Isothiocyanate formation process

Abstract

Process for the Formation of Isothiocyanates. This description relates to a process (200) for producing isothiocyanates (101) from glucosinolates (103), the process comprising the following successive steps: a) forming (201) a first solution by mixing a source of glucosinolates (203) with water (205), the first solution being maintained at a first temperature for a first time, and then optionally cooling the first solution; b) adding a source of enzymes (213) to the first solution, the enzymes being capable of converting glucosinolates to isothiocyanates, thereby forming a second solution; and c) maintaining (219) the second solution at a second temperature for a second time, thereby forming a third solution containing isothiocyanates. Figure for the abstract: Fig. 2A

Description

Title of the invention: Process for the formation of isothiocyanates Technical field

[0001] The present description relates generally to processes for forming chemical compounds suitable for activating, or promoting, the implementation of antioxidant mechanisms in cells of a living organism. The present description relates more particularly to processes for forming isothiocyanates. Prior art

[0002] Conventionally, a cell of a living organism is the site of numerous biological and chemical processes, including oxidation mechanisms which can lead, in the event of cell dysfunction, to its partial or total destruction. In a healthy cell, proteins called "Nrf2" (from the English "nuclear factor erythroid-2-related factor 2") are synthesized by the cell, for example continuously. The Nrf2 proteins are then rapidly captured and destroyed by another protein, called "Keapl" (from the English "Kelch-like ECH-associated protein 1"). The Keapl proteins include receptors capable of detecting an excessive presence of oxidation agents, or oxidizing agents, in the cell. When these receptors detect oxidation, the capture of the Nrf2 proteins by the Keapl proteins is inhibited.The newly synthesized Nrf2 proteins then circulate freely inside the cell, migrate to the cell nucleus, and cause transcription of several hundred genes leading to the release of enzymes and antioxidant substances, thus stopping or slowing down cellular oxidation. In other words, the role of the Keapl proteins is similar, in a figurative way, to that of a valve opening when the oxidizing agents present in the cell have a concentration exceeding a predetermined threshold, so as to release the Nrf2 proteins inside the cell to trigger mechanisms aimed at preventing or limiting its oxidation, the valve remaining closed until the predetermined threshold of concentration of oxidizing agents is reached, in which case the Nrf2 proteins are not released into the cell and the antioxidant mechanisms are not activated.

[0003] Certain pathologies, for example neurodegenerative pathologies such as Parkinson's disease, Alzheimer's disease, Lewy body disease, etc., are the cause of cellular dysfunctions, mainly affecting nerve cells, caused by excessive capture of Nrf2 proteins by Keapl proteins. Excessive oxidation mainly affects dopaminergic neurons, i.e. dopamine producers, and astrocytes. Astrocytes are responsible for keeping neurons healthy by providing nutrients, removing waste, and replacing damaged mitochondria. Astrocytes have the ability to neutralize excessive amounts of oxidizing agents produced by cells through the mechanism described above. Specifically, when neurons are subjected to excessive oxidation, astrocytes produce antioxidants due to the presence of Nrf2 proteins and then transfer the antioxidants to the neurons. In the event of excessive oxidation of a cell affected by one of the aforementioned dysfunctions, the excessive capture of Nrf2 proteins by Keapl proteins in astrocytes tends to inhibit the antioxidant mechanisms activated, or promoted, by the free circulation of Nrf2 proteins within the cell.This results in partial or total destruction of the cell, called neurodegeneration in a case where the cells concerned are nerve cells.

[0004] Chemical compounds, in particular compounds of the isothiocyanate family, are capable of binding to Keapl proteins via the same receptors as those used to detect the presence of substances causing cellular oxidation. In order to effectively counter cellular oxidation, a prerequisite is that the quantity of isothiocyanates capable of binding to the receptors of the Keapl proteins is sufficiently high to exceed the predetermined threshold from which the Nrf2 proteins are free to circulate inside the cell. Below this threshold, the isothiocyanates bound to the receptors of the Keapl proteins are in fact not present in sufficient quantity to allow the free circulation of the Nrf2 proteins, and consequently fail to slow down or stop the mechanisms of cellular oxidation, thus leading to partial or total destruction of the cells.

[0005] Methods for forming chemical compounds suitable for activating, or promoting, antioxidant mechanisms in cells of a living organism have been proposed. Methods for forming isothiocyanates have in particular been proposed.

[0006] However, the known methods for forming isothiocyanates have various drawbacks. In particular, these methods do not make it possible to produce bioavailable isothiocyanates, capable of reaching the bloodstream of a living organism in unchanged form (in other words without being metabolized), and / or in sufficient quantity to activate, or promote, antioxidant mechanisms in cells of a living organism, for example cells of the nervous system of a human being. Summary of the invention

[0007] There is a need to improve all or part of the existing training processes of isothiocyanates. In particular, there is a need to provide a process for forming isothiocyanates making it possible to produce bioavailable isothiocyanates and / or in sufficient quantity to activate, or promote, antioxidant mechanisms in cells of a living organism, in particular cells of the nervous system of a human being.

[0008] For this, one embodiment provides a process for producing isothiocyanates from glucosinolates, the process comprising the following successive steps: a) forming a first solution by mixing a source of glucosinolates with water, the first solution being maintained at a first temperature for a first duration, then optionally cooling the first solution; b) adding a source of enzymes to the first solution, the enzymes being capable of converting glucosinolates to isothiocyanates, whereby a second solution is formed; and c) maintaining the second solution at a second temperature for a second time, whereby a third solution containing isothiocyanates is formed.

[0009] According to one embodiment: - the first temperature is between 70 and 100°C, preferably between 90 and 100°C, more preferably equal to approximately 95°C; and - the first duration is between 1 and 10 min, preferably between 1 and 5 min, more preferably equal to approximately 2 min.

