Use of brassicaceae plants

Abstract

SOLUTION: There is provided a method for producing a conjugate in which an isothiocyanate compound and a thiol group-containing compound are linked by a dithiocarbamate structure, the method comprising: a preparation step of preparing a suspension containing a crushed product of an individual plant or parts thereof of Brassicaceae plant and a thiol group-containing compound or a reaction mixture obtained by subjecting the suspension to solid-liquid separation; and a production step of producing an isothiocyanate-thiol group-containing compound conjugate in the reaction mixture. Consequently, the isothiocyanate-thiol group-containing compound conjugate can be efficiently obtained.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present specification relates to the use of Brassicaceae plants. [Background technology]

[0002] Brassicaceae plants such as broccoli and wasabi are useful as edible plants. Brassicaceae plants are also known to contain glucosinolates, which are precursors of isothiocyanate compounds that induce detoxification, immune activation, etc. When crushed, glucosinolates are converted to isothiocyanate compounds by dissociating glucose with myrosinase contained in the plant itself.

[0003] Isothiocyanate is an unstable compound. Here, Patent Document 1 describes that the ground product of arugula seeds, which is a kind of cruciferous plant, is reacted with yeast extract containing a thiol group-containing compound such as cysteine, and the isothiocyanate and the thiol group-containing compound are bonded with a dithiocarbamate structure to stabilize the compound. Patent Document 1 also describes that the compound is supplied to B16 melanoma and cultured, and the effect of promoting glutathione production in the cells is observed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 002279 Summary of the Invention [Problem to be solved by the invention]

[0005] Isothiocyanates are known to be highly concentrated in seeds, which are propagation materials and are a useful resource for cultivating edible plants in their entirety.

[0006] Patent Document 1 describes a method in which arugula seeds are ground, water is added thereto, and then yeast extract powder or a yeast extract solution is added thereto, and the mixture is allowed to stand at 25°C for 3 hours to produce an erucin-thiol group-containing compound conjugate.

[0007] However, even if a part of a Brassicaceae plant grown into a plant individual comprising roots, stems, leaves, etc. is crushed and reacted with a thiol group-containing compound, even a person skilled in the art could not predict whether an isothiocyanate-thiol group-containing compound conjugate would be obtained. This is because the composition at the time of reaction is different from that of a reaction using seeds. In addition, a method for efficiently producing a conjugate from such a plant individual was not clear.

[0008] On the other hand, in most cases, the inedible parts of cultivated edible plants are generally discarded, and depending on the supply and demand relationship, the edible plants themselves may be discarded. These discarded unused resources have not yet been put to effective use.

[0009] The present specification provides a technique for converting unused parts of Brassicaceae plants into resources and making effective use of them. [Means for solving the problem]

[0010] The present inventors focused on unused parts of individual plants of Brassicaceae plants, which are sometimes discarded. As a result of examining various methods for converting glucosinolates in individual plants of Brassicaceae plants into isothiocyanate-thiol group-containing compound conjugates, they discovered a method for efficiently obtaining the conjugates. This has revealed that unused parts of Brassicaceae plants can be effectively converted into resources as glucosinolates or isothiocyanates, which are functional components. Based on these findings, the present specification provides the following means.

[0011] [1] A method for producing a conjugate in which an isothiocyanate compound and a thiol group-containing compound are bonded via a dithiocarbamate structure, comprising the steps of: A preparation step of preparing a suspension containing a crushed plant body of a Brassicaceae plant or a part thereof and a thiol group-containing compound, or a reaction mixture obtained by solid-liquid separation of the suspension; a generating step of generating an isothiocyanate-thiol group-containing compound conjugate in the reaction mixture; A method comprising: [2] The method according to [1], wherein the preparation step includes: crushing the Brassicaceae plant or a part thereof in the presence of the thiol group-containing compound to obtain a mixed crushed liquid; and subjecting the mixed crushed liquid to solid-liquid separation to obtain the mixed liquid. [3] The method according to [1] or [2], wherein the thiol group-containing compound is one or more compounds selected from the group consisting of cysteine, glutathione, and γ-glutamylcysteine. [4] The method according to any one of [1] to [3], wherein the preparing step uses a yeast extract containing the thiol group-containing compound. [5] The method according to any one of [1] to [4], wherein the generating step is carried out at a temperature of 1° C. or higher and 50° C. or lower. [6] The method according to any of [1] to [5], wherein the Brassicaceae plant is selected from the group consisting of plants of the genus Brassica in the family Brassicaceae and plants of the genus Wasabi in the family Brassicaceae. [7] The method according to [6], wherein the Brassicaceae plant is Brassica oleracea. [8] The method according to [7], wherein the part of the Brassicaceae plant is the leaves and / or stems of Brassica oleracea. [9] The method according to [6], wherein the Brassicaceae plant is Eutrema japonicum.

