MYROSINASE ENZYME FROM BRASSICACEAE PLANT SOURCES

RELATED APPLICATIONS

This application claims priority from NZ589578 dated 29 November 2010, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The application relates to a cancer chemoprotective product. More specifically, the application relates to a product containing both a source of glucoraphanin and/or giucoraphanen compound and myrosinase enzyme and which is stable yet provides chemoprotective activity to a subject on administration.

BACKGROUND ART

Cancer is an illness that affects countless people, either directly where they themselves have the illness or indirectly where a relative or friend may be affected. Given the increasing prevalence of cancer, many therapies are being developing to prevent and/or treat cancer.

In addition to their high nutritional value, cruciferous vegetables (e.g. broccoli, cabbage, kale) are known to have a potential role in cancer chemoprevention, since they are rich in aromatic and aliphatic glucosinolates. Glucosinolates are secondary metabolites in plants from the Brassicaceae family. Research has shown that when glucosinolates are degraded in the stomach of animals via the enzyme myrosinase, their metabolites can counteract the formation of tumour ceils. Some glucosinolate breakdown products, especially some isothiocyanates such as sulforaphane (4-methylsulphinyl-3-butenyl) have shown this special potential role in cancer chemoprevention. In 1992, sulforaphane, a substance produced by the body from a compound in broccoli was shown to trigger the production of phase II enzymes in the body, an important known physiological mechanism in preventing cancer tumour formation.

One existing product that is sold as a chemoprotective agent is a powder containing 10% of glucoraphanin extracted from broccoli sprouts. This product does not contain myrosinase, instead leaving the gut to provide the myrosinase and produce the subsequent downstream isothiocyanates that then engage phase II enzyme production.

A reason for not supplying the GR breakdown metabolites themselves e.g. sulforaphane is that these isothiocyanates lack stability and may quickly further metabolise with the benefits being lost. G on the other hand is storage stable. Brassica Vegetables and Seeds

Vegetables are considered to be an essential part of a balanced diet. The Brassica vegetables are rich sources of dietary fibre, many are good sources of calcium, provitamin A, vitamin C and certain beneficial phytochemicals.

Broccoli is a plant of the cabbage family Brassicaceae (formerly Cruciferae). It is classified as a cultivar group of the species Brassica oleracea. and comprises a large and diverse group of widely consumed vegetables with the key relationships illustrated below:

|— Arabidopsis Campestris Acephala

(Thule cress) (Turnip rap; field mustard) (Kale)

Armoracia Carinata Botrytis

(Horseradish) (Ethiopian mustard) (Cauliflower)

— Brassica Juncea Capltata

(Mustard greens) (Turnip)

|— Capsella

(Shepard's purse) Napus Gemmifera

(Oilseed rape, (Brussels sprouts)

[— Cardaria canola, rutabaga)

(Hoary cress)

Oleracea— Itallca

Cruciferae Eruca (Hoary cress) (Broccoli)

(Arugula)

Rapa— Gongylode

Lepidium (Kohlrabi)

(Peppergrass;cress)

Sabellica

Nasturtium (Collards)

(Watercress) — Chinensis

(Pak Choi)

Raphanus

(Radish; Diakon) Pekinensis

(Chinese cabbage)

|— Sinapis

(Mustardseed) Raplfera

(Turnip)

Thlaspi

(Pennycress)

Within the seed, there usually is a store of nutrients for the seedling that will grow from the embryo. The form of the stored nutrition varies depending on the kind of plant. For example, the major components in Brassica vegetable seeds include lipids, protein, water, enzymes, secondary plant metabolisms, minerals and vitamins. However, brassica family seeds also contain several unusual glucosinolates, acids, proteins or enzymes.

Glucosinolates and Isothiocyanates

One of the common characteristics of brassica vegetables is that they all contain glucosinolates, being sulphur-containing compounds that may be hydrolysed to produce isothiocyanate metabolites. Accurately described, glucosinolates are beta-thioglucoside-N-hydroxysulfates and are primarily found in cruciferous vegetables (for example cabbage, broccoli, broccoli sprouts, brussel sprouts, cauliflower, cauliflower sprouts, bok choy, kale, collards, arugula, kohlrabi, mustard, turnip, red radish and watercress).

Glucosinolates are understood to be key biologically active components in brassicas, described as being antioxidants, boosting immune health, respiratory health and cardiovascular health.

HPLC analysis of broccoli seeds reveals that three aliphatic glucosinolates (glucoraphanin, glucoiberin and glucoerucin) and a group of indol-glucosinolates including 4-hydroxy- glucobrassicin are preformed in the seeds. Myrosinase enzyme breaks the β -thioglucoside bond of the glucosinolate molecules, producing glucose, sulphate, and a diverse group of aglucon products.

