Myrosinase

Myrosinase

Identity, Chemical Classification, and Nomenclature

Myrosinase (EC 3.2.3.1) is the beta-thioglucosidase enzyme responsible for the hydrolysis of glucosinolates, a group of naturally occurring plant metabolites. Its systematic name is thioglucoside glucohydrolase, and it is unique among glycosidases in that whilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. More specifically, myrosinase, a thioglucoside glucohydrolase, is the only enzyme able to hydrolyse glucosinolates, a unique family of molecules bearing an anomeric O-sulfated thiohydroximate function.

Myrosinase exists as a dimer with subunits of 60–70 kDa each. At the structural level, the myrosinase structure reveals a hydrophobic pocket ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic beta-glucosidase.

Myrosinase (thioglucosidase glucohydrolase, EC 3.2.1.147) is a glycoprotein that catalyzes the hydrolysis of glucosinolates. The enzyme is encoded by a multigene family. Myrosinase exists as several forms in Brassicaceae and is encoded by at least three subfamilies of genes, denoted MA, MB, and MC, with the potential for multiple genes in each subfamily. In the model plant Arabidopsis thaliana, six TGG genes encoding classical myrosinases have been found. In oilseed rape (Brassica napus), about 25–30 myrosinase isoforms may be present.

Natural Sources and Botanical Distribution

Myrosinase (EC 3.2.3.1) is the trivial name for the β-thioglucosidase enzyme responsible for the hydrolysis of glucosinolates, a group of sulfur-containing glycosides that occur in all members of the Cruciferae, including the brassica vegetables. The family encompasses a broad range of food plants: important agricultural species include cabbage, broccoli, cauliflower, kale, Brussels sprouts, collard greens, kohlrabi, turnip, rocket salad/arugula, garden cress, watercress, radish, and spices such as horseradish (Armoracia rusticana), wasabi (Eutrema japonicum), and white, brown, and black mustard (Sinapis alba, Brassica juncea, and B. nigra, respectively).

Not all members of the Brassicaceae family contain equal amounts of myrosinase. Sinapis alba is a particularly rich source of myrosinase. In a study carried out by Atle Bones, Sinapis alba seed was found to contain 10 times more myrosinase than that of Brassica napus or Brassica rapa, while earlier work demonstrated that myrosinase activity from crude extracts of Sinapis alba seeds greatly exceeded that of other extract sources, including Brassica juncea, Brassica nigra, Brassica napus, Brassica rapa, and Crambe abyssinica. Other sources of myrosinase include cresses, horseradish, wasabi, and Camelina.

Within the plant body, myrosinase is not distributed uniformly. Myrosinases are mainly localized in specific myrosin cells, observed for the first time in 1884 by Heinricher, who indicated the presence of cells differing in morphology and size compared with neighboring cells and suggested that they contain myrosinase, and accordingly named them as myrosin cells. At the subcellular level, myrosinases have been localized to vacuoles known as myrosin grains, and recently myrosin grains have been shown to form a continuous reticular system denoted the myrosin body.

The Myr I myrosinases are usually localized in specialized myrosin cells in all tissues of Brassica species and A. thaliana, while the substrate glucosinolates are localized in the "aleurone-like" cells in the seedlings and/or "S-cells" in the flower stalk. Glucosinolates and myrosinase are normally harbored in separate compartments within plants, but they come into contact with each other upon tissue disruption from chewing by insects or damage by pathogens, rapidly releasing large amounts of toxic hydrolysis products, typically isothiocyanates and nitriles and their derivatives.

Traditional and Historical Use

While myrosinase as an isolated enzyme is a modern concept, the plants that contain it—and thus the glucosinolate-myrosinase system in its entirety—have an extensive record of traditional use across many cultures. Mustards are members of the Brassicaceae family and are among the earliest cultivated plants. Their seeds are one of the oldest recorded spices, with use and cultivation dating back over 5,000 years. Mustard plants have been widely cultivated and used as spice, medicine, and as a source of edible oils.