[0010] According to one embodiment, the enzyme source contains myrosinase.

[0011] According to one embodiment, the source of enzymes is: - a powder obtained by grinding white mustard seeds; or - a fourth solution obtained by mixing a powder obtained by grinding white mustard seeds and water.

[0012] According to one embodiment: - the second temperature is between 50 and 70°C, preferably between 55 and 65°C, more preferably equal to approximately 60°C; and - the first duration is between 25 and 90 min, preferably between 30 and 50 min, more preferably equal to approximately 35 min.

[0013] According to one embodiment, the source of glucosinolates is a powder obtained by grinding seeds of a plant belonging to the order Capparales, preferably a plant belonging to the Brassicaceae family, more preferably broccoli.

[0014] One embodiment provides a composition in the form of an aqueous solution comprising isothiocyanates and enzymes capable of converting glucosinolates into isothiocyanates, and optionally glucosinolates.

[0015] According to one embodiment, the enzymes are myrosinases.

[0016] According to one embodiment, the glucosinolates are glucoraphanins and the isothiocyanates are sulforaphanes.

[0017] One embodiment provides a composition as described above, for use as a medicament.

[0018] One embodiment provides a composition as described above, for use in the treatment of neurodegenerative diseases, preferably in the treatment of Parkinson's disease.

[0019] According to one embodiment, the composition comprises isothiocyanates having a concentration and bioavailability suitable for: - induce, in the blood of a living organism, preferably a human being, having ingested the composition, a concentration of isothiocyanates exceeding a threshold from which Nrf2 proteins are free to circulate inside cells of the living organism; - reduce or stop oxidative stress in astrocytes and / or neurons of people suffering from a neurodegenerative disease, for example Parkinson's disease; - attenuating, by the action of Nrf2 proteins, an oxidation of mitochondria in cells of a living organism, for example a human being; and / or - alleviate all or part of the symptoms of neurodegenerative diseases, preferably all or part of the non-motor symptoms of Parkinson's disease. Brief description of the drawings

[0020] These characteristics and advantages, as well as others, will be explained in detail in the following description of particular embodiments given without limitation in relation to the attached figures among which:

[0021] [Fig.l] schematically and partially represents a reaction mechanism of an example of a process for forming an isothiocyanate of the type to which, by way of example, described embodiments apply;

[0022] [Fig.2A] is a flowchart illustrating, schematically and in block form, steps of a method for forming isothiocyanates according to one embodiment;

[0023] [Fig.2B] is a flowchart illustrating, schematically and in block form, steps of a variant of the process for forming isothiocyanates of [Fig.2A];

[0024] [Fig.3] is a graph illustrating an example of a curve showing the evolution, as a function of time, of a conversion rate of glucosinolates into isothiocyanates during the implementation of the method of [Fig.2A] or the variant of [Fig.2B];

[0025] [Fig.4] is a timing diagram illustrating, schematically and partially, examples of curves showing the evolution of different characteristic quantities of a cell of a living organism after ingestion of isothiocyanates obtained according to the process of [Fig.2A] or the variant of [Fig.2B];

[0026] [Fig.5] illustrates, schematically and in the form of blocks, an example of a sequence of steps associated with a process of a neurodegenerative disease; and

[0027] [Fig.6] represents, schematically and in the form of blocks, an example of a system adapted to implement the method of [Fig.2A] or the variant of [Fig.2B] according to one embodiment. Description of the embodiments

[0028] The same elements have been designated by the same references in the different figures. In particular, the structural and / or functional elements common to the different embodiments may have the same references and may have identical structural, dimensional and material properties.

[0029] For the sake of clarity, only the steps and elements useful for understanding the described embodiments have been represented and are detailed. In particular, the various biological and chemical mechanisms implemented by a cell of a living organism, for example a human being, have not been detailed. The mechanisms of dopamine production and the oxidation mechanisms in a cell, in particular a dopaminergic neuron, have in particular not been detailed.

[0030] Unless otherwise specified, when referring to two elements connected to each other, this means directly connected without intermediate elements other than conductors, and when referring to two elements connected (in English "coupled") to each other, this means that these two elements can be connected or be connected by means of one or more other elements.

[0031] In the following description, when reference is made to absolute position qualifiers, such as the terms "front", "back", "top", "bottom", "left", "right", etc., or relative position qualifiers, such as the terms "above", "below", "upper", "lower", etc., or to orientation qualifiers, such as the terms "horizontal", "vertical", etc., reference is made, unless otherwise specified, to the orientation of the figures.

[0032] Unless otherwise specified, the expressions “about”, “approximately”, “substantially”, and “of the order of” mean to within 10%, preferably to within 5%.

[0033] [Fig.l] schematically and partially represents a reaction mechanism 100 of an example of a method for forming an isothiocyanate 101 of the type to which, by way of example, described embodiments apply. [Fig.l] more particularly illustrates a method for forming isothiocyanate 101 from a gluco- sinolate 103.

[0034] In the illustrated example, glucosinolate 103 is glucoraphanin (4-methylsulfinylbutyl) and isothiocyanate 101 is sulforaphane (4-methylsulfinylbutylisothiocyanate). In this example, glucoraphanin 103 binds to an enzyme 105. In the illustrated example, enzyme 105 is myrosinase, also called thioglucosidase. In the presence of water, myrosinase 105 makes it possible to cleave, by hydrolysis, a sulfur bond of a glucose group (of empirical formula C6HnO6) of glucoraphanin, thus releasing a glucose molecule 107 (of empirical formula C6Hi2 O6). An unstable reaction intermediate 109, shown in brackets in [Fig.l], is then formed. Reaction intermediate 109 then rearranges to produce either sulforaphane 101 (lower branch, in [Fig.l]) or a sulforaphane nitrile 111 (upper branch, in [Fig.l]).