Depending on the structure of the specific glucosinolates and the existing reaction conditions, isothiocyanates and nitriles usually constitute the majority of these aglucons. One of the reaction products from the enzymatic reaction of glucoraphanin is sulforaphane (SF). Other reaction products are nitriles, such as sulforaphane-nitrile (SF-nitrile) and thiocyanates. The reaction schematic below summarises the reactions occurring:

Upon hydrolysis by the myrosinase the glucosinolate forms glucose and an unstable intermediate (aglucon), thiohydroximate-O-sulfate. This step is pointed out above as step 1 This intermediate aglucon then undergoes further transformation. The structure of the final products being dependent on myrosinase interacting proteins (e.g. epithiospecifier proteins (ESP)), co-factors, presence of metal ions, physical circumstances such as pH-value and temperature. However, a nitrile analogue to sulforaphane, sulforaphane nitrile (step 3) [5- (methylsulfinyl)pentane nitrile] may actually be the pre- dominant hydrolysis product of glucoraphanin. Sulforaphane nitrile has recently been shown not to possess the

anticarcinogenic properties of sulforaphane. Thus the potential health benefit of broccoli as a result of the second reaction product formation, an isothiocyanate called sulforaphane, is compromised by the alternative formation of an inactive nitrile. The reaction to the component sulforaphane, with its high anti-cancer potential, is shown in the schematic above as step 2.

Glucoraphanin and Glucoraphanen

Glucoraphanin (GR), a secondary metabolite of brassica plants, is the most common glucosinolate in broccoli, broccoli sprouts and broccoli seeds. The molecular weight of GR [4- (methylsulfinyl) butyl glucosinolate] is 436.5 and the chemical structure is shown in below:

O

II S -β- D-glucose

S- CH ₂ - CH ₂ - CH ₂ - CH ₂ - C

H ₃ C ^ NOS0 ₃

Glucoraphanin can be split in the presence of myrosinase into glucose, an isothiocyanate (sulforaphane) and a nitrile (sulforaphane-nitrile).

Glucoraphanen is a very similar glucosinolate found in at least daikon and daikon seed (from the species Raphanus sativus). This compound undergoes the same enzymatic reaction in the presence of myrosinase except that the metabolites from glucoraphanin are termed sulforaphene and sulforaphene nitrile.

Sulforaphane and Sulforaphene

Sulforaphane [4-(methylsulfinyl) butyl isothiocyanate] is the aglucon breakdown product of the glucosinolate glucoraphanin, also known as sulforaphane glucosinolate (SGS). Sulforaphane, with a molecular weight of 177.3, is produced from sulforaphane glucosinolate via the action of the enzyme myrosinase (thioglucoside glucohydrolase), an enzyme present in cruciferous vegetables that is activated upon maceration of the vegetables. As mentioned, the enzymatic breakdown reaction of glucoraphanen shows the same reaction steps as glucoraphanin. The isothiocyanate from glucoraphanen is termed sulforaphene. The respective chemical structures are shown below:

O

Sulforaphene

O

I I

Sulforaphane

N =C = S Both compounds have nearly the same chemical structure and similar phase II enzyme induction capacity. The major difference is a double bond in the chemical structure of sulforaphene.

Purified suiforaphane from broccoli is now commercially available for biological research from several companies, but its high price puts limitations on the amount that can be used.

Suiforaphane Nitrile and Sulforaphene Nitrile

Nitriles are other reaction products of the enzymatic glucoraphanin reaction. The reaction of glucoraphanin ends partly in a nitrile called suiforaphane nitrile [5-(methylsulfinyl) pentane nitrile], shown below:

O

II

S - CH ₂ - CH ₂ - CH,- CH, - C≡N

The molecular weight of this reaction product is 145.2. The breakdown conditions during the reaction of glucoraphanin influences the quantity of nitrile formed.

A number of studies have demonstrated that suiforaphane yield from glucoraphanin is low and that a non-bioactive nitrile analogue, suiforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase.

Production of nitriles has been found to be undesirable as these compounds are inactive.

Sulforaphene nitrile has the same properties as suiforaphane nitrile and is the reaction product of glucoraphanen.

Enzymes (Myrosinase)

Myrosinase enzyme is critical to the activation of GR and subsequent production of isothiocyanates, one of which is suiforaphane that is of interest in chemoprotective activity.

