Sulforaphane may release hydrogen sulfide in the body following oral ingestion, suggesting that many of its mechanisms would parallel that of garlic or SAMe (another H₂S releasing supplement)

Sulforaphane appears to be well absorbed, as numerous studies in humans following the consumption of broccoli have noted increased urinary metabolites of Sulforaphane.^[15][16] In humans, bioavailability of sulforaphane appears to be 74% and primarily absorbed in the jejunum.^[17]

Appears to be well absorbed from the intestines after oral administration

In rats, an oral dose of 50umol (8.8mg; which is 58-73mg/kg) appears in the blood within 1 hour and peaks 4 hours after consumption, reaching 20uM; this does is accompanied by a 2.2 hour half-life and reaching close to baseline levels 12 hours after consumption.^[18]

Can be elevated to levels in serum that are used in many in vitro studies, suggesting applicability of those results to practical interventions

Sulforaphane appears to rapidly enter and accumulate into cells^[19] where it is rapidly conjugated, with Glutathione and Glutathione-S-Transferase contributing to its accumulation;^[20][21] Sulforaphane undergoes conjugation with glutathione readily to form dithiocarbamate conjugates such as Glutathione-Sulforaphane, a process accelerated by Glutathione-S-Transferase.^[22] Cellular concentrations may exceed serum concentrations, as one study incubating cultures with 0.028-0.28mM sulforaphane noted cellular concentrations of 4.4-13.3mM, said to be 47-145fold accumulation within 2 hours when counting both Sulforaphane and conjugated Sulforaphane.^[21] This study noted that over the next four hours an 8% decline occurred when the cells remained in medium, but had rapid export of Sulforaphane-Glutathione conjugates when the medium contained no Sulforaphane; this efflux was inhibited potently by MRP protein inhibitors (retaining 54-73% within the cell) and weakly by P-Glycoprotein inhibitors (38-39%; control retained 30%).^[21]

Rapid accumulation into cells, which may exceed serum values; efflux out of cells is mediated mostly by MRP proteins and to a lesser extent by P-Glycoproteins

When fed to rats, sulforaphane is detected in all measure tissues with a significant dose and time interaction in brain, prostate, liver, colon, lung, kidney, and the small intestine mucosa as well as plasma.^[23] The liver, brain, and kidney had peaks at 2 hours after gavage with slight attenuation at 4 hours while the lungs had rapid elimination at 4 hours; all other tissues noted elevations at 2 hours and higher concentrations at 4 hours, with all tissues being depleted of most sulforaphane by 24 hours post-ingestion.^[23] Tissue concentrations varied, however, with brain concentrations being quite low relative to other organs; over 100-fold variability was noted.^[23]

Sulforaphane is able to inhibit CYP1A1 (Aromatase) induction induced by benzo(a)pyrene compounds when incubated at 0.5-2.5uM in HepG2 cells, and is ineffective on CYP1A1/2 in Mcf7 cells when the sulforaphane and the benzopyrene compounds are introduced at the same time; sulforaphane appeared to attenuate the increase in CYP1A induced by previous concentrations of benzo(a)pyrenes.^[24] The maximum inhibition appeared to be 30% of CYP1A at 0.5uM and 14% of CYP1B at 1uM.^[24]

The attenuation of aromatase was deemed secondary to preventing nuclear translocation of the Aryl Hydrocarbon Receptor (AhR) at concentrations of 0.5-1uM.^[24] Sulforaphane is a weak agonist^[25] (less than 10% the potency of the control drug TCDD) yet a non-competitive antagonist of the Aryl Hydrocarbon receptor,^[26] and by preventing binding of more potent agonists to the AhR Sulforaphane reduces genetic transcription of AhR on its reporter (Arnt) and prevents agonist-induced upregulation of aromatase and any cancerous process mediated via AhR.^[27][28][29] This antagonism is not concentration dependent.

