Optimizing Glucosinolates & Sulforaphane

In this Page, I will first look at the Biology of Glucosinolates and Sulforaphane (Section A) and follow up on Techniques to Maximize sulforaphane’s bioavailability (Section B)

Section A

The Biology of the Cruciferous Family of Veggies: Glucosinolates and Sulphoraphane

Glucosinolates and in particular sulphoraphane are important for the body’s metabolic detoxification pathways. (1)

Exposure to toxins and inflammatory foods causes oxidative stress in the body which causes our bodies to produce free radicals faster than we can neutralize them with antioxidants. Antioxidant foods, especially those high in a compound called sulforaphane, give the body the nutrient precursors necessary for the production of glutathione. Cruciferous veggies are the best source of sulforaphane, acting as powerful antioxidants to help spare our cells from damage and reduce oxidative stress. (2) According to a study at Johns Hopkins University, sulforaphane is the most protective antioxidant substance on the planet.

The Brassicaceae or Cruciferous family of vegetables comprises approximately 375 genera and over 3,000 species (Source). Many commonly consumed cruciferous vegetables come from the Brassica genus, including broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, kohlrabi, mustard, rutabaga, turnips, bok choy, and Chinese cabbage. Examples of edible crucifers from other genera within the Brassicaceae family include radish (Raphanus sativus), horseradish (Armoracia rusticana), watercress (Nasturtium officinale), wasabi (Wasabia japonica), and Swiss chard (Beta vulgaris flavescens). (3)

Cruciferous vegetables are unique in that they are a rich source of sulfur-containing compounds called glucosinolates (β-thioglucoside N-hydroxysulfates) that impart a pungent aroma and spicy. Glucosinolates can be classified into three categories based on the chemical structure of their amino acid precursors: aliphatic glucosinolates (e.g., glucoraphanin), indole glucosinolates (e.g., glucobrassicin), and aromatic glucosinolates (e.g., gluconasturtiin) (1). Around 130 glucosinolate structures have been described to date (Source), but only a subset can be found in the human diet. In a cohort of 2,121 German participants in the European Prospective Investigation into Cancer and Nutrition (EPIC study), glucobrassicin, sinigrin, glucoraphasatin (dehydroerucin), glucoraphanin, and glucoiberin were found to contribute most to total glucosinolate intake (Source).

Glucosinolates and their breakdown derivatives (metabolites), especially isothiocyanates and indole-3-carbinol and sulphoraphane, exert a variety of biological activities that may be the most relevant to healthy longevity activation and disease prevention in humans.

Each vegetable, sprout and seed usually contains more than one glucosinolate.  However, certain vegetables, sprouts and seeds may contain a predominant amount of one glucosinolate. An example is the following: Broccoli and broccoli sprouts contain large amounts of glucoraphanin. Mustard seeds and Brussel sprouts contain a large amount of Sinigrin. Garden cress and cabbage contain a large amount of glucotropaeolin. Watercress contains a large amount of gluconasturtiin. The total number of documented glucosinolates from nature can be estimated to around 132, as of 2011.  (Source)  For purposes of this  Page, we will focus on the 4 most important glucosinolates and the ones that have been the subject of the majority of medical research. These 4 glucosinolates include the four mentioned above. 

The Four Key Glucosinolates

Gluconasturtiin, also known as phenethylglucosinolate, is a widely distributed glucosinolate in cruciferous vegetables.  The name is derived from it occurrence in watercress which has the botanical name Nasturtium officinale. Glucoraphanin is a glucosinolate distributed in broccoli, Brussel sprouts, cabbage and cauliflower.  It is also found in large amounts in young sprouts of cruciferous vegetables, like broccoli sprouts. Glucotropaeolin is a phytochemical from Tropaeolum majus, which is commonly known as garden nasturtium, Indian cress or monks cress.  It is also found in cabbage. Sinigrin is widely distributed in the plants of the Brassicaceae such as Brussel sprouts, broccoli, horseradish and black mustard seeds.

Introducing Sulforaphane

Sulforaphane is obtained from cruciferous vegetables such as broccoli, broccoli sprouts, Brussels sprouts, and cabbages. It is produced when the enzyme myrosinase transforms glucoraphanin into sulforaphane upon damage to the plant (such as from chewing), which allows the two compounds to mix and react. When the enzyme myrosinase acts on glucoraphanin, an unstable intermediate is produced.  This unstable intermediate is then acted on by a protein called epithiospecifier protein (ESP) to produce sulforaphane or sulforaphane nitrile.