The three primary commercially cultivated species are Sinapis alba (white mustard or yellow mustard), Brassica juncea (brown mustard), and Brassica nigra (black mustard), whose use in the food and beverage industry has grown immensely due to their nutritional and functional properties. Black mustard plays an important role in traditional medicine since ancient times, either used internally or externally; its oil is also utilized for medicinal remedies. Sinigrin is the major glucosinolate in the seeds of black mustard and can be hydrolysed to allyl-isothiocyanate, giving the characteristic pungent irritating odour.

The pungency produced when mustard seeds are crushed—which activates myrosinase to produce volatile isothiocyanates—was historically the basis for traditional mustard poultices used across European and Asian medicine. Within the history of human settlement in Australia and New Zealand, different types of Brassicaceae mustards have been naturalized, adapted for use as food, incorporated into traditional medicine, and play an important role in agriculture. Mustards have been consumed for centuries as vegetables, and their products used as condiments and as edible and industrial oils. The pungent "mustard oil" produced by the myrosinase-glucosinolate reaction was used as a rubefacient (counter-irritant) and was applied in folk medicine traditions across Europe and Asia for respiratory complaints, joint pain, and as an antimicrobial dressing—applications that modern biochemistry has traced directly to the isothiocyanates generated by myrosinase activity.

It is important to note that in all these traditional applications, the glucosinolate-myrosinase system was used as an integrated, whole-food preparation (seeds, leaves, roots, or poultices) rather than as an isolated enzyme. The concept of myrosinase as a discrete, extractable supplement ingredient is entirely modern.

Common Forms and Supplement Preparations

In the context of dietary supplementation, myrosinase is used not as a primary ingredient in its own right but as an activating enzyme co-delivered with glucosinolate-rich preparations—most commonly glucoraphanin (GR) derived from broccoli seeds or sprouts—to maximize the conversion of GR to sulforaphane (SF). Dietary supplements are used in clinical trials to deliver consistent SFN doses, but myrosinase is often inactivated in available supplements. This has led to the development of preparations that preserve or restore active myrosinase.

Forms encountered in the literature and in practice include:

• Broccoli sprout extracts (BSE) with active endogenous myrosinase: Made from fresh or carefully freeze-dried broccoli sprouts or seeds, in which myrosinase is not heat-inactivated. These are used in a number of clinical studies from Johns Hopkins University and others. The use of such extracts, though effective, is complicated by the fact that sulforaphane is only moderately stable over time, especially in aqueous solution. The reactivity of sulforaphane is exacerbated by the fact that lyophilized extracts are hygroscopic, and as water is adsorbed during protracted storage or formulation, their useful shelf-life is limited.
• Exogenous myrosinase protein (MSP) from mustard seed: Myrosinase isolated from Sinapis alba (white mustard) seeds is used as a separate protein added to glucoraphanin-rich preparations. Study designs have used a crossover treatment of broccoli sprout extract (BSE) containing glucoraphanin alone, or with exogenous myrosinase protein (MSP) added; both treatments have included ascorbic acid.
• Freeze-dried broccoli powder capsules: It has been suggested to make brassica tablets by freeze-drying the vegetable, a method in which the endogenous myrosinase enzymes are not inactivated in the final product. However, the enzymes contained in the tablet are not active at the low pH of stomach acids when the supplement is swallowed before it can convert the glucosinolates into isothiocyanates.
• Enteric-coated preparations: Enteric coating has been shown to enhance conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach.
• Brassica vegetable supplements with added horseradish: An early commercial approach involved adding exogenous myrosinase by incorporating fresh horseradish root. The most preferable source of exogenous myrosinase enzyme for this process is fresh horseradish root. Fresh horseradish is added to fresh, blanched broccoli in the range of 3–10%.

Key Active Compounds and Biochemical Context

Myrosinase does not itself exert direct physiological effects in humans; rather, it functions as a biological catalyst that generates bioactive molecules from otherwise inactive precursors. Understanding its mechanism requires understanding the glucosinolate-myrosinase system as a whole.