[10] The method according to [9], wherein the part of the Brassicaceae plant is one or more species selected from the group consisting of leaves, flowers, petioles and roots of Eutrema japonicum.

[11] The method according to any one of [1] to

[10] , wherein the preparation step uses a yeast extract containing one or more compounds selected from the group consisting of cysteine, glutathione, and γ-glutamylcysteine.

[12] The method according to

[11] , wherein the generating step is carried out at a temperature of 1°C or higher and 50°C or lower. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the contents of isothiocyanate compounds, thiol group-containing compounds, and isothiocyanate-thiol group-containing compound conjugates immediately after preparation of reaction mixtures and control solutions derived from broccoli leaves and florets, respectively. [Diagram 2] FIG. 1 is a graph showing the contents of isothiocyanate compounds, thiol group-containing compounds, and isothiocyanate-thiol group-containing compound conjugates when a reaction mixture derived from broccoli leaves was kept at 4° C. for 0 to 3 days. [Diagram 3] FIG. 1 is a graph showing the contents of isothiocyanate compounds, thiol group-containing compounds, and isothiocyanate-thiol group-containing compound conjugates when a reaction mixture derived from broccoli floret buds was kept at 4° C. for 0 to 3 days. [Figure 4] FIG. 1 is a graph showing the contents of isothiocyanate compounds, thiol group-containing compounds, and isothiocyanate-thiol group-containing compound conjugates immediately after preparation of reaction mixtures and control solutions derived from wasabi leaves and rhizomes, respectively. [Diagram 5] FIG. 1 shows the formation of conjugates when a mixed solution derived from broccoli florets was left standing at room temperature, 37° C., and 50° C. for 3 days. [Figure 6] FIG. 1 shows the formation of conjugates when a mixed solution derived from broccoli leaves was left standing at room temperature, 37° C., and 50° C. for 3 days. [Figure 7A] FIG. 1 shows the production of a conjugate (MSITC-SG) when a mixed solution derived from the rhizome of Wasabi was left standing at room temperature, 37° C., and 50° C. for 3 days. [Figure 7B] FIG. 1 shows the production of a conjugate (AITC-SG) when a mixed solution derived from the rhizome of Wasabi was left standing at room temperature, 37° C., and 50° C. for 3 days. [Figure 8A]FIG. 1 shows the production of a conjugate (MSITC-SG) when a mixed solution derived from wasabi leaves was allowed to stand at room temperature, 37° C., and 50° C. for 3 days. [Figure 8B] FIG. 1 shows the production of a conjugate (AITC-SG) when a mixed solution derived from wasabi leaves was allowed to stand at room temperature, 37° C., and 50° C. for 3 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The disclosure of the present specification relates to the effective utilization of Brassicaceae plants. According to the method disclosed in the present specification, an isothiocyanate-thiol group-containing compound conjugate (hereinafter, simply referred to as a conjugate) can be efficiently obtained from discarded Brassicaceae plants or parts thereof, so that Brassicaceae plants or parts thereof can be effectively utilized as resources as isothiocyanate compounds.

[0014] It is presumed that the method disclosed in the present specification promotes the production of isothiocyanate compounds and conjugates from glucosinolates contained in Brassicaceae plants or parts thereof in the production step, which makes it possible to efficiently obtain conjugates.

[0015] The method for producing the conjugate disclosed in the present specification (hereinafter, simply referred to as the present production method) will be described below. In the present specification, the edible part means a part that is generally used for food, and the non-edible part means a part that is not generally used for food.

[0016] This production method is a method for producing a conjugate. The conjugate is an isothiocyanate compound and a thiol group-containing compound bound together via a dithiocarbamate structure. The structure of the conjugate is represented by the following formula (1).

[0017] [ka]

[0018] In formula (1), R 1indicates the structural portion of the isothiocyanate compound excluding NCS, and R 2 is a thiol group (SH or S) in a thiol group-containing compound. - ) is shown.