[0035] The rearrangement of reaction intermediate 109 into sulforaphane 101 or sulforaphane nitrile 111 is conditioned mainly by the absence or presence of a modifying protein, called "ESP" (from the English "epithiospecifier"), and iron (II) ions (Fe2+). More precisely, the absence of ESP proteins and iron (II) ions causes the rearrangement of the unstable reaction intermediate 109 into sulforaphane 101, while their presence in the reaction medium causes the rearrangement of reaction intermediate 109 into sulforaphane nitrile 111. During the rearrangement of reaction intermediate 109 into sulforaphane 101 or sulforaphane nitrile 111, a monovalent sulfonyl ion is detached, thus forming a fully ionized sulfuric acid molecule (H22+, SO42).

[0036] Glucosinolates, such as glucoraphanin, are secondary metabolites present in particular in plants belonging to the order Capparales, for example plants of the Brassicaceae family (Brassicaceae), also called cruciferous plants. Plants containing glucosinolates also generally contain myrosinase. Thus, destruction of the tissues of a plant belonging to the order Capparales, for example belonging to the Brassicaceae family, leads to contact between the myrosinase and the glucosinolates contained in the plant. This destruction of the tissues occurs, for example, following ingestion by a living organism, for example a human being, of seeds or shoots of the plant. Isothiocyanates, such as sulforaphane, can then be formed according to a process identical or analogous to the process described above in relation to [Fig.l].

[0037] However, the ingestion, by a living being, of plants containing glucosinolates, for example plants belonging to the order Capparales, for example plants belonging to the Brassicaceae family, does not make it possible to produce isothiocyanates, for example sulforaphane, bioavailable and in sufficient quantity to cross the activation threshold of the antioxidant mechanisms of the cells as explained previously. The inventor realized that this is due in particular to the fact that the biological and chemical processes are, in this case, implemented at too low a temperature, for example around 37°C for the human digestive system, thus making these processes much too slow to hope to cross the threshold.

[0038] [Fig.2A] is a flowchart illustrating, schematically and in block form, steps of a method 200 for forming isothiocyanates according to one embodiment.

[0039] In an initial step (block 201), a solution SI is produced by mixing a source of glucosinolates (block 203) with water (block 205). The source of glucosinolates 203 is, for example, a powder obtained by grinding, or pounding, seeds of a plant containing glucosinolates, for example of the type of glucosinolate 103 previously described in relation to [Fig. 1]. The seeds are, for example, chosen so that they contain glucosinolates having, for example, a carbon chain, which may be linear or branched, saturated or unsaturated, and / or a carbon cycle which may be aromatic. Preferably, the carbon chain comprises between two and nine carbon atoms (for example, a carbon chain comprising two, three or four carbon atoms). By "between two and nine", it is meant that the limits two and nine are included.The choice of the source of glucosinolates 203 is made, for example, based on the effectiveness of the isothiocyanates, formed from this source by implementing the method 200, in inhibiting the Keapl protein.

[0040] The seeds originate, for example, from a plant belonging to the order Capparales, for example a plant belonging to the Brassicaceae family. For example, the seeds are broccoli seeds (Brassica oleracea). Alternatively, the seeds may originate from a plant belonging to a family of the order Capparales other than the Brassicaceae, for example the Moringaceae family.

[0041] The solution SI has a temperature T1 of, for example, between 70 and 100°C, for example between 90 and 100°C, for example equal to approximately 95°C. By way of example, the solution SI is produced by mixing the source of glucosinolates 203 with the water 205 previously brought to a boil by a heating device, thus lowering the temperature of the water 205 by a few degrees until reaching the temperature TL. In the case where plant seeds are used as a source of glucosinolates 203, this makes it possible to eliminate all or part of the ESP proteins and / or to render inoperative any enzymes possibly contained by the seeds forming the source of glucosinolates 203. This also makes it possible to promote optimal extraction of the glucosinolates, for example the glucosinolates 103, in the water 205 without initiating their conversion into isothiocyanates.

[0042] The amount of glucosinolate source 203 used to produce the SI solution is for example depending on a quantity of isothiocyanates that it is desired to produce by implementing the method 200. In the case where seeds of a plant of the Brassicaceae family, for example broccoli seeds, are used for oral administration to a living being, for example a human being, of the isothiocyanates produced by the method 200, the mass of glucosinolate source 203 is for example between 0.1 and 10 g, for example between 0.5 and 5 g, for example equal to approximately 2 g.

[0043] The water 205 is, for example, demineralized or deionized water. The use of demineralized or deionized water makes it possible to avoid the presence of ions, in particular iron (II) ions, in the reaction medium when implementing the steps of the method 200. During step 201, the quantity of water 205 is, for example, adjusted according to the mass of glucosinolate source 203. The quantity of water 205 used during step 201 is, for example, approximately fifty times greater, by mass, than the quantity of glucosinolate source 203. The volume of water is, for example, chosen so that a solution containing the isothiocyanates, produced at the end of subsequent steps of the method 200, has a volume compatible with oral administration, in a single dose, to a living being, for example a human being. The volume of water 205 is for example between 10 mL and 1 L, for example between 50 and 500 mL, for example equal to approximately 100 mL.

[0044] For example, during step 201, approximately 2 g of dried broccoli seeds are ground and mixed with approximately 100 mL of demineralized or deionized water previously brought to a boil.

[0045] However, the embodiments described are of course not limited to these examples, and the person skilled in the art is capable, from the indications of the present description, of adjusting the mass of glucosinolate source 203 and the volume of water 205 used during step 201 depending, for example, on the corpulence of a patient to be treated.