Myrosinase (β -thioglucosidase) is present in all plants containing glucosinolates. Many of these plant species are frequently used in the human diet either as vegetables or condiments (cabbage, brussel sprouts, radish, turnip, watercress, mustard). Myrosinase catalyses the hydrolysis of glucosinolates to give D-glucose and an unstable aglycone fragment after cleaving the thioglucosidic bond. The aglycon fragment spontaneously rearranges to give sulphate and a series of sulphur or nitrogen-containing compounds. Myrosinase is located outside the myrosin cells and has the tendency to adhere to membrane surfaces, whereas glucosinolates are found in the myrosin cells. Therefore, myrosinase and glucosinolates do not come in contact unless the seed tissues are damaged.

Experiments where myrosinase is extracted or where seeds containing myrosinase are damaged results in rapid reaction / deactivation and disappearance of myrosinase enzyme.

Fatty Acids (Erucic acid)

Table 1 below shows the distributions of typical fatty acids in broccoli sprouts and seeds. The fatty acid distributions (FADs) of lipid-containing extracts noted were determined by gas chromatography of the fatty acid methyl esters (FAMEs).

Table 1: Typical fatty acid distributions of broccoli sprout and seeds (% total FAMEs).

Broccoli sprout Broccoli seed Broccoli seed

Fatty acid * Total lipid Total lipid Hexane lipid

14:0 0.15 0.05 0.12

14:1 0.01 0.01 0.01

15:0 0.06 0.02 0.03

16:0 6.19 4.02 3.87

16:lwll 1.07 0.08 0.07

16:lw9 2.26 0.46 0.24

16:3 1.08 0.12 0.11

Anteiso-17:0 0.03 0.01 0.01

17:0 0.08 0.02 0.02

18:0 0.56 0.64 0.72

18:lw9 8.34 9.06 8.85

18:lw7 1.78 1.42 1.21

18:2w6 17.28 11.87 11.31

18:3ω3 20.99 13.10 13.10

Anteiso-19:0 0.01 0.01 0.01

20:0 0.32 0.41 0.40

20:lw9 1.81 4.19 4.29

20:lw7 0.72 1.96 1.96

20:2 0.32 0.49 0.50

20:4w6 0.61 0.17 0.14

20:5ω3 0.01 0.01 0.01

21:0 0.02 0.01 0.01

22:0 0.67 0.56 0.57

22:lw9 30.40 45.56 46.89

22:lw7 0.78 1.82 1.52

22:2 0.47 0.95 0.98

24:0 0.60 0.34 0.35

24:lw9 1.27 1.79 1.86

Unidentified 2.09 0.90 0.89

* Values expressed as FAMEs

Based on their potential hazard for human health, the mostly important fatty acid in broccoli seed is erucic acid. Broccoli seeds contain very high concentrations of erucic acid (see Table 2 below) hence the need to avoid erucic acid. Table 2: Erucic acid content in broccoli sprouts and seeds

Sample % Liquid* mg Erucic Acid / 100g ■ ^! **

Sprouts 0.11 320

Seeds (Folch extraction) 28.1 12230

Seeds (Hexan extraction) 26.9 12050

* fresh weight basis

** % lipid x % methyl erucate from FAD x 298.5

(M.W. erucic acid)/312.5 (M.W.methyl erucate)

x 10

In more detail, erucic acid is a monounsaturated ω-9 fatty acid, denoted 22:1 ω-9. Erucic acid is produced naturally across a great range of green plants, but especially so in members of the brassica family, for example broccoli seed. Erucic acid is also known as cis-13-docosenoic acid.

The trans isomer is well established as brassidic acid. Erucic acid exhibits a molar mass of 338.57g, molecular density of 0.860g/cm ³ , a melting point of 33.8°C and a boiling point of 381.5°C. The boiling point of erucic acid is defined as the point of acid degradation. Erucic acid is insoluble in water but soluble in ethanol and methanol. Seeds often contain chemical compounds to discourage herbivores and seed predators. In some cases, these compounds simply taste bad, but other compounds are toxic or break down into toxic compounds within the digestive system.

In the human body, erucic acid is broken down into shorter-chain fatty acids by enzymes (long- chain acyl-coenzyme A (CoA) dehydrogenase). Erucic acid is a natural component of some oils and has been shown to cause fatty deposits in the hearts of test animals, but this effect disappears over time once erucic acid is removed from their diet. The evidence of health effects in animals means that it is possible that frequent and regular consumption of high levels of erucic acid may add to the risk of developing heart disease in humans.

The levels of erucic acid in human foods are restricted, in part, over concerns that it may adversely harm heart tissue. Consequently, the 'Erucic Acid in Food Regulations 1977' (S.I. 1977 No. 691 [as amended]) limit the erucic acid content of foods to no more than 5% of the total fatty acid, in products with more than 5% fat. The US Food and Drug Administration (FDA) set a 2% limit on the erucic acid content.

