Tear gas generating kit

Abstract

The present description discloses a kit and a method with which it is possible to generate a lachrymatory factor gently.　The present disclosure relates to a lachrymatory factor generating kit 1 comprising: (1) a dry PRENCSO composition 2 that includes PRENCSO and a porous carrier thereof, and that is dried; and (2) a dry enzyme composition 3 that includes alliinase, lachrymatory factor synthase (LFS), and a porous carrier thereof, and that is dried. The present disclosure also relates to a method for generating a lachrymatory factor, the method including bringing water into contact with the dry PRENCSO composition 2 and the dry enzyme composition 3.

Description

Technical Field

[0001] The present invention relates to a lachrymatory component generation kit. The present invention also relates to a method for generating a lachrymatory component.

Background Art

[0002] Thiopropanal S-oxide, which is the lachrymatory component (LF) of onions, is produced when the enzyme alliinase acts on the precursor PRENCSO (S-1-propenyl-cysteine sulfoxide) to form sulfenic acid (E-1-propensulfenic acid), and the sulfenic acid is isomerized by the lachrymatory component-forming enzyme (LFS).

[0003] Lachrymatory components are known to be industrially useful compounds. For example, it is known that lachrymatory components can be used as reagents for tear secretion tests and tear secretion test methods, or in other words, for dry eye tests (Patent Document 1). It is also known that lachrymatory components can be used as antibacterial agents (Patent Document 2).

[0004] Since lachrymatory components have low stability and are easily decomposed, when attempting to use lachrymatory components industrially, it is preferable to generate lachrymatory components at the site where they are used.

[0005] Patent Document 3 describes a lachrymatory component generation kit. The lachrymatory component generation kit described in Patent Document 3 includes a dry mixed enzyme produced by absorbing a solution containing alliinase and LFS onto absorbent paper and drying it, and PRENCSO. In Patent Document 3, it is described as a specific embodiment that PRENCSO contained in the kit can be stably stored by making it an acidic solution, and that a lachrymatory component can be generated by dropping an acidic solution of PRENCSO onto the dry mixed enzyme.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] WO2005 / 067907 [Patent Document 2] Japanese Patent Publication No. 2007-267639 [Patent Document 3] Japanese Patent Publication No. 2008-285476 [Overview of the project] [Problems that the invention aims to solve]

[0007] In the method described in Patent Document 3, which involves applying a PRENCSO acidic solution to a dried mixed enzyme to generate a tear-inducing component, it was necessary to measure a specified amount of PRENCSO acidic solution and apply it to the dried mixed enzyme.

[0008] The inventors have also found that in the method described in Patent Document 3, which generates a tear-inducing component by dropping an acidic solution of PRENCSO onto a dry mixed enzyme, the reaction rate is fast and the tear-inducing component is generated rapidly. Therefore, when used for the purpose of exposing a subject's eyes to the generated tear-inducing component to collect tears, the subject experiences pain. The present invention aims to solve these problems. [Means for solving the problem]

[0009] This specification discloses the following specific embodiments as means for solving the aforementioned problems. [1](1) A dry PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal component-producing enzyme (LFS), and a porous carrier on which they are carried, and which is dry. A tear gas generating kit equipped with tear gas components. [2] A tear gas generating kit as described in [1], which combines (1) and (2). [3](1) further comprising sugars, the tear gas generating kit according to [1] or [2]. [4] The tear gas component generating kit according to [3], comprising 5 parts by mass or more of sugars per 1 part by mass of PRENCSO. [5](1) A dry PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal component-producing enzyme (LFS), and a porous carrier on which they are carried, and which is dry. A method for generating tear gas components, including contact with water. [6] The method described in [5], wherein (1) and (2) are combined. [7](1) further comprising sugars, the method according to [5] or [6]. [8] The method according to [7], comprising 5 parts by mass or more of sugars per 1 part by mass of PRENCSO. [9] The method according to any one of [5] to [8], wherein the water is an aqueous solution containing a surfactant.

[0010] This specification includes the disclosures of Japanese Patent Application No. 2021-051943, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0011] According to the tear gas component generating kit and method described above, a tear gas component can be gently generated by contacting it with water. Therefore, when the generated tear gas component is used to collect tears by exposing the subject's eyes to it, the eye pain felt by the subject is suppressed. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the results of the LF (tear gas component) generation comparison test in Experiment 1. [Figure 2] Figure 2 shows the results of the surfactant addition test in Experiment 1. [Figure 3] Figure 3 shows the results of the pain comparison test in Experiment 1. [Figure 4]Figure 4 shows the average values of the residual rates of LF generation amounts of the integrated tear component generation kit 1 (equipped with a lactose-free PRENCSO pad) and the integrated tear component generation kit 2 (equipped with a lactose-containing PRENCSO pad) after storage at 4°C for 1 month, 3 months, 6 months, and 12 months in Experiment 2. [Figure 5] Figure 5 shows the average values of the residual rates of LF generation amounts of the integrated tear component generation kits 1 and 2 after storage at 20°C for 1 month, 3 months, and 6 months in Experiment 2. [Figure 6] Figure 6 shows the average values of the residual rates of LF generation amounts of the integrated tear component generation kits 1 and 2 after storage at 30°C for 1 month, 3 months, 6 months, and 12 months in Experiment 2. [Figure 7] Figure 7 is a plan view of a tear component generation kit in which a sheet-like dry PRENCSO composition and a sheet-like dry enzyme composition are laminated and integrated. [Figure 8] Figure 8 is a cross-sectional view of the tear component generation kit shown in Figure 7 taken along line A-A in Figure 7. [Figure 9] Figure 9 is a plan view of a tear component generation kit in which a sheet-like dry PRENCSO composition and a sheet-like dry enzyme composition are arranged adjacent to each other. [Figure 10] Figure 10 is a cross-sectional view of the tear component generation kit shown in Figure 9 taken along line B-B in Figure 9. [Figure 11] Figure 11 is a plan view of a tear component generation kit including one carrier in which a porous carrier carrying PRENCSO and a porous carrier carrying alliinase and LFS are integrated, a dry PRENCSO composition in which PRENCSO is carried on a part of the one carrier, and a dry enzyme composition in which alliinase and LFS are carried on another part of the one carrier. [Figure 12] Figure 12 is a cross-sectional view of the tear component generation kit shown in Figure 11 taken along line C-C in Figure 11.