[0046] The use of demineralized or deionized water 205 during step 201 makes it possible to avoid or limit the presence of iron (II) ions in the solution SL. Furthermore, producing the solution SI at a temperature above 70°C makes it possible to eliminate the ESP proteins potentially contained by the glucosinolate source 203. During subsequent steps of the method 200, this advantageously makes it possible to avoid or limit the formation of isothiocyanate nitriles, as explained above in relation to [Fig. 1], and to promote the formation of isothiocyanates. Generally speaking, this makes it possible to ensure that the implementation of the method 200 leads mainly to the formation of isothiocyanates, and that the quantity of isothiocyanate nitriles produced is as low as possible.

[0047] The method 200 comprises another step (block 207), subsequent to step 201, waiting for a duration Dl. During this step, the solution SI cools down, for example, by a few degrees upon contact with the ambient air. Step 207, for example, ensures optimal extraction of the glucosinolates 103 contained in the glucosinolate source 203.

[0048] By way of example, the duration D1 is between 1 and 10 min, for example between 1 and 5 min, for example equal to approximately 2 min.

[0049] During another step (block 209), subsequent to step 207, the solution S1 is cooled to a temperature T2. The solution S1 is for example cooled by natural cooling in contact with the ambient air, for example after stopping a heating device that has made it possible to heat the water 205 prior to step 201. As a variant, the temperature of the solution S1 can be lowered by adding a quantity of water, for example demineralized or deionized water, colder than the solution S1, the quantity of water added being for example determined so as to make it possible to reach the temperature T2 after mixing with the solution S1. The solution S1 is then maintained at the temperature T2. The solution S1 is for example further filtered prior to cooling. As a variant, the solution S1 can be filtered after cooling.The temperature T2 is for example between 50 and 70 °C, for example between 55 and 65 °C, for example equal to approximately 60 °C. For example, the solution SI is maintained at the temperature T2 with an accuracy better than plus or minus 5 °C, for example equal to approximately plus or minus 1 °C.

[0050] In another step (block 211), subsequent to step 209, a solution S2 is formed by adding an enzyme source (block 213) to the solution SL. The enzyme source 213 contains, for example, myrosinase molecules 105. For example, the enzyme source is a powder comprising white mustard seeds (biological name “Sinapis alba”, also known as “yellow mustard”), for example a powder obtained by grinding, or pounding, white mustard seeds. The quantity of enzyme source 113 is, for example, approximately ten times lower, by mass, than the quantity of glucosinolate source 203.

[0051] The quantity of enzyme source 213 is for example adjusted so as to make it possible to obtain a reaction for converting glucosinolates into isothiocyanates having first-order kinetics, i.e. limited by the quantity of glucosinolates present in the solution S2. The conversion reaction is then faster than in the case of zero-order kinetics, for example a reaction limited by the quantity of enzymes present in the solution S2. Favoring a rapid conversion reaction advantageously makes it possible to limit the degradation of the isothiocyanates produced from the glucosinolates. This also allows a saving of time when implementing the method 200. For example, a mass equal to approximately 0.2 g of white mustard seed powder is added to the SI solution in step 211, in the case where a mass equal to approximately 2 g of broccoli seeds was used, as a source of glucosinolates 203, in step 201.

[0052] During another step 219, subsequent to step 211, the solution S2 is maintained at the temperature T2 for a duration D2. The duration D2 is for example chosen so as to allow an optimal conversion of the glucosinolates into isothiocyanates. The duration D2 is for example more precisely adjusted according to two antagonistic mechanisms: the production of isothiocyanates from glucosinolates, on the one hand, and the degradation of the isothiocyanates thus produced due to the presence of humidity, on the other hand. By way of example, the duration D2 is between 25 and 90 min, for example between 30 and 50 min, for example equal to approximately 35 min, for example in the case where the temperature T2 is equal to approximately 60°C. Maintaining the temperature T2 for the duration D2 advantageously makes it possible to obtain an optimal yield, for example equal to approximately 90%, of the reaction of conversion of glucosinolates into isothiocyanates as will be explained in more detail below.

[0053] In another step (block 221), subsequent to step 219, a solution S3 containing isothiocyanates is obtained. Solution S3 is more precisely produced after maintaining solution S2 at temperature T2 for duration D2. In a case where the glucosinolate source 203 contains glucoraphanin and where the enzyme source 213 contains myrosinase, solution S3 contains sulforaphane 101 produced, in this example, according to mechanism 100 of [Fig.l].

[0054] The solution S3 can then be ingested by a living being, for example a human being, a few minutes, for example less than an hour, for example less than 10 min, after its production.

[0055] Solution S3 constitutes, for example, a medicinal product in galenic form allowing controlled release of the active ingredient to relieve the symptoms and / or restore and / or protect the neurons of patients suffering from neurodegenerative diseases, for example Parkinson's disease. Solution S3 aims to relieve, among other things, the so-called "non-motor" symptoms of Parkinson's disease, including: constipation, urgent urination (urinary urgency), intense fatigue, loss of balance, brain fog, cognitive disorders, sleep disorders, speech disorders, deterioration of body temperature control, excessive sweating, apathy, depression, anxiety, swallowing disorders, excessive salivation, etc.

[0056] An advantage of the method 200 set out above in relation to [Fig.2A] is that it makes it possible to produce, from the source of glucosinolates 203, bioavailable isothiocyanates, for example bioavailable sulforaphane molecules. The total quantity of isothiocyanates contained in the solution S3, i.e. the total quantity of isothiocyanates produced by the implementation of the method 200, is for example greater, for example at least twenty times greater, than that which would be obtained, for an identical mass of glucosinolate source 203, by direct ingestion of the glucosinolate source 203. Furthermore, the fact of ingesting the solution S3 allows the majority, or even all, of the isothiocyanates contained in this solution to reach the bloodstream after a period of between a few minutes and a few tens of minutes. By way of comparison, in the case of ingestion of the glucosinolate source 203, the isothiocyanates would reach the bloodstream after a longer period, for example equal to approximately 8 hours, and would be produced in a much more progressive manner, for example throughout a phase extending over a period of several hours, or even several tens of hours.In other words, the method 200 makes it possible to achieve, in the bloodstream, a higher concentration of isothiocyanates, for example approximately one hundred times higher, than that which would be achieved after direct ingestion of the source of glucosinolates 203.