As should be appreciated from the above, whilst glucoraphanin provides a stable source of the chemoprotective agent sulforaphane or sulforaphene, conversion to sulforaphane or sulforaphene is dependent on the presence of myrosinase enzyme. Myrosinase enzyme is present in the gut but may be present at differing levels and activities depending on individual metabolism. Controlling and driving the reaction between stable glucoraphanin or

glucoraphanen and myrosinase to produce known and measurable levels of sulforaphane or sulforaphene is desirable. Similarly avoiding other inactive breakdown products, maintaining stability and avoiding undesirable compounds is also advantageous.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents.

For the purpose of this specification, and unless otherwise noted, the term 'comprise' or grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

Further aspects and advantages of the compositions methods and uses thereof will become apparent from the ensuing description that is given by way of example only.

SUMMARY

The application broadly relates to the co-administration of a glucoraphanin and/or

glucoraphanen (GR) extract with a myrosinase enzyme containing plant seed. The resulting coadministered composition delivers a known and measurable amount of chemoprotective agent sulforaphane and/or sulforaphene to the subject on consumption and is storage stable.

In a first aspect there is provided a composition including:

a. an extract containing at least one glucosinolate compound derived from a first plant source; and

b. an extract, plant, or plant part thereof containing myrosinase enzyme derived from a second plant source.

In a second aspect, there is provided a method of converting a glucosinolate compound on delivery to a subject to a bioactive isothiocyanate metabolite by administration of a composition substantially as described above to a subject.

In a third aspect, there is provided a method of inducing phase II enzyme production within a subject by administration of a composition substantially as described above to the subject.

In a fourth aspect, there is provided the use of the composition substantially as described above in the manufacture of a food product that, on eating, induces phase II enzyme production by the subject's metabolism.

Advantages of this combination include being able to react myrosinase enzyme with the glucosinolate compound(s) at the time of administration thereby removing reliance on the subject/patient metabolism to produce the desired isothiocyanate metabolites. Selection of the appropriate amount of myrosinase and selection of raw material sources also ensures that full conversion occurs and that undesirable compounds are not present and/or not produced in the metabolic reaction. The myrosinase source may also contain further chemoprotective agents as well.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the application will become apparent from the following description that is given by way of example only and with reference to the accompanying drawings in which: Figure 1 shows an example of a two layer tablet (A) based on two types of powder containing active myrosinase and glucoraphanin and/or glucoraphanen;

Figure 2 shows an example of a two chamber capsule (B) based on two types of

powder containing active myrosinase and glucoraphanin and/or glucoraphanen; and Figure 3 shows an example of a functional food based on use of a powder

containing glucoraphanin and/or glucoraphanen in a muesli bar with seeds containing active myrosinase.

DETAILED DESCRIPTION As noted above, the application broadly relates to the co-administration of a glucoraphanin and/or glucoraphanen (GR) extract with a myrosinase enzyme containing plant seed. The resulting co-administered composition delivers a known and measurable amount of chemoprotective agent sulforaphane and/or sulforaphene to the subject on consumption and is storage stable. For the purposes of this specification, the term 'extract' refers to the compound processed from a state that occurs in nature.

The term 'plant or plant part thereof refers to a plant itself or parts of the plant including some or all parts of the plant including the flowers, leaves, stems, branches, roots, seeds and combinations thereof. The term 'converting' and grammatical variations thereof refers to the reaction of an inactive precursor compound to a chemoprotective compound or compounds.

The term 'metabolite' refers to products of the reaction of an inactive precursor compound e.g. glucoraphanin to products produced during hydrolysis including sulforaphane. A 'bifunctional inducer' is a molecule which increases activities of both phase I enzymes such as cytochromes P-450 and phase II enzymes and requires the participation of aryl hydrocarbon (Ah) receptor and its cognate Xenobiotic Response Element (XRE). Examples include flat planar aromatics such as polycyclic hydrocarbons, azo-dyes or 2,3,7,8-tetrachloro-dibenzo-p- dioxin (TCDD).

A chemoprotector or chemo-protectant is a synthetic or naturally occurring chemical agent that reduces susceptibility in a mammal to the toxic and neoplastic effects of carcinogens (has chemoprotective effects).

Inducer activity or phase II enzyme inducing activity is a measure of the ability of a compound(s) to induce phase II enzyme activity, particularly that linked to chemoprotective effects. Methods of measuring phase II enzyme inducing activity are described in at least US 7,303,770 incorporated herein by reference.