[0019] The isothiocyanate compound may be any compound having an NCS group, and any known isothiocyanate compound may be used as appropriate. Although not particularly limited, examples thereof include sulforaphane, benzyl isothiocyanate, allyl isothiocyanate, 6-methylsulfinylhexyl isothiocyanate, 4-methylthio-3-butenyl isothiocyanate (radish), phenethyl isothiocyanate, p-hydroxybenzyl isothiocyanate, methyl isothiocyanate, and erucin.

[0020] The thiol group-containing compound is a compound that contains a thiol group (SH or S - ) and can be bonded to an isothiocyanate to produce a bond. The thiol group-containing compound is not particularly limited, but may be, for example, an amino acid having a thiol group or a derivative thereof, a peptide containing a natural amino acid having a thiol group, a protein containing an amino acid having a thiol group, or a thiolated peptide or protein, depending on reactivity and application. More specifically, cysteine ​​or a derivative thereof, or a peptide containing cysteine ​​may be used.

[0021] Such a thiol group-containing compound may be a natural product, an extract, or a chemically synthesized product or a microbially synthesized product. The peptide may be in the L-form, the D-form, or a mixture thereof (e.g., racemic form), but the L-form may be preferred. Examples of the thiol group-containing compound include one or more selected from glutathione, cysteine, cystenylglycine, N-acetylcysteine, and γ-glutamylcysteine, or salts thereof.

[0022] In addition, a composition containing a thiol group-containing compound may be used as the thiol group-containing compound. For example, those derived from fermentation products of the genus Saccharomyces, Schizosaccharomyces, Candida, etc. can be mentioned. These compositions derived from fermentation products can contain, for example, one or more selected from glutathione, cysteine, and γ-glutamylcysteine, or salts thereof.

[0023] As the composition derived from the fermentation product, yeast extracts derived from yeasts belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, the genus Schizosaccharomyces such as Schizosaccharomyces pombe, and the genus Candida such as Candida utilis are commercially available. For example, from the viewpoint of the composition and content of thiol group-containing compounds, yeast extracts derived from yeasts belonging to the genus Saccharomyces or Candida may be suitable. Such yeast extracts are commercially available.

[0024] The salt of the thiol group-containing compound is not particularly limited. Examples of the salt include salts with inorganic bases, salts with inorganic acids, and salts with organic acids, and it is preferable to select a salt that is acceptable for use in medicines, foods, and beverages. Examples of the salt with inorganic bases include alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as calcium and magnesium, and ammonium salts. Examples of the salt with inorganic acids include salts with hydrohalic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, etc. Examples of the salt with organic acids include salts with formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, maleic acid, fumaric acid, and citric acid.

[0025] The combination of the isothiocyanate compound and the thiol group-containing compound constituting the conjugate is not particularly limited and may be appropriately selected as necessary. Examples of the conjugate include the following. Examples of the conjugate include sulforaphane-cysteine ​​conjugate, sulforaphane-glutathione conjugate, sulforaphane-γ-glutamylcysteine ​​conjugate, benzyl isothiocyanate-cysteine ​​conjugate, benzyl isothiocyanate-glutathione conjugate, benzyl isothiocyanate-γ-glutamylcysteine ​​conjugate, allyl isothiocyanate-cysteine ​​conjugate, allyl isothiocyanate-glutathione conjugate, allyl isothiocyanate-γ-glutamylcysteine ​​conjugate, 6-methylsulfinylhexyl isothiocyanate-cysteine ​​conjugate, 6-methylsulfinylhexyl isothiocyanate-glutathione conjugate, 6-methylsulfinylhexyl isothiocyanate-γ-glutamylcysteine ​​conjugate, erucin-cysteine ​​conjugate, erucin-glutathione conjugate, and erucin-γ-glutamylcysteine ​​conjugate.

[0026] This production method can include a preparation step of preparing a mixed liquid by solid-liquid separation of a suspension containing crushed material containing a Brassicaceae plant individual or a part thereof and a thiol group-containing compound, and a production step of producing a conjugate in the mixed liquid.

[0027] (preparation process) In the preparation step, a plant solid or a part thereof of a cruciferous plant is used as an isothiocyanate compound source for the conjugate (hereinafter, also simply referred to as an isothiocyanate source). The isothiocyanate source is typically a plant solid or a part thereof, and is preferably not an isothiocyanate compound extracted from a plant solid or the like, or an extract containing an isothiocyanate compound. The isothiocyanate source may be a raw plant solid or a part thereof, or a refrigerated, frozen, or dried plant solid or the like.