Glucosinolates: The Substrates

Glucosinolates are secondary metabolites occurring in Brassicaceae plants whose hydrolysis may yield isothiocyanates, widely recognized as health-promoting compounds. These sulfur-containing glycosides occur in all members of the Cruciferae, including the brassica vegetables. Over 100 different examples have been isolated and characterized, containing a variety of substituents in the side chain R including allyl (sinigrin), benzyl, and indolyl. Glucoraphanin is the major glucosinolate in broccoli and is the primary precursor of the well-studied isothiocyanate sulforaphane.

The Catalytic Reaction

Enzymic hydrolysis usually occurs when cells are damaged as a result of plant injury or food processing, giving as products β-D-glucose and the aglycone fragment. The aglycone is unstable and reacts further giving the isothiocyanate by means of a Lossen-type rearrangement. More specifically, the hydrolysis leads to the formation of an unstable aglycone intermediate (thiohydroximate-O-sulfonate), glucose, and sulfate. This aglycone undergoes a spontaneous non-enzymatic Lossen rearrangement to yield isothiocyanates (ITCs), thiocyanates, nitriles, oxazolidinethiones, and epithionitriles, depending on the structure of the glucosinolate and the chemical conditions, such as pH, availability of ferrous ions, and the presence of myrosinase-interacting proteins.

The pH of the reaction environment critically determines the nature of the products: the chemical mechanism involves an unstable intermediary (thiohydroxamate-O-sulfonate) that spontaneously decomposes into isothiocyanates or other non-bioactive compounds depending on pH and cofactors. At acidic pH, non-bioactive compounds such as nitriles and thiocyanates are formed, while at neutral pH isothiocyanates are obtained.

Ascorbate as a Cofactor

Ascorbate is a known cofactor of myrosinase, serving as a base catalyst in glucosinolate hydrolysis. For example, myrosinase isolated from daikon (Raphanus sativus) demonstrated an increase in V_max from 2.06 μmol/min per mg of protein to 280 μmol/min per mg of protein on the substrate allyl glucosinolate (sinigrin) when in the presence of 500 μM ascorbate. Sulfate, a byproduct of glucosinolate hydrolysis, has been identified as a competitive inhibitor of myrosinase. This explains why many clinical supplement formulations include ascorbic acid (vitamin C) alongside the glucoraphanin and myrosinase components.

The Catalytic Mechanism at the Molecular Level

Myrosinase is a particular glucosidase which hydrolyzes a variety of plant 1-thio-beta-D-glucosides known as the glucosinolates. This enzyme, which is the only glycosidase able to hydrolyze these naturally occurring thioglucosides, has been found previously to display strong sequence similarities with family 1 O-glycosidases. Myrosinase therefore offers the opportunity to compare the mechanism of enzymatic cleavage of S- vs O-glycosidic bonds. The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst.

Primary Bioactive Product: Sulforaphane

The main mechanism of action of sulforaphane (SF) involves regulation of the nuclear factor erythroid-derived 2-(NF-E2) related factor 2-(Nrf2)-antioxidant response element (ARE) pathway; this in turn upregulates the expression of a range of antioxidant enzymes including HO-1, NQO1, GST, γ-glutamyl cysteine ligase (GCL), and glutathione reductase. Therefore, SF has been considered to act as an indirect antioxidant and a highly potent inducer of phase II cytoprotective enzymes. SF activates the antioxidant and anti-inflammatory responses by inducing the Nrf2 pathway and inhibiting NF-κB. It also has an epigenetic effect by inhibiting HDAC and DNA methyltransferases and modifies mitochondrial dynamics. Moreover, SF preserves proteome homeostasis (proteostasis) by activating the proteasome, which has been shown to lead to increased cellular lifespan and prevent neurodegeneration.

Other Hydrolysis Products

Beyond sulforaphane, the myrosinase system generates a diverse suite of products depending on substrate structure and conditions. The hydrolysis products that result from myrosinase activity on glucosinolates form part of the defense system of the plant against the attack of microorganisms, insects, and herbivores, since they have insecticidal, fungicidal, and bactericidal properties. Indole-3-carbinol (I3C), which derives from the glucosinolate glucobrassicin via myrosinase activity, is separately studied as a chemopreventive agent: I3C is a natural glucosinolate known for its cancer-preventive effects and is already available in commercial preparations as a food supplement.