The term 'bioactive' refers to the compounds or compounds having an activity for example reacting with bioactive compounds, producing a chemoprotective effect or inducing phase II enzyme production.

The term 'subject' and 'patient' are used interchangeably and refer to the animal (including humans unless otherwise specified) to which the composition may be administered.

The term 'grain based food' refers to foods that contain predominantly (greater than 50% wt) of grains. The term 'functional food' refers to foods that have been formulated to confer an added nutritional benefit besides that of eating the food as it normally is formulated. For example, a functional food may be bread containing the composition of the embodiments described below.

In a first aspect there is provided a composition including: a. an extract containing at least one glucosinolate compound derived from a first plant source; and

b. an extract, plant, or plant part thereof containing myrosinase enzyme derived from a second plant source.

The first and second plant sources may be from different plants. This has the advantage of being able to select the desired active compounds and avoid potential harmful or undesired compounds. This point is discussed further below.

The glucosinolate compound or compounds may be glucoraphanin and/or glucoraphanen. Glucoraphanin and glucoraphanen are known in the art to be derived from various Brassicaceae family plants. The activity of at least the key metabolites when reacted with myrosinase enzyme is essentially the same, the main difference being a double bond present in the glucoraphanen. Chemoprotective activities have been detected in certain vegetables that are able to induce the activity of enzymes that detoxify carcinogens (phase II enzymes). One such activity has been detected in broccoli that induces quinone reductase activity and glutathione S-transferase activities in murine hepatoma cells and in the organs of mice. This activity has been purified from broccoli and identified as sulforaphane. In addition, analogues of sulforaphane have been synthesised to determine structure function relationships.

Glucosinolates generally including glucoraphanin and glucoraphanen are relatively stable compounds when stored at room temperature and these can be extracted easily from their raw materials. Their activities, particularly in respect of the myrosinase catalysed breakdown is well understood and are known to be the precursor compounds in the formation of isothiocyanate metabolites sulforaphane and sulforaphene, being known phase II enzyme inducing compounds and therefore cancer preventative (chemoprotective) compounds. Glucoraphanin is typically used ahead of glucoraphanen owing to its greater prevalence in raw materials including broccoli.

The extract from a first Brassicaceae plant family source described above contains substantial quantities of phase II enzyme inducer and is essentially free of phase I enzyme inducers i.e. it is not a bifunctional inducer.

Optionally, the extract may be in the form of a dried powder. Existing methods of production involve the concentration and drying of glucosinolate compounds, the drying methods including spray drying, vacuum drying and/or freeze drying. The water activity of the powder may be less then 0.7. The powder may be ground to have a particle size less than 1 mm although granules of up to 5mm are included within the use of the term 'powder' for the purposes of this specification.

As noted above, the first plant source may be derived from the Brassicaceae family. More specifically, the first plant source may be derived from the species Brassica oleracea. Still more specifically, the first plant source may be broccoli sprouts. As noted above, the Brassicaceae family contain a variety of compounds specific to this family of plants including glucosinolates. Brassica oleracea species include kale, cauliflower, turnip, brussel sprouts, broccoli, kohlrabi and collards to name a few. Broccoli, and in particular the sprouts of broccoli, are known to have some of the higher concentrations of particularly glucoraphanin compounds and they are easy to grow to an economically commercial scale.

US 6,242,018 B1 , incorporated herein by reference, illustrates the improved activity of sprouts versus the mature plant. It should be noted that US 6,242,018 B1 shows that daikon plants have phase II enzyme inducer effects however no teaching or suggestion is made of the myrosinase activity of the daikon. Significant art already exists in relation to brassica extracts and their chemoprotective aspects e.g. US 5,968,567, US 5,968,505, US 5,725,895, US 5,411 ,986, US 6,242,018, US 6,177,122, US 6,521 ,818, US 7,303,770 and US 2009/0247477 all incorporated herein by reference. The composition may contain at least 1 % wt glucosinolate compounds. The composition may contain at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% wt glucosinolate compounds. These levels of glucosinolate are known to be sufficient to induce phase II enzyme induction activity.

Glucoraphanin and/or glucoraphanen may comprise greater than 50% wt of the glucosinolates present in the glucosinolate extract. Glucoraphanin and/or glucoraphanen may comprise greater than 55%, 60%, 65%, 70%, 75%, 80%, 85% wt of the glucosinolates present in the glucosinolate extract. As noted in the art, while glucosinolate compounds may have a variety of activities, glucoraphanin and glucoraphanen are known compounds of interest in respect of in- vivo phase II enzyme induction.

The myrosinase enzyme containing seed may be in the form of a whole seed or partly or fully crushed or powdered.