[0028] Cruciferous plants contain glucosinolates that produce isothiocyanate compounds, and also contain myrosinase that dissociates glucose from the glucosinolates to produce isothiocyanate compounds.

[0029] The Brassicaceae plant is not particularly limited, and any known Brassicaceae plant can be used, such as Brassica sp., Erucca sp., Lepidium sp., Wasabia sp., and Armoracia sp..

[0030] Examples of plants in the Brassicaceae family include broccoli (Brassica oleracea var. italica), cabbage (Brassica oleracea var. capitata), cauliflower (Brassica oleracea var. botrytis), rape (Brassia rapa var. nippo-oleifera), tatsoi (Brassica campestris var. narinosa), mustard (Brassica juncea), takana (Brassica juncea var. intelifolia), mizuna (Brassica nipposinca), kale (Brassica oleracea var. acephala), Brussels sprouts (Brassica oleracea var. gemmifera), bok choy (Brassica rapa var. chinensis), and komatsuna (Brassica rapa var. peruviridis). For example, the inedible parts of broccoli are the leaves and stems. The leaves and stems of broccoli are significant in that they have a large mass. Sulforaphane is an isothiocyanate compound derived from Brassica sp. of the Brassicaceae family.

[0031] [ka]

[0032] Examples of plants of the genus Erucca in the Brassicaceae family include arugula (Erucca sativa), Erucca vesicaria, etc. Examples of isothiocyanate compounds derived from Erucca sp. in the Brassicaceae family include erucin.

[0033] [ka]

[0034] Examples of plants belonging to the genus Lepidium in the Brassicaceae family include maca (Lepidium meyenii), pepper (Lepidium sativum), shepherd's purse (Lepidium apetalum), and maca (Lepidium meyenii). An example of an isothiocyanate compound derived from a plant of the genus Lepidium in the Brassicaceae family, etc., is benzyl isothiocyanate.

[0035] [ka]

[0036] Examples of plants of the Wasabia genus of the Brassicaceae family include Wasabia japonica, etc. Examples of isothiocyanate compounds derived from Wasabia genus of the Brassicaceae family include allyl isothiocyanate and 6-methylsulfinylhexyl isothiocyanate.

[0037] [ka]

[0038] Examples of plants belonging to the genus Armoracia in the Brassicaceae family include Armoracia rusticana, etc. Examples of isothiocyanate compounds derived from the genus Armoracia in the Brassicaceae family include allyl isothiocyanate and 2-phenylethyl isothiocyanate.

[0039] [ka]

[0040] In the preparation step, one or more of these Brassicaceae plants are combined, and the plant individual or a part thereof is used. The present production method is particularly useful when the whole or part of the plant individual is discarded as an edible plant and is not used. In this specification, the plant individual refers to a plant individual that constitutes a whole having organs such as roots, stems, leaves, etc. In addition, in the Brassicaceae plant, for example, sprouts may be removed. In addition, for example, the Brassicaceae plant is intended to mean a plant individual that has grown more advanced than a sprout, and has reached the stage of forming reproductive organs such as inflorescences, flower buds (flower buds and flower stalks), flowers, or precursor organs thereof.

[0041] Furthermore, the term "part of a plant individual" is not particularly limited, and includes structural parts and organs of a Brassicaceae plant depending on the type of the plant. For example, in broccoli, examples include flower buds, flower stalks, leaves, and stems. Of these, the main edible parts are the flower buds and flower stalks. Furthermore, the inedible parts are the leaves, leaf stalks, and flower stalks. Broccoli leaves and / or leaf stalks may be suitable as inedible parts. From the viewpoint of effective utilization of unused resources, leaves and / or leaf stalks are used. Furthermore, the lower part of the flower stalk may be used.

[0042] For example, in the case of wasabi, the main edible parts are the flowers, leaves, petioles, roots, and rhizomes. Of these, the main edible parts are the rhizomes, and the main inedible parts are the flowers, leaves, petioles, and roots. From the viewpoint of effective utilization of unused resources, one or more selected from the inedible parts are used.

[0043] In addition, the crushed product of a cruciferous plant or a part thereof may be the above-mentioned crushed product or crushing liquid, or may be a frozen product obtained by freezing the crushed product or crushing liquid, or a dried product or dried powder obtained by drying the crushed product or crushing liquid by freeze-drying, spray freeze-drying, etc. When preparing a suspension using such a crushed product, an appropriate amount of an aqueous medium is used.

[0044] In the preparation step, a thiol group-containing compound is used as another raw material for the conjugate. The thiol group-containing compound is as described above.