Mechanisms of Action (Downstream Effects)

All mechanisms of action attributed to myrosinase in a human health context are mediated through the isothiocyanates and other products it generates, not through direct enzyme-tissue interaction. The primary mechanistic pathways are as follows:

Nrf2/ARE Pathway Activation

Many putative cellular targets are affected by sulforaphane, although only one, KEAP1-NRF2 signaling, can be considered a validated target at this time. The transcription factor NRF2 is a master regulator of cell survival responses to endogenous and exogenous stressors. Sulforaphane-mediated KEAP1 modification releases NRF2 from degradation and results in NRF2 phosphorylation and nuclear translocation. Within the nucleus, NRF2 binds to promoter regions of target genes which contain antioxidant response element (ARE) sequences, ultimately inducing the transcription of cytoprotective genes encoding phase II detoxification enzymes and antioxidants. Studies using rodents and primary neuron cultures have demonstrated that sulforaphane upregulates key antioxidant enzymes, such as glutathione-S-transferase (GST), NAD(P)H oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1).

Anti-Inflammatory Signaling

The anti-inflammatory activities of NRF2 are complex and include transcriptional upregulation of enzymes encoded by NRF2-target genes, such as leukotriene B4 dehydrogenase, but also suppression of the expression of genes encoding major pro-inflammatory cytokines, such as IL-6 and IL-1β. Recent studies have demonstrated that the Nrf2/ARE pathway is involved in immune and inflammatory processes, and sulforaphane exerts protective effects via Nrf2 activation, which, under basal conditions, is anchored to the cytoplasm by Keap1.

Epigenetic Modulation

SF activates the antioxidant and anti-inflammatory responses by inducing the Nrf2 pathway and inhibiting NF-κB. It also has an epigenetic effect by inhibiting HDAC (histone deacetylase) and DNA methyltransferases and modifies mitochondrial dynamics.

Phase II Enzyme Induction (Chemoprotection)

SF has been considered to act as an indirect antioxidant and a highly potent inducer of phase II cytoprotective enzymes. The resultant detoxification of electrophiles and oxidants can protect against carcinogens, oxidative stress, and inflammation.

Gut Microbiota as an Alternative Myrosinase Source

Sulforaphane is formed by the hydrolysis of glucoraphanin by a plant enzyme called myrosinase, which is inactivated in the stomach of mammals. Since the latter do not have enzymes possessing myrosinase-like activity, glucoraphanin can be metabolized by the gut microbiota to sulforaphane, sulforaphane-nitrile, glucoerucin, erucin, and erucin-nitrile. Emerging evidence suggests that variations in gut microbiota composition significantly influence the efficiency and outcome of glucoraphanin metabolism, while sulforaphane itself may reciprocally modulate gut microbiota composition and functionality.

Scientific Evidence by Area of Use

1. Sulforaphane Bioavailability Enhancement

This is the area with the most robust human clinical evidence for myrosinase's role. The central question is whether co-delivering plant-sourced myrosinase with glucoraphanin meaningfully increases sulforaphane absorption in humans.

A key pharmacokinetic study from Johns Hopkins (Fahey et al., PMC4629881) compared multiple broccoli preparations in humans. A broccoli sprout extract containing glucoraphanin without plant myrosinase showed substantially lower conversion to SF (about 10%), consistent with previously published data. Mean bioavailability of a range of glucoraphanin-rich preparations lacking active myrosinase was roughly 10% of dose, whereas when active myrosinase was included in the dose, bioavailability increased to almost 40%. Both within-subject and between-subject variability was also reduced, and when hydrolysis was accomplished ex-vivo by a short incubation prior to dosing, bioavailability was approximately 90%, confirming that the hydrolysis was most likely mediated by myrosinase present in the dose.