The role of Sulforaphane, as it pertains to Aromatase, is as a selective AhR modulator; preventing strong agonists from activating AhR (and upregulating Aromatase) while weakly activating the AhR itself when no strong agonist is present

The enzyme CYP2A6 (responsible for metabolism of nicotine) may be regulated via Sulforaphane's ability to induce Nrf2, which has been induced up to 1.4fold control levels with 10uM Sulforaphane,^[30] and CYP2A6 has been noted to be increased after 6 days of broccoli sprout consumption at 500g per day by a variable 1.4-5.5 fold increase.^[31] Variances may be due to differing activity of the Antioxidant Response Element (ARE1) that Nrf2 activation works via.^[30]

Sulforaphane can be detected in the neural tissue of rats after oral administration of 5umol (0.88mg; 19.36mg/kg) and 20umol (3.52mg; 77.44mg/kg) at both 2 hours and 6 hours post-ingestion, with correlations between brain concentrations and serum concentrations at all points between 0.72-0.77, with 0.59 at the lower dose at 6 hours when sulforaphane was being depleted from the body; no sulforaphane is detected in the brain at these doses (estimated human equivalence of 1.55mg/kg and 6.2mg/kg; respectively) 24 hours after ingestion.^[23]

Despite this high correlation with serum, neural concentrations of oral sulforaphane seem low; ranging between 0.002-0.003mg/g wet weight in response to the above doses.^[23]

Concentrations in the brain correlate well with concentrations in the blood, but there may be problems in crossing the blood brain barrier as the concentrations in the brain of rats in response to oral sulforaphane seem low

Incubation of nerve cells with sulforaphane appears to be able to offer oxidative protection from high glucose concentrations secondary to induction of Nrf2 and its downstream targets of HO-1 and NOQ1, and antiinflammatory attenuation via inhibition of Nf-kB; both of which were confirmed in vivo when rats were fed 0.5-1mg/kg sulforaphane and the sciatic nerve measured after 6 weeks.^[32]

HDAC inhibition is shown to reduce cocaine cravings in mice (with no influence on sucrose cravings)^[33] which has been noted with Trichostatin A (a common reference used in some sulforaphane studies).^[34] At this moment in time, however, no studies have been conducted assessing the ability of sulforaphane in inhibiting cocaine addiction.

There is mechanistic plausibility for sulforaphane in inhibiting cocaine addiction, but this has not yet been studied

Mechanisms of Sulforaphane related to anti-aging appear to be centralized around inducing the Proteasome activity and reducing cellular build-up of modified proteins; a reduction in the activity of this system induces cellular aging (senesence).^[35]

Sulforaphane has been found to activate the Heat Shock Response via selective overexpression of HSP27 at 7.5-10uM concentration via Hsf1 translocation,^[36] which increases HSP27.^[37] It should be noted that heat-shock was found to not induce Nrf2 activity.^[36]

Activation of the 26S proteasome subunit PSMB5 by Sulforaphane^[38] is a consequence of Nrf2 activation^[39] working via the Genomic Antioxidant Response Element (ARE);^[40] the commonest mechanism of Sulforaphane.^[41][42]

The proteasome activity induced by Sulforaphane appears to be chymotrypsin-like and caspase-like, but not trypsin-like, in multiple cell lines without any apparent apoptosis and to almost 2-fold that of control.^[36][38] As silencing of HSP27 inhibits the increase and potency is correlated to the degree of HSP27 induction in vitro,^[36] it appears that HSP27 modulates Sulforaphane-induced Proteasome activity.

Sulforaphane may also increase MAPK activation (including ERK and p38)^[43] which phosphorylates HSP27, but this phosphorylation is not required for HSP27 to induce proteosome activity.^[36]

Sulforaphane appears to be able to increase the rate of glycerol release into medium (lipolysis) in a concentration-dependent manner up to 10uM concentration, coupled with an increase in Hormone Sensitive Lipase (HSL) mRNA and CPT1A mRNA (approximately 1.6-fold control) and no effect on Perilipin or ATGL mRNA.^[44] Sulforaphane appears to be associated with phorphorylation of HSL at Ser⁵⁶³, which may be indirect through inactivating AMPK via phorphorylating Thr¹⁷² and indirectly activating HSL;^[44][45] Sulforaphane's effects on glycerol release are attenuated when coincubated with AMPK activators, and phosphorylated AMPK was reduced to around 20% of control at 5-10uM Sulforaphane.^[44]

AMPK is able to directly induce glycerol release and cause fat loss, but its inhibition may do the same secondary to increased CPT1A, as evidenced by Compound C (an AMPK inhibitor).^[46]

May have fat burning potential secondary to AMPK, but in the opposite direction that most plant extracts influence AMPK; by inhibition rather than activation. Practical significance is unknown