[0045] In the preparation step, a suspension containing the crushed isothiocyanate source and a thiol group-containing compound or a liquid obtained by solid-liquid separation of the suspension is prepared as a reaction mixture. The crushed isothiocyanate source is not particularly limited, but is usually obtained by shredding, crushing, pulverizing, grinding, or grating the isothiocyanate source with a blender, mixer, grinder, grater, or the like (hereinafter referred to as crushing, etc.). The suspension is typically an aqueous suspension containing water derived from a plant individual or a part thereof or an aqueous medium added separately. If necessary, the isothiocyanate source is shredded to a certain extent, and then an aqueous medium is added as necessary and fed to a blender or the like for processing, thereby obtaining a crushed product obtained by crushing the isothiocyanate source. In the case of an isothiocyanate source with a sufficiently high water content, it may not be necessary to separately add an aqueous medium to obtain the crushed product.

[0046] To obtain the suspension, for example, after obtaining a crushed product of an isothiocyanate source, an aqueous medium and a thiol group-containing compound can be added to the crushed product, if necessary, and mixed to obtain a suspension. If the crushed product contains a sufficient amount of aqueous medium when obtained, it is not necessary to add an aqueous medium separately.

[0047] In addition, to obtain the above suspension, for example, a mixture of an isothiocyanate source, which is a plant individual belonging to the Brassicaceae family or a part thereof before crushing, a thiol group-containing compound, and an aqueous medium added as necessary may be crushed. That is, the isothiocyanate source is crushed in the presence of the thiol group-containing compound. In this way, the above suspension can be obtained at once. In addition, in this way, a rapid reaction between the isothiocyanate compound in the isothiocyanate source and the thiol group-containing compound is expected, and thus the formation of the conjugate is expected to be promoted.

[0048] A typical method for crushing the isothiocyanate source in the presence of a thiol group-containing compound is to stir a mixed solution containing the isothiocyanate source and the thiol group-containing compound with a mechanical crusher such as a mixer for about 1 to 5 minutes.

[0049] The aqueous medium that may be added when preparing the suspension includes water, a buffer solution (e.g., a neutral buffer solution with a pH of 6.5 to 7.5), and a mixture of water and an organic solvent compatible with water. Water or a buffer solution may be preferred. Water is most conveniently used. The amount of the aqueous medium relative to the isothiocyanate source is not particularly limited, but may be, for example, 5 parts by mass or more and 100 parts by mass or less relative to 1 part by mass of the isothiocyanate source used.

[0050] The thiol group-containing compound can be the thiol group-containing compound already described. The amount of use relative to the isothiocyanate source is not particularly limited, but can be appropriately set based on the estimated isothiocyanate compound content in the isothiocyanate source and the SH group content in the thiol group-containing compound. Considering the structure of the bond, for example, it is preferable that the SH group of the thiol group-containing compound is about equimolar or more relative to the isothiocyanate compound.

[0051] The reaction mixture may be prepared by separating the suspension into solid and liquid. The solid-liquid separation may be carried out by known methods such as centrifugation and filtration. Typically, the supernatant may be obtained as the reaction mixture under conditions sufficient to centrifugalize the cell fragments (e.g., 16000 g, 15 minutes). For example, the centrifugation may be performed under a cooled condition of about 5°C or less. If necessary, the supernatant may be filtered through a filter having a pore size of, for example, 0.45 μm to obtain a filtrate as the reaction mixture. For example, the filtrate may be obtained as the reaction mixture by repeatedly filtering the supernatant through filters having different pore sizes.

[0052] The suspension may be directly subjected to a reaction mixture in a conjugate production step, which may promote the production of the isothiocyanate compound and the conjugate. Also, the suspension may be separated into a solid and a liquid, which may be subjected to a reaction mixture in a conjugate production step, which may promote the production of the isothiocyanate compound and the conjugate.

[0053] In the above preparation steps, the steps of preparing the suspension and obtaining the reaction mixture are not particularly limited, but may be carried out at a temperature of 1° C. to 30° C., depending on the process time. Alternatively, the temperature may be, for example, 5° C. to 25° C. or 10° C. to 20° C. Inactivation of enzymes such as myrosinase can be avoided or appropriately suppressed.

[0054] (generation process) The formation step is a step of forming a conjugate in a reaction mixture. The reaction itself for obtaining a conjugate from an isothiocyanate compound and a thiol-containing compound is a known method, and is described, for example, in ZHANG, Y., et al., Carcinogenesis, 2000, Vol. 21, No. 6, pp. 1175-1182, etc.