If epithiospecifier protein (ESP) is abundant in the plant and is active, it will convert this unstable intermediate to a sulforaphane nitrile.  This sulforaphane nitrile has no anti-cancer activity. (Source) In the case epithiospecifier protein (ESP) is not abundant and is low-active, then it will convert this unstable intermediate into sulforaphane.  Sulforaphane has anti-cancer activity.

  “Recent identification of a sub-population of tumor cells with stem cell-like self-renewal capacity that may be responsible for relapse, metastasis, and resistance, as a potential target of the dietary compound, may be an important aspect of sulforaphane chemoprevention. Evidence also suggests that sulforaphane may target the epigenetic alterations observed in specific cancers, reversing aberrant changes in gene transcription through mechanisms of histone deacetylase inhibition, global demethylation, and microRNA modulation. Critical Issues: In this review, we discuss the biochemical and biological properties of sulforaphane with a particular emphasis on the anticancer properties of the dietary compound. Sulforaphane possesses the capacity to intervene in multistage carcinogenesis through the modulation and/or regulation of important cellular mechanisms. The inhibition of phase I enzymes that are responsible for the activation of pro-carcinogens, and the induction of phase II enzymes that are critical in mutagen elimination are well-characterized chemopreventive properties. Furthermore, sulforaphane mediates a number of anticancer pathways, including the activation of apoptosis, induction of cell cycle arrest, and inhibition of NFκB”. (Source)

Image result for sulforaphane nitrile

Figure 1.  ESP converts into sulforaphane and sulforaphane nitrile  (Source)

Decreasing Epithiospecifier Protein Activity

A research paper entitled “Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli”, published in Phytochemistry in 2004, examined the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture.(Source)

Metabolism and Bioavailability of Glucosinolates

he hydrolysis of glucosinolates, which is catalyzed by a class of enzymes called myrosinases (β-thioglucosidases), leads to the formation of breakdown compounds, such as thiocyanates isothiocyanates, indoles, oxazolidine-2-thiones (e.g., goitrin), epithionitrile, and nitrile. In intact plant cells, myrosinase is physically separated from glucosinolates. Yet, when plant cells are damaged, myrosinase is released and comes in contact with glucosinolates, catalyzing their conversion into highly reactive metabolites. In plants, thiocyanates, isothiocyanates, epithionitrile, and nitrile are defensive compounds against pathogens, insects, and herbivores (Source). When raw cruciferous vegetables are chopped, glucosinolates are rapidly hydrolyzed by myrosinase, generating metabolites that are then absorbed in the proximal intestine. In contrast, boiling cruciferous vegetables before consumption inactivates myrosinase, thus preventing the breakdown of glucosinolates.

“When cruciferous are cooked before consumption, myrosinase is inactivated and glucosinolates transit to the colon where they are hydrolyzed by the intestinal microbiota. Numerous factors, such as storage time, temperature, and atmosphere packaging, along with inactivation processes of myrosinase are influencing the bioavailability of glucosinolates and their breakdown products. This review paper summarizes the assimilation, absorption, and elimination of these molecules, as well as the impact of processing on their bioavailability”. (Source)

A small fraction of intact glucosinolates may be absorbed in the small intestine, but a large proportion reaches the colon (Source). Of note, boiling cruciferous vegetables has also been found to reduce their glucosinolate content to a much greater extent than steam cooking, microwaving, and stir-frying do (Source). Nonetheless, when cruciferous vegetables are cooked, bacterial myrosinase-like activity in the colon is mainly responsible for glucosinolate degradation, generating a wide range of metabolites (Source).

A neutral pH may favor the formation of isothiocyanates from glucosinolates. Once absorbed, isothiocyanates, such as glucoraphanin-derived sulforaphane, are conjugated to glutathione in the liver, and then sequentially metabolized in the mercapturic acid pathway. Sulforaphane metabolites — sulforaphane-glutathione, sulforaphane-cysteine-glycine, sulforaphane-cysteine, and sulforaphane N-acetylcysteine — collectively known as dithiocarbamates, are ultimately excreted in the urine (Source).