[0057] [Fig.2B] is a flowchart illustrating, schematically and in block form, steps of a variant 200' of the method 200 for forming isothiocyanates of [Fig.2A]. The variant 200' of [Fig.2B] comprises elements in common with the method 200 of [Fig.2A]. These common elements will not be described again below. The variant 200' of [Fig.2B] differs from the method 200 of [Fig.2A] in that, in the variant 200', step 211 is substituted by another step (block 211'), subsequent to step 209, during which a solution S4 (block 215) is mixed with the solution SL. A solution S2', analogous to the solution S2, is then obtained.

[0058] The solution S4 is for example formed, prior to step 211', by mixing the enzyme source (block 213) with water (block 217). The water 217 is for example demineralized or deionized water, for example for reasons identical or similar to those explained previously, in relation to step 201 of the method 200, for the water 205. The solution S4 constitutes an enzyme source similar to the enzyme source 213.

[0059] For example, solution S4 is prepared at least two hours, for example between several hours and several days, before the implementation of step 211'. Solution S4 is then mixed, during step 211', with solution S1 cooled to temperature T2 at the end of step 209. Alternatively, the temperature and volume of water 217 used to prepare solution S4 may be chosen so as to allow solution S1 to be cooled to temperature T2 by mixing solutions S1 and S4, step 209 then being omitted for example in this case. Preparing solution S4 allows the enzymes naturally present in white mustard seeds to convert the glucosinolates naturally contained in these seeds into isothiocyanates. Thus, advantageously, the absence of the original glucosinolates allows the enzymes contained in white mustard seeds to be more available, when mixing the S4 solution with the SI solution, to convert the glucosinolates present in the SI solution into isothiocyanates.

[0060] Alternatively, solution S4 may be dried, for example at room temperature, to produce a powder containing enzymes and substantially free of glucosinolates. The powder is, for example, subsequently mixed with solution S1 to form solution S2 in a manner analogous to that previously discussed in connection with step 211 of method 200 of [Fig.2A].

[0061] In the example shown, step 219 of maintaining at temperature T2 for a duration D2 is implemented, from solution S2', for example in a manner analogous or identical to that which was previously explained in relation to method 200.

[0062] At the end of the method of variant 200', during a step subsequent to step 219, a solution S3' is obtained (block 221'). The solution S3' is for example analogous or identical to the solution S3 obtained by implementing the method 200.

[0063] The variant 200' has advantages similar to those which have been previously detailed in relation to [Fig.2A] for the method 200.

[0064] [Fig. 3] is a graph 300 illustrating an example of a curve 301 of evolution, as a function of time (abscissa, in minutes), of a conversion rate of glucosinolates into isothiocyanates (ordinate, in percentage) during the implementation of the method 200 of [Fig. 2A] or of the variant 200' of [Fig. 2B].

[0065] Curve 301 illustrates more precisely the variations, as a function of time, of the rate or yield of conversion of the glucosinolates of the solution S1 into isothiocyanates present in the solution S3, during the implementation of the method 200 previously explained in relation to [Fig.2A], or in the solution S3', during the implementation of the variant 200' previously explained in relation to [Fig.2B], in a case where: - the source of glucosinolates 203 consists of a powder obtained from approximately 2 g of crushed broccoli seeds; - the source of enzymes 213 consists of a powder obtained from approximately 0.2 g of crushed white mustard seeds; and - the temperature T2 is equal to approximately 60°C.

[0066] As illustrated in [Fig. 3], the conversion rate of glucosinolates to isothiocyanates increases rapidly, in a substantially linear manner as a function of time, during the first minutes of the reaction of step 219 of process 200 or 200'. The conversion rate then increases less rapidly (in other words, the conversion rate decreases) and then exhibits an asymptotic appearance from a duration equal to approximately 35 min, in this example. In this example, by setting the duration D2 of holding at temperature T2 during step 219 to a value greater than 35 min, for example equal to about 45 min, it is possible to ensure that the majority of glucosinolates 103 contained in solution SI have been converted into isothiocyanates 101 in solution S3 or in solution S3'.

[0067] It is possible to monitor the temporal evolution of the conversion rate of glucosinolates 103 into isothiocyanates 101 indirectly, for example by chemical titration of the sulfuric acid produced during the reaction, for example by using a strong base such as sodium hydroxide to neutralize the sulfuric acid produced. For example, in the case where broccoli seeds are used as a source of glucosinolates 203, the amount of sulfuric acid produced during the implementation of step 219 of method 200 or 200' is of the order of 25 to 40 pmol per gram of broccoli seeds, and can be determined with an accuracy of the order of 2 to 4%. However, this method is relatively slow to implement.

[0068] Alternatively, it is possible to estimate the conversion rate of glucosinolates into isothiocyanates by measuring the pH. However, the change in pH during the implementation of step 219 of the method 200 or 200' is non-linear. Furthermore, the pH measurement may be disturbed by the presence of weak acids and bases in the source of glucosinolates 203, for example in a case where broccoli seeds are used as a source of glucosinolates 203. Thus, the relationship between the pH and the conversion rate of glucosinolates into isothiocyanates may prove difficult to establish.

[0069] Surprisingly, the inventor realized that it was possible to estimate the evolution of the conversion rate of glucosinolates into isothiocyanates indirectly, by measuring the ionic conductivity of the solution during step 219 of the method 200 or 200'. The conductivity measurement has the advantage of being rapid, reliable and easy to implement.