[0055] In this production method, the reaction mixture obtained as described above is allowed to stand or, for example, stirred to promote the production of the isothiocyanate compound and the conjugate. The temperature of the production step is not particularly limited as long as it results in an increased amount of the conjugate, and is appropriately set depending on the process time.

[0056] The temperature in the production step may be, for example, a lower limit temperature of 1°C, for example, 2°C, for example, 3°C, for example, 5°C, for example, 10°C, for example, 15°C, for example, 20°C, for example, 25°C, or for example, 30°C. The temperature in the production step may be, for example, an upper limit temperature of 50°C, for example, 45°C, for example, 40°C, for example, 35°C, for example, 30°C, for example, 25°C, for example, 10°C, for example, 8°C, or for example, 7°C. The temperature range in the production step can be set by appropriately combining these lower and upper limits, but for example, 2°C to 20°C, 2°C to 15°C, 2°C to 10°C, 2°C to 8°C, or 3°C to 7°C, etc., may be effective in promoting the formation of the bond in the edible part. For example, setting the temperature to 1°C or higher and 50°C or lower, 1°C or higher and 50°C or lower, 5°C or higher and 45°C or lower, 10°C or higher and 40°C or lower, 15°C or higher and 40°C or lower, 20°C or higher and 30°C or lower, or 25°C or higher and 40°C or lower, may be effective in promoting the formation of combinations between edible and inedible parts.

[0057] Depending on the temperature in the production step, the reaction mixture can be heated if necessary. In addition, the production step does not need to be at a constant temperature throughout the production step, and multiple temperatures can be combined as appropriate. For example, at the beginning of the production step, a first production sub-step can be performed at a first temperature, and then a second production sub-step can be performed at a second temperature lower than the first temperature.

[0058] The generation time in the generation step depends on the generation temperature, but can be, for example, from 2 hours to 180 hours, for example, from 10 hours to 160 hours, from 20 hours to 140 hours, for example, from 30 hours to 100 hours, for example, from 30 hours to 80 hours, or for example, from 36 hours to 72 hours.

[0059] The formation temperature and formation time are appropriately set so that the amount of the conjugate formed in the reaction mixture is the largest. In the present production method, for example, the formation temperature is 15°C to 40°C and the formation time is 10 hours to 200 hours or 30 hours to 90 hours, or the formation time is 25°C to 40°C and the formation time is 10 hours to 200 hours or 30 hours to 90 hours, to efficiently obtain the conjugate.

[0060] After the production step is completed, the reaction mixture containing the produced conjugate can be used as is, or it can be concentrated, or it can be dried by evaporating water by freeze-drying or the like. If necessary, the conjugate can be separated and purified by chromatography or the like. Alternatively, it can be separated into solid and liquid.

[0061] The obtained conjugate can be used for various purposes. In the state of a conjugate, an unstable isothiocyanate compound can be stabilized as a conjugate with a thiol group-containing compound. Therefore, a conjugate and a composition containing the conjugate in various forms can be produced. In addition, even for oral intake by animals such as humans, it is expected that the isothiocyanate compound can be stably delivered to the body, and can be decomposed by metabolism by intestinal bacteria or the action of gamma-glutamyl transferase in the body, and can contribute to detoxification and immunoactivation in the body as an isothiocyanate compound. EXAMPLES

[0062] In the following, examples are given as specific examples to more specifically explain the disclosure of the present specification. The following examples are intended to explain the disclosure of the present specification, but are not intended to limit the scope thereof. EXAMPLES

[0063] In this example, the production state of sulforaphane and a conjugate of sulforaphane and a thiol group-containing compound in the leaves (inedible parts) and flower buds (edible parts) of broccoli (Brassica oleracea var. italica) was confirmed. That is, 1 g of each of broccoli leaves and flower buds cut into 1 cm pieces was mixed with 1 g of yeast extract (Hithion Extract YH-15, Mitsubishi Corporation Life Sciences Co., Ltd.) in 50 mL of water, and then crushed in a mixer for 3 minutes to obtain a suspension. This suspension was centrifuged (16000 g, 4° C., 15 minutes) to recover the supernatant, and the supernatant was filtered through a 0.45 μm filter (GL Science) to remove insoluble matter and prepare a filtrate as a reaction mixture (hereinafter simply referred to as a mixture). Furthermore, a control solution without yeast extract was prepared by the same procedure as above, except that yeast extract was not added.