 Bioavailability

The composition and content of glucosinolates in cruciferous vegetables are relatively stable, yet depend on the genus and species and can vary with plant growing and post-harvest storage conditions and culinary processing (Source). Since most cruciferous vegetables are cooked prior to eating, bacterial myrosinase-like activity in the gut rather than plant myrosinase is responsible for the initial step in glucosinolate degradation. In a feeding study involving 45 healthy subjects, the mean conversion rate of glucosinolates (of which 85% was glucoraphanin) to dithiocarbamates over a 24-hour period was estimated to be around 12% with wide variations among participants (range, 1.1 to 40.7%) (Source). In contrast, 70%-75% of ingested isothiocyanates were found to be metabolized to dithiocarbamates. Therefore, following the ingestion of cooked cruciferous vegetables, the conversion of glucosinolates into isothiocyanates by gut bacteria appears to be a limiting step in the generation of dithiocarbamates (Ibid). However, differences in individuals’ capacity to metabolize glucosinolates have not been linked to differences in gut microbiota composition (9).

The Macrobiota Connection

Crucifers contain very high concentrations of glucosinolates (GS; β-thioglucoside-N-hydroxysulfates). Although not themselves protective, GS are converted by coexisting myrosinases to bitter isothiocyanates (ITC) which defend plants against predators. Coincidentally, ITC also induce mammalian genes that regulate defenses against oxidative stress, inflammation, and DNA-damaging electrophiles. Consequently, the efficiency of conversion of GS to ITC may be critical in controlling the health-promoting benefits of crucifers. If myrosinase is heat-inactivated by cooking, the gastrointestinal microflora converts GS to ITC, a process abolished by enteric antibiotics and bowel cleansing.  (Source)

Heating Broccoli for a short time at 60 Celsius

Researchers experimented with cooking broccoli at different temperatures and at different times periods.  They then measured the point at which the epithiospecifier protein is destroyed. What they learned was that they only had to heat the broccoli for a short time in order to destroy the epithiospecifier protein thus yielding more sulforaphane and little to no sulforaphane nitrile.

Specifically, to maximize the sulforaphane in broccoli, they found that heating it for 10 minutes at 140 degrees Fahrenheit (60 degrees Celsius). This can translate to steaming broccoli lightly for about 3 to 4 minutes.

When they heated the broccoli for 10 minutes at 158 degrees Fahrenheit (70 degrees Celsius) it not only destroyed the epithiospecifier protein but also the sulforaphane content. So cooking too much destroys this important compound. (Ibid)

Broccoli sprouts

Broccoli sprouts are germinated from broccoli seeds for about 3 days minimum and 5 days maximum. Broccoli sprouts that are 3 days old have very concentrated sources of glucoraphanin.  It is estimated that broccoli sprouts have 10 to 100 times more glucoraphanin by weight than mature broccoli plants. (Source)

Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens.

“Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety.”  (Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10367-72.)

Three-days old broccoli sprouts are concentrated sources of glucoraphanin, which is the precursor to sulforaphane. Fresh broccoli sprouts contain 10 to 100 times more glucoraphanin by weight than mature broccoli plants.  Fresh broccoli sprouts can contain at least 73 mg of glucoraphanin (also called sulforaphane glucosinolate) per 1-oz serving(Source)

The conclusion that three-day-old broccoli sprouts are highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats has been established for a long time. But the drug companies are not interested in testing this nutrient in humans. Yet, other forms of evidence shows that small quantities of crucifer sprouts protects against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety. (Source)

The activity of the epithiospecifier protein (ESP) in broccoli sprouts fluctuates based on the number of days the sprouts have grown.  (Source) ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. Thus, the optimal amount of days to grow broccoli sprouts is probably 5 days since the amount of glucoraphanin in broccoli seeds remains more or less constant as those seeds germinate and grow into mature plants. (Source)

Heating Broccoli Sprouts at 70 Celsius  will Increase Sulforaphane and maximize its anti-cancer effects

When the researchers heated broccoli sprouts for 10 minutes at 158 degrees Fahrenheit (70 degrees Celsius) in water, it minimized the epithiospecifier protein and maximized the sulforaphane content.

Heating broccoli sprouts in water under these exact specifications will increase the sulforaphane content for maximum anti-cancer benefit. Steaming is the preferred mode of light cooking.

Freezing Broccoli Sprouts Increases Sulforaphane Yield

A study from 2015 published in the journal RSC Advances by researchers from the College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China and the College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, People’s Republic of China, investigated whether freezing broccoli sprouts would have an effect on glucoraphanin and ascorbic acid content, myrosinase activity, sulforaphane and sulforaphane nitrile formation. (Source) In this study, the researchers froze broccoli sprouts at −20 °C (DF-20), −40 °C (DF-40) and −80 °C (DF-80) or stored at −20 °C (LN-20), −40 °C (LN-40) and −80 °C (LN-80) after being frozen in liquid nitrogen for 5 min or always frozen in liquid nitrogen (LN).