A more recent randomized crossover clinical study (Scientific Reports, 2026) specifically evaluated exogenous myrosinase from mustard seed added to broccoli seed extract. Glucoraphanin plus myrosinase, on average, doubled the bioavailability of sulforaphane (39.8 ± 3.1%) compared to glucoraphanin alone (18.6 ± 3.1%), and increased the conversion rate in the first 8 h (25.4% ± 2.7%) compared to glucoraphanin alone (8.0% ± 2.7), based on measurement of urinary metabolites. The recovery in the first 8-hour urine collection following administration of an oral bolus of glucoraphanin from BSE with vitamin C was 3.2-fold higher with than without added myrosinase (p = 0.0005). To the authors' knowledge, this was the first human study to simultaneously investigate a well-defined myrosinase source, broccoli seeds as a source of glucoraphanin, and prediction of gut microbial responsiveness to glucoraphanin.

An earlier pilot study examined a myrosinase-treated broccoli sprout extract in healthy adults. Researchers evaluated SF absorption from a myrosinase-treated broccoli sprout extract and were the first to report effects of twice-daily, oral dosing on SFN exposure in healthy adults. These studies clearly demonstrated differences in SFN bioavailability from whole foods and dietary supplements. Despite having lower SFN bioavailability compared to broccoli sprouts, the myrosinase-treated extract may be an acceptable SFN source for use in clinical trials to study certain chemopreventive mechanisms of SFN. The data demonstrated maintenance of plasma SFN levels using a twice-daily dosing schedule, which may be important for increasing or prolonging certain chemopreventive benefits associated with SFN consumption.

Evidence strength: The evidence that plant-sourced myrosinase substantially increases sulforaphane bioavailability in humans is consistent and well-documented across multiple pharmacokinetic studies in healthy adults. This is the best-supported application of myrosinase in a supplement context.

2. Cancer Chemoprevention

Cruciferous vegetables present antimicrobial, antifungal, antioxidant, anti-inflammatory, and anti-cancer activities, with chemo-preventive and chemotherapeutic effects against different cancer types. A diet rich in cruciferous vegetables is generally associated with a lower risk of cancer. According to an umbrella review of 41 systematic reviews and meta-analyses of 303 observational studies, there is suggestive evidence for beneficial associations in gastric cancer, lung cancer, endometrial cancer, and all-cause mortality. Cruciferous vegetables contain glucosinolates, which are under research for their potential for cancer prevention. Glucosinolates are hydrolyzed to isothiocyanates by myrosinase. ITCs are being investigated for their chemopreventive and chemotherapeutic effects.

Since the re-discovery of sulforaphane in 1992 and the recognition of the bioactivity of this phytochemical, many studies have examined its mode of action in cells, animals, and humans. Broccoli, especially as young sprouts, is a rich source of sulforaphane, and broccoli-based preparations are now used in clinical studies probing efficacy in health preservation and disease mitigation. Reviews summarize the chemical biology of sulforaphane as an inducer of NRF2 signaling and its efficacy as an inhibitor of carcinogenesis, as well as findings from clinical trials using a suite of broccoli sprout preparations on a series of short-term endpoints reflecting a diversity of molecular actions.

Evidence strength: Epidemiological and observational evidence linking cruciferous vegetable consumption (and thus the myrosinase-glucosinolate system) to reduced cancer risk is suggestive but not conclusive. Mechanistic evidence in cell and animal models is strong. Clinical trials using broccoli sprout preparations (which inherently include myrosinase) have primarily established pharmacodynamic biomarker endpoints, not cancer incidence outcomes. Direct attribution to myrosinase specifically, as distinct from the sulforaphane it produces, is not possible. Evidence for cancer prevention in humans from myrosinase-containing supplements remains preliminary.

3. Antioxidant and Anti-Inflammatory Effects

Sulforaphane has been shown to induce anti-oxidative mechanisms and protect against cell inflammatory stress via activation of the NRF2 transcription factor. Under basal conditions, NRF2 is anchored to the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1). Through interactions with the cysteine residues of Keap1, sulforaphane may induce the release of NRF2 allowing for its nuclear localization, where it binds to the antioxidant response element (ARE) in the promoter region of a variety of different genes. This cascade upregulates the body's intrinsic antioxidant enzyme production.