The seed or parts thereof may be dried. Drying methods may include spray drying, vacuum drying and/or freeze drying. The water activity of the seed or part thereof after drying may be less then 0.7. The seed may be ground to have a particle size less than 1 mm although granules of up to 5mm are included within the use of the term 'powder' for the purposes of this specification.

Whole plant seed containing myrosinase enzyme may be advantageous. Forming extracts requires processing which inevitably requires resources in terms of labour, equipment and energy. Powders can be useful in that they have a high surface active area but, in the case of myrosinase, are not stable when mixed directly with a glucosinolate compound and often reactions may occur before the time of administration. Administration of plant seeds (whole or in coarse parts) keeps the myrosinase intact and prevents metabolites being produced prior to administration.

As should be appreciated, powdered seed derived myrosinase may still be used but ideally needs to be kept separate to the glucosinolate extract until ready for use. The alternative of incorporating a seed (whole or in part) containing myrosinase avoids stability issues as myrosinase is very stable when in the form of a seed and can be mixed with a glucosinolate powder without risk of reaction occurring. In addition seeds are comparatively stable, occur naturally (i.e. minimal processing required) and they can be 'activated' by the user on administration e.g. by chewing the seeds and breaking apart the structure to release myrosinase enzyme.

The extract, plant, or plant part thereof may not contain erucic acid. As discussed in the background, erucic acid is an undesirable fatty acid present in some plant materials. The presence of erucic acid may prevent certain plant materials from being used as a myrosinase source. For example, broccoli seeds contain both myrosinase enzyme and high quantities of erucic acid. For broccoli seeds to be useful as myrosinase source, the seeds would need to be processed to remove or reduce the quantity of erucic acid. This is undesirable as additional processing increases costs due to extra labour, equipment and energy expenses. Plant materials that contain myrosinase and do not contain erucic acid are therefore preferable.

Further, the extract, plant, or plant part thereof may contain insufficient epithiospecifier protein (ES protein) to cause more than 10% production of isothiocyanate nitriles. More specifically, the extract, plant, or plant part thereof contains insufficient epithiospecifier protein (ES protein) to cause more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % production of isothiocyanate nitriles. As noted in the background, ES protein causes the production of isothiocyanate nitriles that have been found to be inactive chemo protective agents. Selection of materials that contain myrosinase enzyme and that do not contain ES protein are therefore desirable to limit or avoid the production of isothiocyanate nitriles. This consequently maximises for example the production of sulforaphane (active) and minimises the production of sulforaphane-nitrile (inactive).

The myrosinase enzyme containing plant seed may be derived from the species Raphanus sativus. One particular plant source for myrosinase enzyme that the inventors have identified is the variety of Raphanus sativus var. longipinnatus or 'daikon'. Daikon is a mild-flavoured large white East Asian radish. There are many varieties of daikon. Daikon is rich in myrosinase enzyme. Daikon is also advantageous as it does not include erucic acid and does not contain ES protein. As a result, daikon provides rich source of myrosinase without the drawbacks of containing erucic acid (harmful) and ES protein (unwanted as it promotes production of inactive compounds).

While removing the compounds responsible for this radish-like taste and heat or adding flavourings to mask the taste and heat may be possible, the taste effect may be desirable as it provides the consumer with positive feedback of a reaction occurring. Many nutritional formulations are sold with supporting literature explaining the uses and advantages of the formulations but often the actual effects are subtle or difficult to see, particularly over the short term. The combination of a glucosinolate and daikon has the advantage of providing a very real and noticeable feedback to the consumer that something is indeed happening when the composition is taken orally.

The composition may contain sufficient myrosinase enzyme to complete at least 90% conversion of the glucosinolate compound(s) to isothiocyanate metabolites. In further embodiments, the composition may contain sufficient myrosinase to complete at least 91 %, 92%, 93%, 94%, 05%, 96% conversion of the glucosinolate compound(s) to isothiocyanate metabolites. As should be apparent, to make the most of the raw material glucosinolate, it may be desirable to convert the majority or all of the glucosinolate into active isothiocyanate metabolites to maximise the chemoprotective aspect of the composition. In one embodiment, all of the glucosinolate compounds present as either glucoraphanin or glucoraphanen may be fully converted to sulforaphane or sulforaphene and potentially other compounds. Conversion to sulforaphane analogues may also occur. Examples of sulforaphane analogues may be found in at least US 2009/0247477 A1.