Mode for Carrying Out the Invention

[0013] One or more embodiments of the invention disclosed herein are (1) A dry PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal component-producing enzyme (LFS), and a porous carrier on which they are carried, and which is dry. This relates to a tear gas generating kit equipped with a tear gas component.

[0014] One or more other embodiments of the invention disclosed herein are: (1) A dry PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal component-producing enzyme (LFS), and a porous carrier on which they are carried, and which is dry. The present invention relates to a method for generating tear gas components, including contact with water.

[0015] According to the kit and method disclosed herein, a tear gas component (LF) (thiopropanal S-oxide) can be gently generated by a simple operation of contacting it with water. Furthermore, since there is no need to strictly control the amount of water added, it is easy to implement in situations where the tear gas component is used.

[0016] In this specification, "dried" and "being dry" are not limited to a completely dry state in which there is no water at all in the dried PRENCSO composition and the dried enzyme composition, but include a state in which they contain about 0% to 20% by mass of water. That is, each composition may contain preferably 20% by mass or less, more preferably 10% by mass or less, more preferably 3% by mass or less, and more preferably 2% by mass or less of water relative to the total amount of each composition. The water content of the dried PRENCSO composition and the dried enzyme composition can be measured by completely drying these compositions and determining the ratio of the mass reduced by complete drying to the total mass of the composition before complete drying.

[0017] A preferred embodiment of the dried PRENCSO composition will be described below. PRENCSO (S-1-propenyl-cysteine ​​sulfoxide) may be artificially synthesized or extracted from natural products. It is preferable to use PRENCSO that has been purified by ion exchange resin or reverse-phase chromatography.

[0018] In the above-mentioned dried PRENCSO composition, a porous carrier having water absorption properties is preferred, a porous carrier containing a hydrophilic polymer such as cellulose and having water absorption properties is more preferred, and a paper having water absorption properties is particularly preferred. The shape of the porous carrier is not particularly limited, but a sheet shape is preferred. A dried PRENCSO composition containing a porous carrier supporting PRENCSO can be obtained by allowing a PRENCSO-containing solution to be absorbed into the porous carrier and then drying it. The aforementioned dried PRENCSO composition preferably further contains an acid such as hydrochloric acid. Patent Document 3 describes that PRENCSO is stabilized in an acidic aqueous solution.

[0019] The dried PRENCSO composition preferably further contains sugars. The dried PRENCSO composition containing sugars is suitable for long-term storage because the decomposition of PRENCSO during storage is suppressed compared to the case without sugars. Furthermore, a tear gas component generating kit equipped with the dried PRENCSO composition containing sugars is preferable because the reduction in the amount of tear gas component generated after long-term storage is suppressed compared to the case without sugars.

[0020] The type of sugar is not particularly limited; for example, one or more selected from monosaccharides, disaccharides, polysaccharides, and sugar alcohols can be used. Monosaccharides, disaccharides, polysaccharides, and sugar alcohols each also include esters with fatty acids.

[0021] Examples of monosaccharides include one or more selected from glucose and fructose. Examples of disaccharides include one or more selected from trehalose, sucrose, sucrose esters, maltose, and lactose. Examples of polysaccharides include dextrin. Examples of sugar alcohols include one or more selected from mannitol, isomaltulose (trade name Palatinose®), and reduced isomaltulose.

[0022] In the dried PRENCSO composition, the ratio of PRENCSO to sugars is not particularly limited, but preferably contains 5 parts by mass or more, more preferably 10 parts by mass or more, more preferably 50 parts by mass or more, and more preferably 100 parts by mass or more of sugars per 1 part by mass of PRENCSO. The upper limit of the ratio is not particularly limited, but typically contains preferably 1000 parts by mass or less, more preferably 500 parts by mass or less, and more preferably 200 parts by mass or less of sugars per 1 part by mass of PRENCSO. The amount of sugars in the ratio refers to the total amount of multiple types of sugars if multiple types of sugars are included.