Evidence strength: Mechanistic evidence in cell and animal models is extensive and well-characterized. Human clinical evidence for anti-inflammatory outcomes specifically attributable to myrosinase-enabled sulforaphane is more limited and often confined to biomarker studies rather than clinical endpoints.

4. Neuroprotection and Neurodegeneration

Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables. SFN activates the antioxidant and anti-inflammatory responses by inducing the Nrf2 pathway and inhibiting NF-κB. It also has an epigenetic effect by inhibiting HDAC and DNA methyltransferases and modifies mitochondrial dynamics. Moreover, SFN preserves proteome homeostasis (proteostasis) by activating the proteasome, which has been shown to lead to increased cellular lifespan and prevent neurodegeneration.

Increased neuroinflammation and oxidative stress resulting from heightened microglial activation is associated with age-related cognitive impairment. The objectives of one study were to examine the effects of the bioactive sulforaphane on the Nrf2 pathway in BV2 microglia and primary microglia, and to evaluate proinflammatory cytokine expression in LPS-stimulated primary microglia from adult and aged mice. SFN increased Nrf2 DNA-binding activity and upregulated Nrf2 target genes in BV2 microglia, while reducing LPS-induced interleukin (IL-)1β, IL-6, and inducible nitric oxide synthase (iNOS).

Evidence strength: Evidence in this area is predominantly from in vitro and animal models. Human clinical evidence for neuroprotection via myrosinase-generated sulforaphane is emerging but not yet established. Studies in autism spectrum disorder using broccoli sprout extract (which relies on myrosinase activity) are ongoing.

5. Cardiovascular Protection

Sulforaphane (SFN) is an ITC shown to possess anticancer activities by both in vivo and epidemiological studies. Recent data have indicated that the beneficial effects of SFN in CVD are due to its antioxidant and anti-inflammatory properties. Oxidative stress plays a major role in the pathophysiology of cardiac disorders. Several studies have highlighted the cardinal role played by the overproduction of reactive oxygen or nitrogen species in the pathogenesis of ischemic myocardial damage and consequent cardiac dysfunction.

Evidence strength: Human clinical trials specifically investigating myrosinase-containing preparations for cardiovascular outcomes are lacking. Evidence is preclinical (cell and animal) and is derived from studies of sulforaphane rather than myrosinase directly.

6. Liver Health

In most published clinical studies, broccoli sprout extracts or glucoraphanin-rich preparations were used, and administration of these formulations showed significant inter-individual variation in bioavailability of SFN. Among these factors, the myrosinase-catalyzed conversion of glucoraphanin has demonstrated large differences in effect among individuals and is considered an important factor that affects the bioavailability of SFN.

Evidence strength: Liver-related outcomes in clinical trials of broccoli sprout extracts (including myrosinase-active preparations) include exploratory endpoints such as liver enzyme markers. Direct, robust human evidence for liver disease treatment or prevention via myrosinase supplementation is not established.

7. Gut Health and Microbiota Interactions

Since the latter do not have enzymes possessing myrosinase-like activity, glucoraphanin can be metabolized by the gut microbiota to sulforaphane, sulforaphane-nitrile, glucoerucin, erucin, and erucin-nitrile. Emerging evidence suggests that variations in gut microbiota composition significantly influence the efficiency and outcome of glucoraphanin metabolism, while sulforaphane itself may reciprocally modulate gut microbiota composition and functionality. Sulforaphane is assumed to alleviate intestinal inflammation and oxidative stress, maintaining intestinal homeostasis and gut barrier integrity. The role of sulforaphane in breaking the vicious cycle of oxidative stress and gut dysbiosis is reported, demonstrating the potential of dietary isothiocyanates to support gut barrier function.