Other studies indicate Nrf2, a target of sulforaphane that is antioxidative in nature (by activating the genetic antioxidant response elemant ARE^[47]), appears to be involved in the regulation of adipocytes (or possibly just thought to be so due to contradicting evidence)^[48] with both knockout^[49] and overexpression^[50] reducing adipocyte growth (which may be accompanied by adverse health events such as insulin resistance^[50]). Downstream of Nrf2, a protein called NAD(P)H:quinone oxidoreductase (not to be confused with NADPH oxidase, a target of Spirulina) which, when activated up to 10.5-fold higher with Sulforaphane at 5-20uM can reduce triglyceride accumulation in fat cells independent of fat cell differentiation.^[51]

Possible anti-obesity effects, but this is also understudied with unknown practical significance

Possibly secondary to its ability to act as a Histone Deacetylase Inhibitor, Sulforaphane may repress Myostatin transcription and attenuate negative feedback on Myostain suppression in porcine satellite cells.^[52] It has been noted elsewhere in liver fibrosis pathology that neither NQO1 and HO-1 (downstream of Nrf2) but Nrf2 itself disrupted signalling from TGF-β to Smad proteins, via inhibiting nuclear accumulation of Smad3;^[53] one of the proteins which mediate the actions of Myostatin in the nucleus.^[54] However, application of these mechanisms to satellite cells may not be valid as Sulforaphane appears to regulate TGF-β/Smad signalling differentially depending on the cellular conditions.^[55][10]

The aforementioned study in pigs noted that both Sulforphane and the reference drug 5-aza-2′-deoxycytidine suppressed Myostatin, but not trichostatin A (another Histone Deacetylase Inhibitor).^[52] Sulforaphane at 5, 10, and 15uM concentration (achieveable with oral intake) failed to increase Follistatin concentrations (natural antagonist to Myostatin) but increases in Smad7 and Smurf1 mRNA was noted, with most significance at 5uM.^[52] miRNAs that increase Myostatin signalling did not appear to be downregulated, with only suppression in miR-29a and miR-29b.^[52]

One study in skeletal muscle satellite cells noting an attenuation of Myostatin signalling (an anabolic event), possibly due to interactions with Sulforaphane's HDAC or Nrf2 inhibitor actions; practical relevance for muscle hypertrophy unclear

Interestingly, this study noted a significant reduction in MyoD mRNA levels at 10uM concentration (5uM not tested) in these porcine satellite cells, coupled with diminished binding to the Myostatin promoter via hypoacetylation of the binding site.^[52]

In an animal model of type 1 diabetes (streptozotocin-induced) oral doses of 0.1, 0.25, or 0.5mg/kg sulforaphane for three days prior to injections of streptozotocin noted that, in the week following injections, that Sulforaphane was able to attenuate the changes to liver weight and changes in body weight and wholly normalized changes in blood glucose and insulin sensitivity.^[56]

Sulforaphane appears to act on the prototypical anti-inflammatory mechanism of inhibiting NF-kB translocation, a mechanism which disrupts inflammatory signals to the nucleus.^[57] In a study using Macrophages (RAW 264.7 cell line), LPS-induced inflammation was attenuated with Sulforaphane with IC₅₀ values on inhibiting NO release, TNF-α release, and PGE₂ production being 0.7uM, 7.8uM, and 1.4uM; respectively.^[57]

The mechanism of sulforaphane inhibiting NF-kB translocation does not appear to be via directly influencing the inhibitory unit IκB-β (which holds NF-kB in an inactive position^[58]]^[59]) but instead attenuates the production of IκB-α. Sulforaphane may also directly prevent NF-kB from forming complexes when in the nucleus.^[57]

Anti-inflammatory effects of Sulforaphane are mediated by preventing NF-kB from translocating to the nucleus, which disrupts pro-inflammatory signals from the cytosol and serum from acting in the nucleus

Sulforaphane appears to be an inhibitor of thioredoxin reductase in LPS-stimulated macrophages, which may be upstream of its effects on NF-kB^[60] as thioredoxin may increase NF-kB activity in pro-inflammatory conditions.^[61] Sulforaphane appears to be synergistic in this regard with the agent CDNB,^[60] an irreversible thioredoxin reductase inactivator.^[62]

In non-immune cells not stimulated with LPS, thioredoxin can be increased secondary to activation of the anti-oxidant response element (ARE) from Nrf2 activation; induction of thioredoxin has been seen with injections of 0.5mg/kg sulforaphane (physiologically relevant concentrations).^[63]

Thioredoxin reductase is also a molecular target of pyrroloquinoline quinone, but interactions with Sulforaphane are unknown.