[0023] The method for producing the dried PRENCSO composition preferably involves absorbing a solution containing PRENCSO onto a porous carrier and drying it. In this embodiment, PRENCSO and other components present in the solution are supported on the porous carrier. If the dried PRENCSO composition contains sugars, the solution can be a solution containing PRENCSO and sugars. The solution is preferably an aqueous solution, more preferably an acidic aqueous solution further containing an acid such as hydrochloric acid, and particularly preferably an acidic aqueous solution with a pH of 3.5 or less. The drying method is not particularly limited, but freeze-drying or hot-air drying are examples.

[0024] Freeze-drying typically involves pre-freezing the sample at -80°C to -20°C, followed by vacuum drying. Any suitable temperature can be used for the drying temperature. Freezing methods include, but are not limited to, immersion in liquid nitrogen, placement in a freezer at -4°C to -80°C, and placement in a dry ice and alcohol freezing bath. The drying temperature should be higher than the freezing temperature, preferably below 80°C, more preferably below 65°C, and most preferably in the range of 20°C to 50°C. Hot air drying can be carried out at a temperature of, for example, 40°C to 80°C, preferably around 60°C.

[0025] Next, preferred embodiments of the dried enzyme composition will be described. As the alliinase, alliinase derived from plants of the Alliaceae family can be used, with garlic-derived alliinase being particularly preferred. The alliinase may be purified or crudely purified alliinase. Crudely purified alliinase is, for example, extracted from garlic by a simple method. A simple method for preparing crudely purified alliinase is to "add acid to an extract of garlic that has been hydrated and crushed, precipitate the alliinase at its isoelectric point, collect the precipitate, and redissolve it." Note that the material from which the alliinase is extracted is not limited to garlic.

[0026] The above-mentioned dried enzyme composition may further contain an alliinase protectant. Examples of alliinase protectants include sugars, particularly preferably disaccharides. Examples of disaccharides include one or more disaccharides selected from sucrose, trehalose, maltose, lactose, or cerbiose. In addition to sugars, the alliinase protectant may further contain a salt. Examples of salts include any salt, such as KCl, MgCl2, and NaCl, with monovalent metal salts being preferred, and specifically NaCl being preferred. Furthermore, the alliinase protectant may also contain pyridoxal phosphate. That is, the alliinase protectant preferably consists of (a) sugars, or (b) sugars and a salt and / or pyridoxal phosphate.

[0027] As LFS, LFS and its mutant enzymes derived from plants belonging to the Alliaceae family (preferably onions) produced by genetic engineering, for example, LFS described in Republished Publication No. 02 / 020808 and Republished Publication No. 03 / 074707 can be used. Particularly preferred as LFS is LFS prepared by the genetic engineering method described in Republished Publication No. 02 / 020808 (WO02 / 020808). Alternatively, LFS isolated from Alliaceae plants, especially onions, can also be used.

[0028] LFS may be purified LFS or crudely purified LFS. Crudely purified LFS can be exemplified by LFS extracted from onions using a simple method. A simple method for preparing crudely purified LFS is to "treat the extract of onions that have been hydrated and crushed with an anion exchange resin, adsorb the onions, and then elute them." The material from which the LFS is extracted is not limited to onions.

[0029] In the aforementioned dried enzyme composition, the ratio of alliinase to LFS is not particularly limited, but the amount of alliinase per 1 μg of LFS can be, for example, 0.02 units to 200 units, more preferably 0.5 units to 50 units. The LFS referred to here may be the rLFS used in the examples, and its specific activity is 6.0 × 10⁻⁶. 6 It is measured in area / μg.

[0030] The porous carrier contained in the dried enzyme composition is preferably a porous carrier having water absorption properties, more preferably a porous carrier containing a hydrophilic polymer such as cellulose and having water absorption properties, and particularly preferably paper having water absorption properties. The shape of the porous carrier is not particularly limited, but it is preferably in sheet form.

[0031] The method for producing the dried enzyme composition preferably involves absorbing a solution containing alliinase and LFS onto a porous carrier and drying it. The solution containing alliinase and LFS is preferably an aqueous solution, and more preferably an aqueous solution further containing the above-mentioned alliinase protective agent. The mixing ratio of alliinase and LFS in the solution may be the same as the mixing ratio in the dried PRENCSO composition.

[0032] The drying method is not particularly limited, and examples include freeze-drying or hot-air drying. Preferred embodiments of freeze-drying and hot-air drying are described with respect to the dried PRENCSO composition.

[0033] Next, preferred embodiments of the kit and method relating to this disclosure will be described. In the method for generating a tear gas component and the tear gas component generating kit according to this disclosure, it is preferable that the dried PRENCSO composition and the dried enzyme composition are integrated. If the pre-integrated dried PRENCSO composition and the dried enzyme composition are used, there is no need to mix the two compositions at the site where the tear gas component is to be used, and the tear gas component can be generated by bringing only water into contact with it. The tear gas component generating kit according to this disclosure, comprising the dried PRENCSO composition and the dried enzyme composition, wherein the dried PRENCSO composition and the dried enzyme composition are integrated, may be referred to as a "tear gas component generating device" or a "tear gas component generating composition".

[0034] In a more preferred embodiment of the configuration in which the dried PRENCSO composition and the dried enzyme composition are integrated, the dried PRENCSO composition and the dried enzyme composition are integrated in a way that allows water to permeate them. In an even more preferred embodiment, the dried PRENCSO composition and the dried enzyme composition are bonded and integrated via adhesive means such as an adhesive or a water-permeable double-sided tape. In one embodiment of these embodiments, it is particularly preferable that the porous carrier supporting the PRENCSO and the porous carrier supporting the alliinase and LFS are both in sheet form, and that the dried PRENCSO composition and the dried enzyme composition are laminated and integrated.