An important inter-individual variability factor has been noted: administration of glucoraphanin as an oral precursor of SF results in highly variable conversions of GR to SF metabolites among volunteers, ranging from 1–40%, whereas 70–90% of oral SF is consistently converted to urinary dithiocarbamate metabolites in all subjects who have been studied. This variability is largely determined by the composition of an individual's gut microbiome and its myrosinase-like bacterial activity. Adding exogenous plant myrosinase reduces this inter-individual variability by bypassing dependence on gut bacteria.

Evidence strength: Mechanistic and in vitro evidence is strong. Human clinical data specifically focused on gut health outcomes from myrosinase supplementation are limited and preliminary.

Body Systems and Health Areas Associated with Myrosinase Activity

• Gastrointestinal system: Site of myrosinase-catalyzed glucoraphanin conversion; gut microbiome modulation by sulforaphane products; intestinal barrier function.
• Hepatic system: Sulforaphane from myrosinase-catalyzed reactions is studied for phase II enzyme induction and liver detoxification support.
• Oncology / chemoprevention: Isothiocyanate generation via myrosinase is the basis for glucosinolate-related cancer prevention research.
• Immune system: Via NF-κB suppression and Nrf2-mediated cytokine modulation by sulforaphane.
• Cardiovascular system: Antioxidant and anti-inflammatory mechanisms relevant to cardiac protection in preclinical models.
• Neurological system: Neuroprotective effects of sulforaphane studied in neurodegeneration and neuroinflammation models.
• Endocrine / metabolic: Exploratory research on sulforaphane and type 2 diabetes, obesity-related inflammation; not yet established in clinical evidence.

Dosage Forms and Dosages Reported in Studies

No specific standardized dosage has been established for myrosinase as an isolated supplement. The dosages reported in clinical research pertain to the glucoraphanin/glucosinolate content of preparations that include active myrosinase. The following dosage information is reported from primary sources:

• Bioavailability crossover study (Scientific Reports, 2026): The study design used a crossover treatment of broccoli sprout extract (BSE) containing glucoraphanin alone, or with exogenous myrosinase protein added. Both treatments included ascorbic acid. Glucoraphanin plus myrosinase, on average, doubled the bioavailability of sulforaphane (39.8 ± 3.1%) compared to glucoraphanin alone (18.6 ± 3.1%).
• Twice-daily dosing study (Fahey et al., PMC4394840): Researchers evaluated SF absorption from a myrosinase-treated broccoli sprout extract and were the first to report effects of twice-daily, oral dosing on SFN exposure in healthy adults.
• GR dose in pharmacokinetic comparison studies (PMC4629881): Mean bioavailability of GR preparations lacking active myrosinase was roughly 10% of dose, whereas when active myrosinase was included in the dose, bioavailability increased to about 40%. When hydrolysis was accomplished ex-vivo prior to dosing, bioavailability was closer to 90%.
• Proton pump inhibitor interaction pilot study (PMC6682992): A broccoli seed and sprout extract rich in glucoraphanin and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor.
• Horseradish supplement formulations (patent literature): Fresh horseradish is added to fresh, blanched broccoli in the range of 3–10%, and more preferably in the range of 3–8%, as a source of exogenous myrosinase.

Sulforaphane itself, generated by myrosinase from glucoraphanin, is excreted substantially via the urine: oral SF from extracts of broccoli sprouts or seeds is converted to urinary DTC metabolites, and between 70% and 90% of the dose is consistently excreted in the urine of all subjects who have been studied.

Safety Considerations and Notable Interactions

Thermal Inactivation

A critical safety-relevant property is the enzyme's sensitivity to heat, which determines how much active myrosinase is actually present in any preparation or food. The glucosinolate-myrosinase system in Brassica plants can be thermally altered on multiple levels due to heating or cooking. Heat may result in total or partial inactivation of myrosinase, seeping of glucosinolates or their metabolites into the cooking medium, loss of enzymatic cofactors, and thermal degradation or volatilization of metabolites. The degree of change can be correlated to the method and duration of cooking, the extent of cellular disruption, and the structure and stability of the glucosinolate precursors.