Some cross-over from Sulforaphane's antioxidant actions may influence inflammatory properties due to some proteins that act in both pathways

Rheumatoid Arthritis is characterized by inflammation and rapidly proliferating synoviocytes, and commonly seen as a treatment target.^[64]

Sulforaphane appears to be able to, in vitro in cultered synoviocytes, suppress TNF-α induced inflammation and proliferation when pre-incubated (secondary to activating Nrf2) and induce apoptosis in cultures already stimulated with TNF-α.^[65]

Sulforaphane appears to act as a Histone Deacetylase inhibitor, a mechanism that can induce expression of p21^Cip1/Waf1 via deacetylating histones H3 and H4.^[66][67]

The potency of sulforaphane at 15uM appears to be as effective as the standard drug trichostatin A at 100ng/mL, with additive benefits when both were coincubated;^[67] as they do not influence Histone Deacetylase protein content nor β-catenin (content or translocation), it appears they work by depressing the TOPflash promoter resulting in up to a doubling of acetylation as measured by histone H3 and H4.^[67]

Sulforaphane itself does not work in this regard, but appears to work via metabolites, with sulforaphane-cysteine and sulforaphane-NAC being effective in a concentration dependent manner.^[67]

Sulforaphane acts as a Histone Deacetylase inhibitor at concentrations that can be achieved in serum

Oral administration of Sulforaphane can reach the prostate at concentrations between 0.07-0.1mg/g wet weight between 2-6 hours after ingestion of (human dose equivalent extrapolated from mice) 1.55-6.2mg/kg; although in a manner that does not correlate with serum values.^[23]

Sulforaphane is confirmed to reach the prostate following oral ingestion, and it appears to bioaccumulate in the concentrations required for its mechanisms of action

Sulforaphane is able to release free hydrogen sulfide (H₂S) in prostatic cancer cells, and 5-50µM sulforaphane causes concentration dependent H₂S release (via cystathionine γ-lyase) and reductions in cellular viability in a manner that is partially blocked by scavenging H₂S.^[14] H₂S, as well as sulforaphane, activated all three major MAPKs (ERK, JNK, p38) and inhibiting the activation of these MAPKs blocked the effects of H₂S.^[14]

It seems that the activatin of MAPKs in prostatic cancer cells from sulforaphane is due to sulforaphane releasing hydrogen sulfide (H₂S)

Histone deacetylase 6 (HDAC6) is a protein that appears to disrupt a cytoplasmic chaperone called HSP90,^[68] and this disruption dysregulates the androgen receptor and attenuates signalling through the androgen receptor at 10-20µM concentration;^[69] this is mediated via HSP90 hyperacetylation from inhibiting HDAC6.^[70] Transcriptional activiy was not ablated with sulforaphane, indicating that inhibition came post-transcription and was thought to be through a reduction in Androgen Receptor content in both LNCaP and VCaP prostatic cancer cell lines,^[69] as well as BPH-1 and PC-3 cells^[71] most of which mimick the elevation of androgen receptor during prostate cancer.^[72]

Mechanistically, sulforaphane inhibits the activation of HDAC6 which then reduces the ability of androgens to signal through the prostate. Since androgens can act to make prostatic cancer cells survive, this is an antisurvival mechanisms

Interestingly, there is a synthetic analogue of Sulforaphane called D,L-Sulforaphane that is currently being investigated for its usage against prostate cancers; which appears to be promising.^[73][74]

Possibly secondary to these mechanisms, Sulforaphane has been found to inhibit cancerous cell growth and induce apoptosis of cancer cells in vitro.^[75]

One study assessing possible nutrient interactions noted, with curcumin and EGCG form green tea catechins tested in LNCaP cells alongside Sulforaphane, that pairing either of two two nutraceuticals with Sulforaphane showed efficacy in reducing cell proliferation relative to control and combining all three trended to be more significant.^[76]

Nonsignificant additive beneficial effects of Curcumin, EGCG, and Sulforaphane in reducing prostatic cell growth

One study using Caco-2 cells noted that incubation of sulforaphane increased mRNA content of the TGF-β receptor, its two subunits (receptors I and II), and enhanced SMAD2/3 signalling when this receptor was stimulated which contributed to apoptotic effects in these cells as assessed by phosporylation and SBE₄luc transluciferase activity.^[55] Previously, broccoli extracts have been implicated in suppressing Smad2 phosphorylation and contest these results.^[10]

In response to oral sulforaphane, kidney concentrations of sulforaphane appear to range from 0.06-0.07mg/g wet weight betwen 4-6 hours after ingestion; suggesting that oral supplementation can load in the kidneys.^[23]