[0035] Furthermore, in another embodiment of the kit and method according to this disclosure, the dried PRENCSO composition and the dried enzyme composition are arranged adjacent to each other. In the kit according to this embodiment, when water is added to at least one of the dried PRENCSO composition and the dried enzyme composition, the water diffuses throughout, thereby generating a tear-inducing component.

[0036] Furthermore, in yet another embodiment of the kit and method according to the present disclosure, a porous carrier supporting PRENCSO and a porous carrier supporting alliinase and LFS are integrated into a single carrier, wherein PRENCSO is supported on a portion of the single carrier, and alliinase and LFS are supported on another portion of the single carrier. In the kit according to this embodiment, the portion of the single carrier supporting PRENCSO is a dry PRENCSO composition, and the other portion of the single carrier supporting alliinase and LFS is a dry enzyme composition. In the kit according to this embodiment, when water is added to the single carrier, the water diffuses throughout, thereby generating a tear gas component.

[0037] Figures 7 and 8 show an example of a tear gas generating kit in which a dried PRENCSO composition and a dried enzyme composition are laminated and integrated. In this example, the tear gas generating kit 1 consists of a sheet-shaped dried PRENCSO composition 2 and a sheet-shaped dried enzyme composition 3 that are laminated and integrated. In this example of the tear gas generating kit 1, it is preferable that the dried PRENCSO composition 2 and the dried enzyme composition 3 are laminated in such a way that water can permeate through their boundary 4. Although not shown, adhesive means such as an adhesive or water-permeable double-sided tape may be interposed between the dried PRENCSO composition 2 and the dried enzyme composition 3 at the boundary 4.

[0038] Figures 9 and 10 show an example of a tear gas generating kit in which a dried PRENCSO composition and a dried enzyme composition are arranged adjacent to each other. In this example, the tear gas generating kit 1 has a sheet-shaped dried PRENCSO composition 2 and a sheet-shaped dried enzyme composition 3 arranged adjacent to each other on one surface. In this example of the tear gas generating kit 1, it is preferable that the boundary 5 between the dried PRENCSO composition 2 and the dried enzyme composition 3 is permeable to water. At the boundary 5, the dried PRENCSO composition 2 and the dried enzyme composition 3 may or may not be bonded.

[0039] Figures 11 and 12 show an example of a tear gas component generating kit comprising a single carrier comprising a porous carrier supporting PRENCSO and a porous carrier supporting alliinase and LFS, a dry PRENCSO composition in which PRENCSO is supported on a portion of the single carrier, and a dry enzyme composition in which alliinase and LFS are supported on another portion of the single carrier. In this example, the tear gas component generating kit 1 comprises a dry PRENCSO composition 2, which is a portion of a sheet-shaped carrier 10 in which PRENCSO is supported, and a dry enzyme composition 3 in which alliinase and LFS are supported on another portion of the sheet-shaped carrier 10.

[0040] In the method for generating tear gas components and the tear gas component generating kit according to this disclosure, the water that is brought into contact with the dried PRENCSO composition and the dried enzyme composition to generate the tear gas components may be pure water or an aqueous solution containing other components. Examples of other components include surfactants. When an aqueous solution of surfactant is brought into contact with the dried PRENCSO composition and the dried enzyme composition, the tear gas components tend to be generated more easily than when pure water is used. As the surfactant, nonionic surfactants or amphoteric surfactants are preferred, and nonionic surfactants are particularly preferred. Examples of nonionic surfactants include polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, etc. The surfactant is preferably added to the water to a final concentration of 0.005% to 1% by mass. The tear gas component generating kit according to this disclosure may further include an aqueous solution containing a surfactant as a component.

[0041] The method for generating tear gas components and the tear gas component generating kit described herein are not limited to human subjects. Specifically, they can also be applied to mammals such as monkeys, dogs, cats, horses, cattle, sheep, rabbits, and mice.

[0042] There are no specific limitations on the method of exposing the eyes to LF (tear gas component), but the most efficient method is to place a tear gas component generating kit in a tear gas cup, fill it with LF, and then tightly seal it over one of the subject's eyes so that the opening covers the eye. Alternatively, since it is sufficient for the volatile LF to enter the eye, tear gas can also be produced by placing the tear gas component generating kit below the eye. The tear gas component generating kit can be placed below the eye by directly attaching it below the eye or by using a device such as goggles designed to position the tear gas component generating kit below the eye. [Examples]

[0043] <Experiment 1> 1. How to prepare PRENCSO pads 1.1.Materials 20mg / ml PRENCSO solution 0.1N hydrochloric acid Sugars (lactose) Thin circular filter paper (φ8mm, thickness 0.7mm) -80 degree freezer freeze dryer

[0044] 1.2. Method (1) A 20 mg / ml PRENCSO solution was diluted with a mixture of ultrapure water and 0.1 N hydrochloric acid (15:1, v / v) to a final concentration of PRENCSO of 0.67 mg / ml to prepare a 0.67 mg / ml PRENCSO hydrochloric acid solution. (2) A 20 mg / ml PRENCSO solution was diluted with a mixture of ultrapure water and 0.1 N hydrochloric acid (15:1, v / v) to a final concentration of PRENCSO of 1 mg / ml to prepare a 1 mg / ml PRENCSO hydrochloric acid solution. (3) A 1 mg / ml PRENCSO lactose hydrochloride solution was prepared by diluting a 20 mg / ml PRENCSO solution and lactose with a mixture of ultrapure water and 0.1 N hydrochloric acid (15:1, v / v) so that the final concentration of PRENCSO was 1 mg / ml and the final concentration of lactose was 100 mg / ml. (4) Each of the above solutions was applied in 30 μl portions to circular filter paper and frozen at -80°C. (5) After freezing, the pads were dried in a freeze dryer for 14 hours or more to form the PRENCSO pads.