[0064] Then, immediately after preparation of each mixture and control solution, sulforaphane, yeast extract-derived GSH, and sulforaphane-glutathione conjugate (sulforaphane-SG) were collected and detected by LC-MS. Specifically, the reaction mixture was introduced into an LC-MS system (Waters), the cone voltage was set to 30 eV, and the compounds were detected by specifying their molecular weights in SIR mode. The analysis conditions were as shown in Table 1. The results are shown in Figure 1.

[0065] [Table 1]

[0066] As shown in Figure 1, a comparison of the leaf-derived control solution and the flower bud-derived control solution revealed that sulforaphane was more abundant in the flower buds. In addition, a comparison of the leaf-derived mixed solution and the flower bud-derived mixed solution revealed that a large sulforaphane-SG peak was detected in the flower bud-derived mixed solution, but a conjugate peak was also detected in the leaf-derived mixed solution. The GSH peak was smaller in the flower bud-derived mixed solution and larger in the leaf-derived mixed solution, indicating that the coexistence of GSH promoted the production of sulforaphane-SG in the leaf-derived mixed solution as well. EXAMPLES

[0067] In this example, we observed the time course of sulforaphane-SG production in broccoli floret-derived mixed solutions and leaf-derived mixed solutions prepared in the same manner as in Example 1. Specifically, these mixed solutions were allowed to stand at 4°C for up to 3 days, and sulforaphane, GSH, and conjugates were collected and detected by LC-MS in the same manner as in Example 1. The results are shown in Figures 2 and 3.

[0068] As shown in Figure 2, in the leaf-derived mixed solution, a slight increase in sulforaphane-SG was observed over time, along with the change in GSH over time. In addition, as shown in Figure 3, in the flower bud-derived mixed solution, a clear increase in sulforaphane and sulforaphane-SG over time was observed, along with the change in SGH over time. In addition, when the change in sulforaphane over time in the flower bud-derived control solution at 4°C was also observed, no change in the amount of sulforaphane was observed.

[0069] From the above, it was found that in the leaf-derived mixed solution and the flower bud-derived mixed solution, the amount of sulforaphane-SG tends to increase from day 0 to day 3 due to the coexistence of GSH, and that sulforaphane-SG can be generated in the flower bud-derived mixed solution by storing these mixed solutions at 4°C for about 70 hours. In addition, since the amount of sulforaphane also increased over time, it was found that the amount of sulforaphane-SG can be further increased by allowing a sufficient amount of GSH to coexist. EXAMPLES

[0070] In this example, the contents of sulforaphane, GSH, and sulforaphane-SG were confirmed for a broccoli floret-derived mixed solution (before freeze-drying) prepared in the same manner as in Example 1, and a solution obtained by redissolving the floret-derived mixed solution in water after freeze-drying. As a result, no change in the contents due to freeze-drying was observed. EXAMPLES

[0071] In this example, a rhizome-derived mixed solution, a leaf-derived mixed solution, and a control solution were prepared from 1 g each of rhizomes (edible part) and leaves (inedible part) of wasabi (Eutrema japonicum) in the same manner as in Example 1. Immediately after preparation of each mixed solution and the control solution, the compounds contained therein (allyl isothiocyanate (AITC), 6-methylsulfinylhexyl isothiocyanate (6-MSITC), GSH, AITC-SG, 6-MSITC-SG) were detected using LS-MS in the same manner as in Example 1. The results are shown in FIG. 4.

[0072] As shown in Figure 4, AITC was not detectable in either the root-derived or leaf-derived mixed solution, but AITC-SG was detectable. 6-MSITC was detectable in the root-derived control solution, and 6-MSITC was not detectable in the root-derived mixed solution, but 6-MSITC-GS was detectable. Small amounts of AITC-GS and 6-MSITC-GS were detectable in the leaf-derived mixed solution.

[0073] From the above, it was found that in the case of mixed solutions derived from wasabi rhizomes and leaves, the presence of GSH made it possible to efficiently extract the isothiocyanates contained in these mixed solutions in the form of conjugates. EXAMPLES

[0074] (Consideration of temperature in the conjugate production process) In this example, the amount of conjugate produced was confirmed when the production process was carried out by leaving the mixture at three different temperatures, room temperature (generally 22 to 23°C), 37°C, and 50°C, for three days. The edible parts (flower buds) and non-edible parts (leaves) of broccoli were crushed for three minutes in the presence of yeast extract in accordance with Example 1 to obtain a suspension. These suspensions were prepared as a flower bud-derived mixed solution and a leaf-derived mixed solution, respectively. After these mixed solutions had been left at a predetermined temperature for three days, they were centrifuged (16000g, 4°C, 15 minutes) in accordance with Example 1 to collect the supernatant, and the supernatant was filtered with a 0.45 μm filter (GL Science). The amount of sulforaphane-SG produced in the filtrate from which insoluble matter had been removed was detected by LC-MS in the same manner as in Example 1.