[0070] The fastest hydrolysis kinetics, i.e. the case in which the maximum conversion rate of glucosinolates 103 to isothiocyanates 101 is reached after the shortest time, is for example obtained for a temperature T2 of between 55 and 65°C, for example equal to approximately 60°C. In this case, the maximum conversion rate of glucosinolates 103 to isothiocyanates 101 is for example reached after a time of between 30 and 45 min, for example equal to approximately 35 min.

[0071] For a temperature T2 between 50 and 55°C, the reaction is slower than when the temperature T2 is between 55 and 60°C. For example, the maximum conversion yield is reached after approximately 50 min in the case where the temperature T2 is equal to approximately 50°C.

[0072] For a temperature T2 above 65°C, the enzymes contained in the source 213 degrade at least partially and thus lead to a slower reaction. Above 70°C, the enzymes are rapidly degraded, consequently increasing the reaction time and reducing the maximum conversion yield by more than half. For example, the maximum conversion yield is equal to: - approximately 70%, in the case where the temperature T2 is equal to approximately 70°C, the maximum yield then being reached after a time equal to approximately 40 min; and - approximately 35%, in the case where the temperature T2 is equal to approximately 75°C, the maximum yield then being reached after a time equal to approximately 30 min.

[0073] Being able to achieve the highest possible conversion yield in the shortest possible time advantageously makes it possible to have a maximum quantity of bioavailable isothiocyanates before they have had time to degrade.

[0074] [Fig.4] is a chronogram 400 illustrating, schematically and partially, examples of evolution curves, as a function of time (t, abscissa) of different characteristic quantities of a cell of a living organism, for example a human being, after ingestion of the isothiocyanates 101 obtained according to the method 200 of [Fig.2A] or according to the variant 200' of [Fig.2B].

[0075] [Fig.4] more precisely includes: - a curve 401 of evolution, as a function of time t, of a concentration of isothiocyanates, for example sulforaphane (SFN), in the cell; - a curve 403 of evolution, as a function of time t, of a rate of Keapl proteins still active, that is to say not inhibited by isothiocyanates; - a curve 405 of evolution, as a function of time t, of a concentration of Nrf2 proteins free to circulate in the cell; and - a curve 407 of evolution, as a function of time t, of a concentration of antioxidant substances (Antiox.) in the cell.

[0076] At a time t0, the solution S3 or the solution S3' is for example ingested by a living organism, for example a human being.

[0077] Between time t0 and a time t1, subsequent to time t0, the concentration of isothiocyanates 101 increases until reaching, at time t1, a threshold S_SFN. In addition, between times t0 and t1, the quantity of Keapl proteins capable of capturing Nrf2 proteins and not inhibited by isothiocyanates decreases until reaching, at time t1, a threshold S_Keapl. The threshold S_Keapl corresponds for example to the predetermined threshold from which the Nrf2 proteins are free to circulate inside the cell due to the inhibition of the Keapl proteins by the isothiocyanates. Below the S_Keapl threshold, for example between times t0 and tl, the concentration of isothiocyanates is not sufficient to inhibit the excess Keapl proteins and thus allow the free circulation of Nrf2 proteins, and therefore fails to slow down or stop cellular oxidation mechanisms.Below the S_Keapl threshold, Nrf2 proteins are present in excess relative to Keapl proteins. active, i.e. capable of capturing Nrf2 proteins, an increasing proportion of Keapl proteins being inhibited by isothiocyanates. The S_Keapl threshold thus corresponds to a dynamic equilibrium situation in which the kinetics of capture, by Keapl proteins, of newly synthesized Nrf2 proteins counterbalances the kinetics of synthesis of Nrf2 proteins. Above the S_Keapl threshold, active, i.e. uninhibited, Keapl proteins are present in excess of Nrf2 proteins. The S_SFN threshold corresponds, for example, to a concentration of isothiocyanates 101 allowing the S_Keapl threshold to be reached.

[0078] For example, the instants t0 and tl are separated by a duration of the order of a few tens of minutes.

[0079] Between time t1 and a time t2, subsequent to time t1, the concentration of isothiocyanates 101 continues to increase, passes through a maximum then decreases until reaching, at time t2, the threshold S_SFN. Between times t1 and t2, the concentration of isothiocyanates remains higher than the threshold S_SFN. Furthermore, between times t1 and t2, the quantity of uninhibited Keapl proteins decreases, passes through a minimum then increases until reaching, at time t2, the threshold S_Keapl. Crossing, at time t1, the threshold S_Keapl causes, between times t1 and t2, an increase in the concentration of Nrf2 proteins capable of circulating freely inside the cell, thus activating mechanisms aimed at slowing down or stopping cellular oxidation. This thus leads, from time t1, to an increase in the production of antioxidant species.

[0080] For example, the instants t1 and t2 are separated by a duration of the order of a few tens of minutes.

[0081] For comparison, [Fig.4] also illustrates, by a curve 409, an evolution as a function of time t of a concentration of isothiocyanates, for example sulforaphane SFN, in the cell following an ingestion of isothiocyanate precursors, for example identical or analogous to the source of glucosinolates 203. These precursors are for example ingested in the form of capsules or tablets. In this case, the concentration of isothiocyanates 101 increases from time t0 to reach a maximum at time t3, after time t2. Curve 409 differs from curve 401 in particular in that curve 409 always remains below the threshold S_SFN corresponding to the threshold S_Keapl from which the antioxidant mechanisms of the cell are activated.This is because the ingestion of precursors, for example the glucosinolate source 203, causes a slower release of isothiocyanates 101 in the body, i.e. over a longer period, than in the case of ingestion of solution S3 or solution S3'. In the example shown, the total quantity of isothiocyanates produced by ingestion of the precursors, corresponding to the area under the curve 409, is lower, for example twenty times in . lower than that produced by ingestion of solution S3 or solution S3', corresponding to the area under curve 401. Furthermore, the maximum value of curve 409, reached at time t3, is lower than the threshold S_SFN, i.e. the ingestion of isothiocyanate precursors does not trigger a release of Nrf2 proteins in the cells.