The composition may include a ratio of approximately 60% wt to 80% wt glucoraphanin and/or glucoraphanen to approximately 20% wt to 40% wt myrosinase enzyme. More specifically, the ratio may be approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% wt glucoraphanin and/or glucoraphanen to approximately 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35% wt myrosinase enzyme. It should be appreciated that the exact ratio may vary depending on the amount of carriers and other components within which the composition is mixed and hence should not be seen as limiting. However, the inventors have found that this ratio may be an optimum to ensure full conversion of the glucoraphanins and/or gluraphanens whilst avoiding waste of myrosinase by including an excessive amount of myrosinase.

The inventors have found that the composition is stable. The term 'stable' in respect of this specification means that the speed and degree of reaction of the composition, in converting glucosinolates compounds to their isothiocyanates remains the approximately same (within approximate^ 5% of a hypothetical maximum conversion) between an un-stored composition and the same composition stored at room temperature in an air tight atmosphere. The composition may be storage stable for a time period of at least 2-months, 4-months, 6-months, 8-months, 10-months, or 12-months.

The composition may be formulated for transdermal or oral administration. The composition may be formulated using known pharmaceutically acceptable carriers and substances to form a pill or capsule, a liquid drink and various solids. The term 'pharmaceutically acceptable carriers' is defined in detail in US 2009/0247477 incorporated herein by reference.

The composition may be a food product containing the composition. Functional foods are common on the market where the food product contains an added nutritional benefit, in this case being the induction of chemoprotective phase II enzymes. The inventors envisage that functional foods may be one key way to administer the composition. Alternatively, the composition may be in the form of a pill or capsule.

The food product may be a dietary composition. The term 'dietary composition' is any ingestible preparation including sulforaphane, isothiocyanates, glucosinolates or analogues thereof. For example sulforaphane, isothiocyanates, glucosinolates or analogues thereof may be mixed with a food product. The food product can be dried, cooked, boiled, lyophilised, or baked. Breads, teas, soups, cereals, salads, sandwiches, sprouts, vegetables, animal feed, pills and tablets are among the vast range of different foods contemplated.

Selected non-limiting examples of dietary compositions include a muesli bar, a cereal, and a biscuit or cookie. Low water activity foods may be preferable to avoid risk of microbial growth during storage. In one embodiment, the water activity may be less than approximately 0.7.

In a second aspect, there is provided a method of converting a glucosinolate compound on delivery to a subject to a bioactive isothiocyanate metabolite by administration of a composition substantially as described above to a subject.

It should be appreciated that although the gut of a subject may complete the enzymatic conversion, a person (or animals) gut metabolism varies and it would be preferable to deliver a combination at the time of administration that provides a known does and controlled reaction. Use of myrosinase enzyme delivered with glucosinolate ensures that the reaction occurs and ensures that the production of isothiocyanates occurs to the full extent possible or desired.

In a third aspect, there is provided a method of inducing phase II enzyme production within a subject by administration of a composition substantially as described above to the subject. In the above aspect, the phase II enzymes may be chemoprotective agents.

In a fourth aspect, there is provided the use of a composition substantially as described above in the manufacture of a food product that, on eating, induces phase II enzyme production by the subject's metabolism

In the above methods and use, the act of chewing the composition in the subject's mouth results in release of myrosinase enzyme and subsequent conversion of glucoraphanin or

glucoraphanen to sulforaphane or sulforaphene. Conversion typically occurs in part or in full in the subject's mouth immediately prior to swallowing meaning the subject obtains a measurable and well defined dose that is not reliant on the subject's individual metabolism to perform the conversion. Since individual metabolisms vary in the amount of myrosinase present, the above methods and use reduce the variation in dose that is inherent to individual variation.

In the above methods and use, the animal may be a human. Alternatively, the animal may be a non-human animal.

The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relates, such known equivalents are deemed to be incorporated herein as of individually set forth,

Where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

The above described compositions and related methods and uses are now described by reference to working examples illustrating embodiments and variations thereof. WORKING EXAMPLES

The application is now described with reference to examples illustrating embodiments of the composition.

EXAMPLE 1

In this example, sources of myrosinase were tested for their suitability. Seeds from the Brassicaceae family were analysed to find an ideal source of active myrosinase.

An ideal source of myrosinase was found in ground daikon powder. The daily intake of ground daikon powder does not have any health disadvantages. for humans as daikon does not contain any erucic acid. In addition daikon does not contain ES protein avoiding the undesired reaction of glucosinolates to inactive nitrile metabolites.

Ground broccoli seed is also well known as a rich source of myrosinase but shows two major disadvantages. Firstly, ground broccoli seed contains levels of erucic acid sufficient to not meet dietary regulations. Secondly, broccoli seed contains ES protein which reduces the amount of active metabolites produced e.g. sulforaphane as opposed to inactive nitrile metabolites.