Sulforaphane, at an oral dose of 0.5mg/kg bodyweight in mice for 3 months, was able to significantly attenuate the progression of renal disease (as assessed by kidney weight the albumin:creatinine ratio, normalizing the difference between diabetic and control mice by 41% and 37.7% respectively) in diabetic mice only when it was being consumed; with benefits ceasing upon cessation of Sulforaphane.^[77] These effects were mediated by Nrf2 activation preventing oxidative and inflammatory stressors,^[77] which also appears to underlie protection of the kidneys from organotoxins (with similar potency as curcumin),^[78] unilateral ureteral obstruction,^[79] diabetic nephropathy,^[77][80] ischemia/reperfusion,^[81] and cisplatin.^[82]

Appears to be an effective kidney protective compound during periods of toxin or physical stressors to the kidneys, with protection only existing during periods of Sulforaphane consumption

One study using streptozotocin to induce type 1 diabetes noted that sulforaphane at 0.05-0.5mg/kg of the diet prior to injections exacerbated some damage to the liver, increasing AST (but not ALT) and total cholesterol (but not HDL-C nor hepatic Triglycerides; serum TGs was beneficially reduced to control levels).^[56]

Sulforaphane appears to be synergistic with Curcumin in regards to anti-inflammatory actions in Macrophages, as the level of antiinflammatory effects exerted by either alone is matched by their combination when concentrations are dropped to 40%.^[83] This synergism is not at the level of proinflammatory mRNA induction, but rather at the level of inducing antioxidative protein mRNA of HO-1 and NQO1.^[83]

One other study measuring apoptosis in pancreatic cancer cells (PaCa-2 and Panc-1) in vitro noted synergism with Sulforaphane, Curcumin, and Aspirin when at 5uM, 2.5uM, and 25uM; respectively.^[84]

Phenethyl Isothiocyanate (PEITC) is another compound found in Brassica vegetables alongside Diindoylmethane and Sulforaphane (SFN), and appears to be synergistic with the latter. In a study on cultured macrophages (RAW 264.7, immune cells) it was found that PEITC was equally effective as curcumin in isolation on inhibiting inflammation from the macrophage (measured by Nitric Oxide; IC₈₀ of 5uM each) while SFN was more potent with an IC₈₀ of 1uM; the synergism between PEITC and SFN at these concentrations was more potent than the synergism betwen SFN and Curcumin, and the combination with 40% of the concentration (2uM PEITC and 0.4uM SFN) was as effective as either alone at higher concentrations.^[83]

Neoglucobrassicin is another glucosinolate from broccoli alongside PEITC and Sulforaphane, with relatively low contents in Broccoli sprouts and higher levels in mature Broccoli where it is similar to that of Sulforaphane.^[85][86] In vitro, Neoglucobrassicin and its metabolites appear to compete with and inhibit signalling of Sulforaphane via Nrf2, when assessing glutathione induction.^[87] These metabolites do not stimulate Nrf2 themselves, and inhibition ranges from absolute to practically insignificant in vitro depending on the promoter measured.^[87]

Practical relevance unknown, but may reduce the benefits of Sulforaphane on Nrf2 (where it mediates a good deal of antioxidant effects)

Glucoraphanin gets hydrolyzed by the myrosinase enzyme and then produces one of two metabolites, either 5-methylsulfinylpentane nitrile (via the ESP enzyme) or sulforaphane. While heating at low temperatures can enhance sulforaphane production from inactivating ESP^[8] higher temporatures also inhibit myrosinase and prevent either metabolite from being formed.^[9] Due to this balancing act (of which practical cooking techniques tend to denature myrosinase), the addition of mustard to the cooking of broccoli has been investigated as mustard (sinapsis alba) has a form of myrosinase that is much more heat resistant^[88][89] whereas broccoli tends to have a heat sensitive form.^[89][90] Due to this, when mustard seed powder is mixed with broccoli powder (1-2%; or 12-25mg per 250mg broccoli powder) the sulforaphane availability is increased when cooked, and the losses that would normally occur beyond 60°C instead occur above 90°C and cooking at a temperature that normally abolishes sulforaphane formation (8-12 minutes of boiling sous vide) has three-fold more sulforaphane when it is mixed with mustard seed.^[91]

The mixture of mustard seeds alongside broccoli powder during cooking can increase sulforaphane bioavailability since mustard has a more heat-resistant form of the enzyme that makes sulforaphane. That being said, the study mixed powders of both foods rather than cooking with the whole food products; this information may not apply to placing mustard seeds alongside broccoli florets