[0082] For example, the times t2 and t3 are separated by a duration of the order of several hours, or even several tens of hours.

[0083] Between time t2 and a time t4, subsequent to time t2, the concentration of antioxidant species increases until reaching a maximum, at time t4.

[0084] For example, the times t2 and t4 are separated by a duration of the order of several days.

[0085] Between time t4 and a time t5, subsequent to time t4, the concentration of antioxidant species decreases. Furthermore, between time t4 and time t5, the concentration of isothiocyanates 101 decreases until it reaches a value substantially equal to that which preceded the ingestion of the isothiocyanates at time t0, and no longer manages to inhibit the Keapl proteins which then find themselves again in excess compared to the Nrf2 proteins. In this case, all the newly synthesized Nrf2 proteins are captured and then degraded. A new ingestion of solution S3 or solution S3' then makes it possible, for example, to return to a situation similar or identical to that of time t0.

[0086] For example, the ingestion of solution S3 or solution S3' by a living being, for example a human being, suffering from a neurodegenerative disease, for example Parkinson's disease, is repeated once every 4 to 10 days, for example once or twice a week.

[0087] [Fig.5] illustrates, schematically and in the form of blocks, an example of a sequence of steps associated with a process of a neurodegenerative disease, for example Parkinson's disease.

[0088] During an initial step (block 501), a cell of a living organism, for example a human being, exhibits a supposedly normal oxidation. The cell is for example a nerve cell, or neuron, of the living organism. In this case, the cell comprises for example a body and up to two million axons. The cell is for example more precisely a dopaminergic neuron.

[0089] During another step (block 503), subsequent to the initial step 501, the oxidation of the cell increases. For example, the increase in the oxidation of the cell comes from oxidative stress (or oxidative pressure). Oxidative stress corresponds to a state in which the concentration of oxidizing chemical species is higher than normal and manages to damage parts of the cell. Oxidative stress is for example linked to cellular aging. Oxidative stress can be alternatively or complementary caused by exposure to toxic substances, or toxins, such as herbicides, for example the compound known under the trade name "paraquat" (l,r-dimethyl-4,4'-bipyridinium), glyphosate (N-(phosphonomethyl)glycine), etc. or chlorinated substances, for example trichloroethylene (C2HC13).

[0090] During another step (block 505), subsequent to step 503, mitochondria of the cell are oxidized. The oxidation of mitochondria comes for example from the fact that the neurodegenerative disease from which the organism is suffering promotes the capture of Nrf2 proteins by Keapl proteins, thus tending to inhibit the antioxidant mechanisms activated, or promoted, by the free circulation of Nrf2 proteins inside the cell.

[0091] Oxidation of the cell's mitochondria leads to an increase in oxidative stress, consequently leading (arrow 507) to an increase in cell oxidation during step 503. The mechanisms at work during steps 503 and 505 form a vicious circle leading to an acceleration of cell oxidation.

[0092] During another step (block 509), subsequent to step 505, the oxidation of the mitochondria leads to a loss of energy inside the cell.

[0093] During another step (block 511), subsequent to step 509, all or part of the axons of the cell are destroyed.

[0094] During another step (block 513), subsequent to step 511, the cell dies.

[0095] This leads (arrow 515), in a case where the cell is a dopaminergic neuron, to a drop in dopamine in the living organism.

[0096] Steps 503, 505 and 509 are reversible steps, while steps 511 and 513 are irreversible steps.

[0097] The ingestion, by the living organism suffering from a neurodegenerative disease, of the solution S3 produced according to the method 200 previously described in relation to [Fig.2A], or of the solution S3' produced according to the variant 200' previously described in relation to [Fig.2B], advantageously makes it possible to slow down or stop the cellular oxidation cycle, symbolized in [Fig.5] by the arrow 507 between the blocks 503 and 505, thus preventing the subsequent steps 509, 511, 513 and 515 from occurring.

[0098] [Fig.6] represents, schematically and in the form of blocks, an example of a system 600 adapted to implement the method 200 of [Fig.2A] or the variant 200' of [Fig.2B] according to one embodiment.

[0099] In the example shown, the system 600 comprises a container 601 adapted to contain a reaction medium 603. The reaction medium corresponds for example to the solutions S1, S2 or S2', and S3 or S3', previously described in relation to FIGS. 2A and 2B. In this example, the system 600 further comprises a weighing device 605. The weighing device 605 makes it possible for example to measure the mass of the glucosinolate source 203, the mass of water 205 and / or the mass of the source of enzymes 213 placed in the container 601.

[0100] In the illustrated example, the system 600 further comprises a stirring and heating device 607. The stirring and heating device 607 makes it possible, for example, to maintain stirring, for example at a constant speed, the reaction medium 603 contained in the container 601 during the implementation of the steps of the method 200 or 200'. In addition, the stirring and heating device 607 makes it possible, for example, to bring the water 205 to a boil prior to the production of the solution S1 and to maintain the reaction medium 603 (solution S2 or S2') at the temperature T2 for the duration D2 during the step 219.

[0101] In the example shown, the system 600 further comprises a temperature sensor 609 making it possible to measure the temperature of the reaction medium 603.