EXAMPLE 2

Once a myrosinase source was identified, shelf life tests were completed on the individual extracts to analyse the glucoraphanin content and myrosinase activity over different storage times and temperatures (4°C, 20°C, 40°C).

The glucoraphanin powder exhibited a shelf life longer than 6-months. Analysed powders produced up to two years previous still show the same GR amount as would be observed directly after production of the extract.

The shelf life of myrosinase in the ground powder extract was analysed after storage at 4°C, 20°C, 40°C (storage in the dark). A small decrease in the myrosinase activity was detected over 3-months. This decrease wasn't significant in comparison to the activity in fresh ground powder and the myrosinase extract was still ideal for the proposed use to catalyse the glucosinolate hydrolysis reaction.

EXAMPLE 3

After having developed a HPLC measurement for the analyses of sulforaphane, the conversion of glucoraphanin based on ground broccoli and daikon seed was analysed to confirm that indeed, hydrolysis and conversion occurred as expected.

Conversion with daikon myrosinase showed the highest amount of sulforaphane and little sulforaphane nitrile production if any.

By comparison, incubation trials with broccoli seed as the myrosinase source showed a high dependence on the incubation temperature due to the presence of ES protein. Higher incubation temperatures exhibited an increase in the amounts of sulforaphane however lower temperatures appear to steer the reaction towards nitrile formation that are inactive.

EXA PLE 4 The inventors conducted trials to determine the optimum ratio of glucosinolate to myrosinase in order to result in full or as full as possible conversion of glucosinolate to isothiocyanate metabolites.

The optimum amounts varied dependent on the quantity of other components in the mixture. An ideal range to achieve optimum conversion appears to lie between 20% wt and 40% wt myrosinase to 60% wt to 80% wt glucoraphanin powder. One result measured the level as being an approximately 30:70 ratio myrosinase to glucoraphanin.

EXAMPLE 5

The inventors conducted trials to determine the shelf stability of a mixture of glucosinolate and myrosinase.

Two powdered extracts (one of glucoraphanin and one of myrosinase) were mixed and stored at temperatures of 4°C, 20°C and 40°C). All samples exhibited a loss in the glucoraphanin content post storage that was expected given the propensity of the reaction to proceed once myrosinase is present. The same experiment was repeated using glucoraphanin extract mixed with myrosinase containing seeds from daikon plants. In this case there was no loss in activity irrespective of temperature or duration of storage. Keeping the myrosinase in an intact form such as in the seed appears to keep the myrosinase from reacting with the glucoraphanin extract.

EXAMPLE 6

Referring to Figures 1 and 2, capsule (Figure 1 ) and pill (Figure 2) embodiments are described. As noted, there is a risk of the myrosinase extract reacting with glucosinolate powder and forming unstable metabolites if mixed before administration thereby reducing the

chemoprotective effects of the composition.

Figure 1 illustrates a capsule generally indicated by arrow A with two different chambers 10, 20 that may be taken by a user as one capsule A yet contains two separate sections 10,20, one with glucosinolate extract 10 and the other with myrosinase extract 20. The two sections may be separated via an interlayer 30.

Figure 2 illustrates a tablet generally indicated by arrow B containing two layers 40, 50 separated by an interlayer 60. As with the capsule A of Figure 1 , each layer 40,50 may contain the extract separate to the other e.g. the first layer 40 contains glucosinolate extract while the second layer 50 contains myrosinase extract. Again the user may take the combined tablet B in one step and the two sections 40,50 dissolve and react together.

EXAMPLE 7

Referring to Figure 3, a grain based food is illustrated in the form of a muesli/granola bar generally indicated by arrow C. The bar contains glucosinolate powder 100 homogenised throughout the bar C. The bar C also contains myrosinase containing seeds 200 interspersed throughout the bar C as well. The seeds 200 may be from the daikon plant. The glucosinolate powder 100 may be a 10% wt glucoraphanin extract derived from broccoli spouts. The bar may contain a ratio of approximately 1 :2 to 1 :5 seeds to glucosinolate extract powder. Seeds may be included in the bar sufficient to form approximately 5-20% wt of the bar.

The bar itself may be made from known muesli bar ingredients including grains, honey, yoghurt and so on. Ideally, when formulated, the bar has a relatively low water activity sufficient to prevent microbial growth as is normal in manufacture of edible bars.

When the bar is eaten, the user chews the seeds breaking up the seeds and releasing myrosinase enzyme in the user's mouth. The myrosinase reacts with the glucosinolates present thereby delivering a known dose of sulforaphane or sulforaphene to the user immediately prior to ingestion or swallowing meaning the user receives a known and measurable dose.

Aspects of the composition, methods and use thereof have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.