[0075] In addition, the edible part (rhizome) and the inedible part (leaves) of wasabi were crushed in the presence of yeast extract for 3 minutes in accordance with Example 4 to obtain a suspension. The suspension was prepared as a rhizome-derived mixed solution and a leaf-derived mixed solution, respectively. After these mixed solutions were left at a predetermined temperature for 3 days, they were centrifuged (16000g, 4°C, 15 minutes) in accordance with Example 1 to collect the supernatant, which was then filtered through a 0.45μm filter (GL Science), and the filtrate from which insoluble matter had been removed was subjected to detection of AITC-SG and 6-MSITC-SG using LS-MS in accordance with Example 4.

[0076] In addition, for both broccoli and wasabi, yeast extract (yeast extract (containing glutathione) manufactured by FAP Japan Co., Ltd.) was used instead of the yeast extract (YH-15) used in Example 1 as a source of glutathione, a thiol group-containing compound.

[0077] The evaluation results of the amount of binders produced in the mixed solution derived from broccoli florets (edible part), broccoli leaves (inedible part), wasabi rhizomes (edible part), and wasabi leaves (inedible part) are shown in Figures 5 to 8, respectively.

[0078] As shown in Figure 5, the amount of the conjugate produced increased in the mixture derived from broccoli flower buds. 37°C was favorable.

[0079] As shown in Figure 6, the amount of the conjugate produced also increased in the mixed solution derived from broccoli leaves. Room temperature to 37°C tended to be favorable.

[0080] As shown in Figures 7A and 7B, in the mixed solution derived from wasabi rhizomes, the amount of conjugate produced increased at room temperature to 50°C for both 6-MSITC-SG and AITC-SG.

[0081] As shown in Figures 8A and 8B, in the mixed solution derived from wasabi leaves, for 6-MSITC-SG, the amount of conjugate produced tended to increase at all temperatures from room temperature to 37°C.

[0082] From the above results, it was found that the compound was effective not only in edible parts but also in non-edible parts at room temperature to 37°C in terms of the amount of conjugates formed.

Claims

1. A method for producing a conjugate in which an isothiocyanate compound and a thiol group-containing compound are bound to each other via a dithiocarbamate structure, comprising the steps of: A preparation step of preparing a suspension containing a crushed plant body of a Brassicaceae plant or a part thereof and the thiol group-containing compound, or a reaction mixture obtained by solid-liquid separation of the suspension; a generating step of generating an isothiocyanate-thiol group-containing compound conjugate in the reaction mixture; A method comprising:

2. The method according to claim 1 , wherein the preparing step comprises crushing the Brassicaceae plant or a part thereof in the presence of the thiol group-containing compound to obtain the suspension.

3. 2. The method according to claim 1, wherein the thiol group-containing compound is one or more compounds selected from the group consisting of cysteine, glutathione, and γ-glutamylcysteine.

4. The method according to claim 1 , wherein the preparing step uses a yeast extract containing the thiol group-containing compound.

5. The method according to claim 1 , wherein the generating step is carried out at a temperature of 1° C. or more and 50° C. or less.

6. The method of claim 1 , wherein the cruciferous plant is selected from the group consisting of Brassica spp. and Wasabi spp.

7. The method of claim 6, wherein the cruciferous plant is Brassica oleracea.

8. 8. The method of claim 7, wherein the part of the Brassicaceae plant is the leaves and / or stems of Brassica oleracea.

9. The method of claim 1 , wherein the Brassicaceae plant is Eutrema japonicum.

10. The method according to claim 9, wherein the part of the Brassicaceae plant is one or more selected from the group consisting of leaves, flowers, petioles and roots of Eutrema japonicum.

11. The method according to claim 1, wherein the preparing step uses a yeast extract containing one or more compounds selected from the group consisting of cysteine, glutathione, and γ-glutamylcysteine.

12. The method according to claim 11 , wherein the generating step is carried out at a temperature of 1° C. or more and 50° C. or less.

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