The results showed the following: glucoraphanin content was not significantly affected by freezing ; myrosinase activity was enhanced; sulforaphane yield  was increased by 1.54–2.11 fold; sulforaphane nitrile formation decreased; ascorbic acid content was decreased.

By freezing fresh broccoli sprouts, sulforaphane can be significantly increased by on average 1.825 times its original value when fresh and not frozen. The only downside of the freezing process is the decrease of ascorbic acid. But that can always be remedied with vitamin C supplementation of even better with a whole food diet rich in ascorbic acid.

How To use Frozen Broccoli Sprouts

It is recommended to use the frozen broccoli sprouts directly from the freezer into the smoothie jar. To get maximum effect, they should not be allowed to thaw.

Raw Cruciferous Vegetables: small amounts are enough.

Each of the vegetables, sprouts and seeds contain the enzyme myrosinase, which is activated when the vegetable, sprout or seeds is damaged (chopped or chewed) in the presence of water.  The glucosinolate converts to an isothiocyanate (or thiocyanate) through the enzymatic activity of myrosinase.  These isothiocyanates are the defensive substances of the plantThus glucosinolates are the precursors to isothiocyanates through the breakdown of the enzyme myrosinase.

Myrosinase activity on the glucosinolate also continues in the gastrointestinal tract through intestinal bacteria which allows for some further formation and absorption of isothiocyanates. (Source)  To release this enzyme, it is best to chew abundantly on some organic cruciferous.

Polymorphism Variable

There is also a genetic variant with regard the proper activation of the detox pathways and the use of cruciferous vegetables.  In one study, cruciferous vegetable intake was inversely associated with RCC risk, and this risk was modified by GSTT1 and GSTM/T1 combined genotypes. Risk was only significantly elevated when intake was low. (Source). In yet another study, higher cruciferous vegetable intake reduced lung cancer risk, but only among individuals with the GSTM1 present genotype.  (Source) However, in these studies, the cancer risk was only significant when the intake of cruciferous veggies was low, like once a month. By eating these vegetables multiple times a week under the conditions indicated above, cancer risks significantly subside.

Conclusion

While the entire family of cruciferous plants, including mustard seeds are valuable insofar as sulforaphane is concerned, both broccoli and especially broccoli sprouts have the biggest therapeutic bang provided the cooking, freezing and proper chewing (or chopping) of its raw form in order to release myrosinase is holistically performed.

For a healthy lifespan, there are few other families than the Cruciferous family that can help us to achieve a optimized detoxification, one that is all the more necessary that we are each day bombarded with toxicants.

Ch Joubert (H.M Institute Director)

Reference and Precision Notes

(1). Sulphoraphane is one of the important nutrients that is needed for the body’s metabolic detoxification pathways. There are 6 Phase II detoxification pathways in the body.  Each conjugation pathway serves a specific purpose of detoxifying certain toxins and requires specific nutrients to function.  These 6 detoxification pathways include: Glutathione conjugation Methylation Sulfation Acylation/Glycation Acetylation Glucuronidation These 6 conjugation pathways are found primarily in the liver, but also in various other locations within the body, including, but not limited to the kidneys, intestine, lung, skin, prostate, spleen, gastric mucosa CNS. (Source) After a toxin (xenobiotic) has gone through the process of becoming hydrophilic (water-soluble) through reactions overseen by Phase I enzymes, its intermediate molecules are then conjugated with endogenous hydrophilic enzymes. These endogenous enzymes include: glucuronic acid (glucuronyl transferases) sulfate (sulfotransferases) glutathione (glutathione transferases) amino acids (amino acid transferases) acetyl group (N-acetyl transferases) methyl group (N- and O-methyltransferases) (Source)
(2). Cruciferous vegetables include broccoli sprouts, broccoli, cauliflower, Brussels sprouts, cabbage and kale, and in addition to providing sulforaphane, they are some of the richest food sources of glutathione. Brussels sprouts are the richest in glutathione, followed by cauliflower, broccoli (particularly the flowers, not the stem), cabbage, kale, bok choy, cress, mustard, horseradish, turnips, rutabagas, and kohlrabi. Brussels sprouts have also been shown to rejuvenate liver cell function.
(3). International Agency for Research on Cancer. Cruciferous vegetables. Cruciferous vegetables, isothiocyanates and indoles. Lyon, France: IARC; 2004:1-12.
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