[0102] In the example illustrated in [Fig.6], the weighing device 605, the stirring and heating device 607 and the temperature sensor 609 are connected, or linked, to a processing unit 611, or processing circuit 611. The processing unit 611 is for example configured to implement the sequence of steps of the method 200 or 200', in particular to apply a ramp for increasing the temperature of the water 205 prior to the formation of the solution S1 and to regulate, during the duration D2, the temperature T2 of the reaction medium 603 around a set value. For example, the processing unit 611 is a microcontroller or a microprocessor.

[0103] In the example shown, the processing unit 611 is linked, or connected, to a human-machine interface (HMI) 613. The human-machine interface 613 comprises for example one or more elements chosen from a display screen, for example a touch screen, indicator lights, push buttons, switches, etc. The human-machine interface 613 is for example configured to allow a user to control the implementation of the steps of the method 200 or 200' by the system 600 in an at least partially automated manner.

[0104] Although this has not been detailed in [Fig.6], the system 600 may further comprise other elements or circuits, for example memory circuits, a power connector, communication buses, etc. These elements or circuits have been symbolized, in [Fig.6], by a single functional block 615 (FCT).

[0105] Various embodiments and variants have been described. Those skilled in the art will understand that certain features of these various embodiments and variants could be combined, and other variants will occur to those skilled in the art. In particular, those skilled in the art are able to adapt the mass of glucosinolate source 203 used for implementing the method 200 or 200' depending on a concentration of glucosinolates 103 present in this source. Thus, the lower the concentration of glucosinolates 103, the greater the mass of glucosinolate source 203 used.

[0106] Furthermore, the temperatures T1 and T2 and the durations DI and D2 can be adjusted, for example depending on the source of glucosinolates 203 and the source of enzymes 213. These adjustments are within the reach of the person skilled in the art from the indications of the present description.

[0107] Furthermore, although the embodiments described take as an example a case in which the enzyme is myrosinase, this example is not limiting and the embodiments apply more generally to any type of enzyme capable of converting glucosinolates into isothiocyanates.

[0108] Finally, the practical implementation of the embodiments and variants described is within the reach of the person skilled in the art from the functional indications given above. In particular, the programming of the processing unit 611 for the implementation of the method 200 and the variant 200' is within the reach of the person skilled in the art from the indications of the present description.

[0109] Furthermore, the person skilled in the art is capable, based on the indications of the present description, of adapting in particular the quantities, durations and temperatures involved in the implementation of the method 200 or 200' to produce a solution S3 or S3' comprising isothiocyanates having a concentration and bioavailability adapted to: - induce, in the blood of a living organism, for example a human being, having ingested solution S3 or S3', a concentration of isothiocyanates exceeding a threshold from which Nrf2 proteins are free to circulate inside cells of the living organism; - reduce or stop oxidative stress in astrocytes and / or neurons of people suffering from a neurodegenerative disease, for example Parkinson's disease; - attenuate, by the action of Nrf2 proteins, an oxidation of mitochondria in cells of a living organism, for example a human being, leading for example to reversing the vicious circle, symbolized by arrow 505 of process 500 of [Fig.5], in cells of a living organism suffering from a neurodegenerative disease, for example Parkinson's disease; and / or - alleviate all or part of the symptoms of neurodegenerative diseases, for example Parkinson's disease, for example all or part of the non-motor symptoms of Parkinson's disease.

Claims

Claims

1. A method (200; 200') for producing isothiocyanates (101) from glucosinolates (103), the method comprising the following successive steps: a) forming (201) a first solution (SI) by mixing a source of glucosinolates (203) with water (205), the first solution being maintained at a first temperature (Tl) for a first time (Dl), and then optionally cooling the first solution; b) adding a source of enzymes (213; 215) to the first solution, the enzymes being capable of converting glucosinolates into isothiocyanates, whereby a second solution (S2; S2') is formed; and c) maintaining (219) the second solution at a second temperature (T2) for a second time (D2), whereby a third solution (S3; S3') containing isothiocyanates is formed.

2. Method (200; 200') according to claim 1, wherein: - the first temperature (Tl) is between 70 and 100°C, preferably between 90 and 100°C, more preferably equal to approximately 95°C; and - the first duration (Dl) is between 1 and 10 min, preferably between 1 and 5 min, more preferably equal to approximately 2 min.

3. The method (200; 200') of claim 1 or 2, wherein the enzyme source (213; 215) contains myrosinase (105).

4. Method (200; 200') according to claim 1, 2 or 3, wherein the source of enzymes (213; 215) is: - a powder obtained by grinding white mustard seeds; or - a fourth solution (S4) obtained by mixing a powder obtained by grinding white mustard seeds and water (217).

5. Method (200; 200') according to any one of claims 1 to 4, wherein: - the second temperature (T2) is between 50 and 70°C, preferably between 55 and 65°C, more preferably equal to approximately 60°C; and - the first duration (D2) is between 25 and 90 min, preferably between 30 and 50 min, more preferably equal to approximately 35 min.

6. Method (200; 200') according to any one of claims 1 to 5, in which the source of glucosinolates (203) is a powder obtained by grinding seeds of a plant belonging to the order Capparales, preferably a plant belonging to the Brassicaceae family, more preferably broccoli.

7. Composition (S3; S3') in the form of an aqueous solution comprising isothiocyanates (101) and enzymes (105) capable of converting glucosinolates (103) into isothiocyanates, and optionally glucosinolates.

8. Composition (S3; S3') according to claim 7, wherein the enzymes (105) are myrosinases.

9. Composition (S3; S3') according to claim 7 or 8, wherein the glucosinolates (103) are glucoraphanins and the isothiocyanates (101) are sulforaphanes.

10. Composition (S3; S3') according to claim 7, 8 or 9, for use as a medicament.

11. Composition (S3; S3') according to claim 7, 8 or 9, for its use in the treatment of neurodegenerative diseases, preferably in the treatment of Parkinson's disease.

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