Studies in cabbage found that myrosinase was most stable after stir-frying, with up to 65% residual activity. Steaming and microwaving resulted in over 90% loss of myrosinase activity in some accessions. Stir-frying resulted in the greatest decrease in glucosinolate concentration, resulting in up to 70% loss. Steamed cabbages retained the highest glucosinolates after cooking, up to 97%. Broccoli myrosinase has the lowest thermal stability compared to other myrosinase sources, while it is highest in the case of rapeseed myrosinase. Thermal inactivation of green cabbage myrosinase started at 35 °C.

Gastric Acidity and Proton Pump Inhibitors

A clinically important interaction involves gastric acid and acid-suppressing medications. Researchers examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin, to convert glucoraphanin to sulforaphane. A broccoli seed and sprout extract rich in glucoraphanin and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate glucoraphanin bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. This means that the acidic gastric environment partially inactivates myrosinase before it can act on glucoraphanin, and that individuals with naturally lower gastric acidity (or those taking acid-suppressing drugs) may paradoxically see enhanced sulforaphane conversion from myrosinase-containing preparations.

Notably, there were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality on conversion. Greater body mass correlated with reduced conversion efficiency.

Inter-Individual Variability in Gut Microbial Conversion

The inclusion of active myrosinase is critical in clinical trials because although myrosinase-producing gut bacteria can convert unhydrolyzed glucosinolates to their cognate isothiocyanates, the conversion rate is highly variable and subject to inter-individual differences in gut bacteria populations. Adding exogenous plant-sourced myrosinase reduces, but does not fully eliminate, this variability.

Product Formulation and Stability Concerns

The use of broccoli sprout extracts, though effective, is complicated by the fact that sulforaphane is only moderately stable over time, especially in aqueous solution. The reactivity of sulforaphane is exacerbated by the fact that lyophilized extracts are hygroscopic, and as water is adsorbed during protracted storage or formulation, their useful shelf-life is limited unless chemically stabilized, kept cold, or made frequently during the study. It is difficult and expensive to stabilize and formulate sulforaphane for extended clinical trials and particularly for long-term interventions.

Mild Gastrointestinal Side Effects

A high incidence of mild stomach upset has been observed in a clinical trial of α-cyclodextrin inclusion of SFN. This is attributed to the isothiocyanate products of myrosinase activity rather than to the enzyme itself.

Thyroid Considerations

Some glucosinolate hydrolysis products other than sulforaphane—specifically oxazolidinethiones and nitriles—have been associated with goitrogenic effects at very high intakes. However, this is a function of specific glucosinolate precursors (particularly in rapeseed/canola) rather than myrosinase per se. The enzyme indiscriminately catalyzes hydrolysis of all glucosinolates present, and the nature of the products depends on the substrate profile, pH, and cofactors present.

Exclusion Criteria in Clinical Trials

Exclusion criteria in at least one rigorous myrosinase clinical trial included diarrhea or oral antibiotic intake within the previous 4 weeks, and use of proton pump inhibitors (PPIs), antacids, or other relevant medications. This reflects the known interactions of these factors with myrosinase activity and glucosinolate metabolism.

Summary of Evidence Strength

The most rigorously established function of myrosinase in a supplement context is as a bioavailability enhancer for glucosinolate-derived isothiocyanates, particularly sulforaphane. Multiple human pharmacokinetic studies demonstrate that including active myrosinase in a glucoraphanin-containing supplement substantially increases urinary sulforaphane recovery—approximately 4-fold in some studies compared to myrosinase-free preparations. All other health outcomes associated with myrosinase are mediated via the downstream molecules it generates (primarily sulforaphane and other isothiocyanates), and the evidence strength for those outcomes varies from preliminary (neurological, cardiovascular) to suggestive from epidemiology but not yet established in intervention trials (cancer chemoprevention) to mechanistically well-characterized but lacking large clinical trials (antioxidant/anti-inflammatory effects). No clinical evidence supports attributing any health effect directly to myrosinase protein itself, as distinct from its enzymatic products.

References

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