[0045] 2. Method for preparing enzyme pads (1) Preparation of LFS Lacrimal-inducing enzyme (LFS) was produced by genetic engineering using the method described in WO02 / 020808.

[0046] The summary is as follows: A plasmid expressing the LFS protein (pGEX-4T-3-E2-3-1) was introduced into E. coli using the competent method to obtain transformants. The obtained transformants were cultured with shaking at 37°C in LB medium containing 100 μg / ml ampicillin. When isopropyl-β-thiogalactopyranoside (IPTG) was added to the medium to induce production, a fusion protein of glutathione S-transferase (GST) and E2-3-1 (hereinafter referred to as GST-E2-3) accumulated in the bacterial cells.

[0047] The transformed cells were cultured as described above, and after collecting the cells by centrifugation, they were sonicated. The supernatant recovered by centrifugation was passed through a glutathione Sepharose 4 Fast Flow column (Amersham Pharmacia) to adsorb the GST fusion protein onto the column. After washing the column, the fusion protein was eluted with an elution buffer containing reduced glutathione to obtain a purified E2-3 fusion protein sample.

[0048] The fusion protein sample was passed through a HiTrap Desalting column (Amersham Pharmacia) to remove reduced glutathione, and then adsorbed onto a glutathione Sepharose 4 Fast Flow column. After washing the column, it was filled with a buffer containing thrombin and protease treatment was performed at room temperature for 2 hours to cleave the GST tag from the fusion protein. Recombinant E2-3, with the GST tag removed, was eluted from the column, and Benzamidine Sepharose 6 was added to the eluate and mixed. Thrombin in the eluate was removed by centrifugation to obtain the recombinant E2-3 sample (this is referred to as rLFS).

[0049] (2) Preparation of alliinase (2-1) Crush the garlic and precipitate it in acid. First, the mixer jug ​​was placed in the refrigerator to cool. The rotor was also set in a cryogenic centrifuge, and the temperature was set to 4°C for cooling. Equal amounts of buffer A were added to 100g of garlic cloves and ground in the mixer. A double layer of gauze was secured to the mouth of the jug, which was placed on ice, with a rubber band. The ground material was poured onto the gauze and filtered. After obtaining a sufficient amount of filtrate, the ground material on the gauze was wrapped in the gauze and squeezed. The obtained filtrate was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. While stirring the collected supernatant on ice, acetic acid was added while monitoring the pH until the pH reached 4.0. Once the pH reached 4.0, it was left to stand for 5 minutes. The sample from which the precipitate had formed was centrifuged at 12000 rpm for 10 minutes at 4°C, and the centrifugal pellet was collected (using buffer A).

[0050] The collected pellet was diluted to 50 ml to 100 ml (buffer A) and allowed to stand at 5°C for 30 minutes. The sample was centrifuged at 12000 rpm for 10 minutes at 4°C. The pH of the recovered supernatant was adjusted to 6.5 using a 1N sodium hydroxide aqueous solution. This was used as the crude alliinase extract.

[0051] (2-2) Hydroxyapatite column treatment The crude alliinase extract (centrifugation supernatant) described above was applied to a hydroxyapatite column equilibrated with buffer A. It was adsorbed onto the hydroxyapatite column as a yellow band. The hydroxyapatite column with the applied sample was washed with 300 ml of buffer A. After washing, 300 ml of buffer C was passed through the hydroxyapatite column to eluate it. The eluate was fractionated into 10 ml portions using a fraction collector, and the fraction containing the yellow eluate was collected.

[0052] (2-3) Purification of alliinase using a ConA column The calcium and magnesium ion concentrations in the sample solution were increased by adding 1 / 20th the volume of a 20 mM calcium chloride and magnesium chloride solution to the collected fraction (20 ml). The sample was then applied to a regenerated ConA column equilibrated with starting buffer (the recommended buffer described in the ConA Sepharose 4B manual). The ConA column was washed with 50 ml of starting buffer after the sample application. Elution was then performed by flowing 50 ml of ConA elution buffer (the recommended buffer described in the ConA Sepharose 4B manual) through the washed ConA column. The eluate was fractionated into 2 ml portions using a fraction collector, and the fraction containing the yellow eluate was collected.

[0053] (2-4) Method for concentrating purified alliinase Ten ml of the yellow eluate (alliinase solution) obtained by ConA column purification was placed in CENTRIPLUS CONCENTRATORS (up to 15 ml, No. 4421) (Millipore). The CENTRIPLUS CONCENTRATORS were placed in a centrifuge cooled to 4°C and centrifuged at 3000 rpm for 30 minutes. After checking the contents once, the centrifuge was repeated at 3000 rpm for another 30 minutes. The activity of the concentrated alliinase solution was measured according to the method shown below to confirm that it was at the required concentration.

[0054] The above-mentioned Buffer A (pH 7.0) (50 mM Buffer A for alliinase purification) was prepared as follows: A 50 mM dipotassium hydrogen phosphate solution (5.22 g dissolved in 600 ml) and a 50 mM potassium dihydrogen phosphate solution (3.4 g dissolved in 500 ml) were prepared. The two solutions were mixed while monitoring the pH, and the pH was adjusted to 7.0. To 9 vol. of the resulting buffer, 1 vol. of glycerol was added and mixed thoroughly. To 1 L of the resulting glycerol-containing buffer, 5.3 mg of pyridoxal phosphate was added and mixed. The resulting buffer was labeled and stored in a low-temperature laboratory (10°C).

[0055] Furthermore, Buffer C (pH 7.0) (500 mM Buffer C for alliinase purification) was prepared as follows: A 500 mM dipotassium hydrogen phosphate solution (43.6 g dissolved in 500 ml) and a 500 mM potassium dihydrogen phosphate solution (34.0 g dissolved in 500 ml) were prepared. The two solutions were mixed while monitoring the pH, and the pH was adjusted to 7.0. To 9 vol. of the resulting buffer, 1 vol. of glycerol was added and mixed thoroughly. To 1 L of the resulting glycerol-containing buffer, 5.3 mg of pyridoxal phosphate was added and mixed. The resulting buffer was labeled and stored in a low-temperature laboratory (10°C).

[0056] (2-5) Method for measuring alliinase activity The activity of alliinase was calculated by measuring the amount of pyruvate produced when alliinase breaks down PRENCSO.

[0057] Using 100 mM phosphate buffer (pH 6.5), 0.16 mg / ml pyridoxal phosphate solution, 3.8 mg / ml NADH solution, 6.0 μg / ml lactate dehydrogenase (LDH) solution, and 20 mg / ml PRENCSO solution were prepared. The alliinase solution to be used as the sample was then diluted 100-fold using the aforementioned pyridoxal phosphate solution. Next, 100 μl of 0.16 mg / ml pyridoxal phosphate solution, 100 μl of 3.8 mg / ml NADH solution, 100 μl of 6.0 μg / ml LDH solution, and 100 μl of 20 mg / ml PRENCSO solution were added to 2500 μl of 100 mM phosphate buffer (pH 6.5) and mixed. To this mixed solution, 100 μl of the alliinase solution to be used as the sample was added to initiate the enzymatic reaction, and the change in absorbance of the reaction solution over time was measured for 3 minutes immediately after the start of the reaction. From the obtained absorbance change results, the absorbance decrease rate was calculated and confirmed to fall within the range of 0.02 to 0.19. If the absorbance decrease rate did not fall within this range, the dilution ratio of the alliinase solution was changed and the measurement was repeated. From the obtained absorbance decrease rate, i.e., the amount of absorbance change per minute, the alliinase activity (Unit) was calculated.

[0058] (3) Preparation of enzyme pads 120 μl of 0.02 mg / ml rLFS solution was mixed with 120 μl of 50 U / ml alliinase solution (10% trehalose, 1% NaCl, 25 μM pyridoxal phosphate). 30 μl of this mixture was absorbed onto thin circular filter paper (φ8 mm, thickness 0.7 mm), and then pre-frozen at -80°C under atmospheric pressure. After freezing, freeze-drying was performed under reduced pressure at -10°C to obtain enzyme pads.

[0059] 3. Method for preparing a tear gas generating kit Each of the prepared PRENCSO pads and enzyme pads was bonded together using tape adhesive (KOKUYO Dotliner) to create an integrated tear gas component generating kit.

[0060] 4. Comparison test of LF (tear gas component) generation amount 4.1.Materials Integrated tear gas generating kit (equipped with a PRENCSO pad prepared by impregnating it with a 0.67 mg / ml PRENCSO hydrochloric acid solution) Conventional tear gas generating kits (kits consisting of an enzyme pad and a 0.67 mg / ml PRENCSO hydrochloric acid solution) 1.5ml tube 4ml vial with septum cap Glass syringe (with needle) Vortex Mixer 0.45 μm filter 1 ml syringe

[0061] 4.2. Equipment Waters Alliance HPLC

[0062] 4.3. High-Performance Liquid Chromatography (HPLC) Analysis Conditions Column ODS (Sensu Science) Detection at 254nm Oven temperature 35℃ Injection volume: 20 μl Flow rate 0.6ml / min Mobile phase (MeOH: pH 3.3, TFA water = 30%:70%, isocratic) Sample cooler 5°C

[0063] 4.4. Method (1) LF generation using conventional tear gas component generating kits (1-1) As a conventional tear gas component generating kit described in Japanese Patent Publication No. 5383075, an enzyme pad without a PRENCSO pad attached was placed in a 4 ml vial, 30 μl of 0.67 mg / ml PRENCSO hydrochloric acid solution was added, and the vial was sealed tightly. (1-2) 30 seconds to 3 minutes after the addition of the above solution, 500 μl of methanol was added to the vial using a glass syringe, and the mixture was stirred with a vortex mixer for 30 seconds to stop the reaction. (1-3) 200 μl of the supernatant was filtered and transferred to an HPLC vial. (1-4) The LF peak area was determined by HPLC at a detection wavelength of 254 nm and a retention time of approximately 9.9 minutes.

[0064] (2) LF generation using an integrated tear gas component generating kit (2-1) An integrated tear gas component generating kit was used, which consisted of a PRENCSO pad prepared by impregnating it with a 0.67 mg / ml PRENCSO hydrochloric acid solution and an enzyme pad that were bonded together. (2-2) The integrated tear gas component generating kit was placed in a 4 ml vial, and 60-100 μl of water was added from either the PRENCSO pad side or the enzyme pad side, and the vial was sealed tightly. (2-3) One minute and two minutes after the addition of water, 500 μl of methanol was added to the vial using a glass syringe, and the mixture was stopped by mixing with a vortex mixer for 30 seconds. (2-4) Filter 200 μl of the supernatant and transfer it to an HPLC vial. (2-5) The LF peak area was determined by HPLC at a detection wavelength of 254 nm and a retention time of approximately 9.9 minutes.

[0065] 5. Surfactant addition test 5.1.Materials 0.05%Tween20 water

[0066] 5.2. Method (1) An integrated tear gas component generating kit was used, which consisted of a PRENCSO pad prepared by impregnating it with a 0.67 mg / ml PRENCSO hydrochloric acid solution and an enzyme pad that were bonded together. (2) The integrated tear gas component generating kit was placed in a 4 ml vial, and 80 μl of water or 0.05% Tween 20 water was added from either the PRENCSO pad side or the enzyme pad side, and the vial was sealed tightly. (3) One minute after adding water or 0.05% Tween 20 water, 500 μl of methanol was added to the vial using a glass syringe and mixed with a vortex mixer for 30 seconds to stop the reaction. (4) The supernatant was filtered and transferred to an HPLC vial. (5) The LF peak area was determined by HPLC at a detection wavelength of 254 nm and a retention time of approximately 9.9 minutes.

[0067] 6. Comparative pain tests 6.1.Materials Integrated tear gas generating kit 1 (equipped with a PRENCSO pad prepared by impregnating it with a 1 mg / ml PRENCSO hydrochloric acid solution) Integrated tear gas generating kit 2 (equipped with a PRENCSO pad prepared by impregnating it with a 1 mg / ml PRENCSO lactose hydrochloride solution) Conventional tear gas generating kits (kits consisting of an enzyme pad and a 1 mg / ml PRENCSO hydrochloric acid solution) Tear gas cup (a bottomed container with an opening shaped to cover one eye) double-sided tape

[0068] 6.2. Subjects Men and women in their 30s, 40s, and 60s

[0069] 6.3. Method (1) In the conventional tear gas component generation kit, one enzyme pad was attached to the bottom of the tear gas cup with double-sided tape, and 30 μl of 1 mg / ml PRENCSO hydrochloric acid solution was dropped onto it to generate the tear gas component (LF). (2) One integrated tear gas component generating kit 1 or 2 was attached to the bottom of the tear gas cup with double-sided tape so that the side and bottom of the PRENCSO pad were in contact with each other, and 80 μl of water was dropped from the side of the enzyme pad to generate LF. (3) The opening of the tear gas cup was sealed with a lid and the tear gas cup was filled with LF for 1 minute. (4) Next, the tear gas cup with the lid removed was placed in close contact with one of the subject's eyes, with the opening covering the eye, and the eye was exposed to LF for 120 seconds. The subject evaluated the intensity of the pain felt after the start of exposure based on the following 5-point scale. The test was conducted on a total of 5 people (subjects A to E), and the average value was calculated. (5) After 120 seconds of exposure, tears were collected in a tear gas cup and the amount of tears was measured.

[0070] [Table 1]

[0071] 7.Results 7.1. Comparison Test of LF (Tear Gas Component) Generation Amount Figure 1 shows the results of the comparative test of LF (tear gas component) generation. When water was dropped onto an integrated tear gas generating kit, which consisted of a PRENCSO pad and an enzyme pad, the amount of LF (Luminous Flux) generated was in the range of 30-58% of the amount of LF generated when PRENCSO solution was dropped onto the enzyme pad of a conventional tear gas generating kit. It was confirmed that LF is generated relatively gently in the integrated tear gas generating kit.

[0072] 7.2. Surfactant Addition Test The results of the surfactant addition test are shown in Figure 2.

[0073] When water or 0.05% Tween20 water was dropped onto the integrated tear gas generating kit from the PRENCSO pad side, no difference was observed in the amount of LF (Low-Frequency) generated, regardless of whether Tween20 was added or not.

[0074] On the other hand, when 0.05% Tween20 water was added to the integrated tear gas generating kit from the PRENCSO pad side, the amount of LF (Low-Frequency Liquid) generated was significantly greater compared to when water was added from the same side. The above results indicate that adding a surfactant to the water dropped into the integrated tear gas component generating kit promotes the generation of LF (Low-Factor Pulse).

[0075] 7.3. Comparative pain tests Figure 3 shows the results of the pain intensity evaluation in the comparative pain test. The graph in Figure 3 shows the change in pain intensity felt by subjects over time, with the horizontal axis representing the exposure time to the eyes (seconds) and the vertical axis representing the pain intensity at each point in time (average of evaluation scores from a total of 5 subjects), for cases where an integrated tear gas component generating kit 1 (PRENCSO pad does not contain lactose) was used to generate LF (integrated kit) and when a conventional tear gas component generating kit was used (conventional kit).

[0076] When using the integrated tear gas generating kit 1 (PRENCSO pad does not contain lactose) to generate LF, the pain intensity was 7.4 at its maximum, 5.3 over 120 seconds, and 5.8 from 20 seconds to 120 seconds after the start of exposure. Although not shown in the figure, the pain intensity when using the integrated tear gas generating kit 2 (PRENCSO pad contains lactose) to generate LF was similar to that when using the integrated tear gas generating kit 1. On the other hand, when using the conventional tear gas generating kit, the pain intensity remained above 6.9 after 20 seconds from the start of exposure, with an average of 8.0 over 120 seconds and an average of 8.4 from 20 seconds to 120 seconds after the start of exposure.

[0077] The amount of tears collected was equivalent when using the integrated tear gas component generating kits 1 and 2, and when using the conventional tear gas component generating kit.

[0078] The results above indicate that the method of exposing the eyes to LF generated by the integrated tear gas component generating kits 1 and 2 to induce tearing and collect tears is less painful and less burdensome for subjects compared to the method using conventional tear gas component generating kits.

[0079] <Experiment 2> 1. Integrated tear gas generating kit (1) A PRENCSO pad, prepared by impregnating it with a 1 mg / ml PRENCSO hydrochloric acid solution according to the procedure described in "1. Method for preparing PRENCSO pads" of Experiment 1, and an enzyme pad, prepared according to the procedure described in "2. Method for preparing enzyme pads" of Experiment 1, were combined according to the procedure described in "3. Method for preparing a tear gas component generating kit" of Experiment 1 to create an integrated tear gas component generating kit 1. (2) A PRENCSO pad, prepared by impregnating it with a 1 mg / ml PRENCSO lactose hydrochloride solution according to the procedure described in "1. Method for preparing PRENCSO pads" of Experiment 1, and an enzyme pad, prepared according to the procedure described in "2. Method for preparing enzyme pads" of Experiment 1, were combined according to the procedure described in "3. Method for preparing a tear gas component generating kit" of Experiment 1 to create an integrated tear gas component generating kit 2.

[0080] 2. Preservation Test (1) The prepared integrated tear gas component generating kits 1 and 2 were each placed in 1.5 ml tubes, sealed, and then placed in aluminum pouches and sealed. (2) The samples were stored in constant temperature baths at 4°C, 20°C, and 30°C. (3) The amount of LF generated was determined for integrated tear gas component generating kits 1 and 2 that were stored in a constant temperature bath at each temperature for a predetermined period of up to 12 months. For each of integrated tear gas component generating kits 1 and 2, the percentage of the amount of LF generated after storage for the predetermined period at each temperature compared to the amount of LF generated before storage at each temperature was defined as the LF generation retention rate (%).

[0081] (4) The amount of LF generated was determined by the following procedure. One integrated tear gas component generating kit 1 or 2 was placed in a 4 ml vial with a septum so that the side and bottom of the PRENCSO pad were in contact, and 80 μl of water was added dropwise from the side of the enzyme pad before sealing the vial tightly.

[0082] One minute after the water was added, 500 μl of methanol was added to the vial using a glass syringe, and the mixture was stopped by mixing it with a vortex mixer for 30 seconds.

[0083] 200 μl of the supernatant was filtered through a 0.45 μm filter, and the filtrate was transferred to an HPLC vial.

[0084] The LF peak area was determined using HPLC at a detection wavelength of 254 nm and a retention time of approximately 9.9 minutes. The HPLC analysis conditions were as described in "4.3. High-Performance Liquid Chromatography (HPLC) Analysis Conditions" of "4. LF (Tear Gas Component) Generation Comparison Test" in Experiment 1.

[0085] 3.Results For each of the integrated tear gas generating kits 1 and 2, the residual LF generation rate of three samples was determined for each storage time at each temperature, and the mean and standard deviation were calculated.

[0086] Figure 4 shows the average residual LF (Lactic Acid Focus) levels of integrated tear gas component generating kits 1 and 2 after storage at 4°C for 1 month, 3 months, 6 months, and 12 months (error bars represent the standard deviation).

[0087] Figure 5 shows the average value (error bars represent standard deviation) of the remaining LF (Lactic Acid Focus) generation rate of integrated tear gas component generating kits 1 and 2 after storage at 20°C for 1 month, 3 months, and 6 months.

[0088] Figure 6 shows the average residual LF (Lactic Acid Focus) levels of integrated tear gas component generating kits 1 and 2 after storage at 30°C for 1 month, 3 months, 6 months, and 12 months (error bars represent the standard deviation).

[0089] Compared to integrated tear gas generating kit 1, which has a PRENCSO pad that does not contain lactose, integrated tear gas generating kit 2, which has a PRENCSO pad that contains lactose, was confirmed to have a higher retention rate of LF (Lactic Acid Focusing) after storage at various temperatures.

[0090] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. (1) A dried PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal enzyme (LFS), and a porous carrier on which they are supported, and which is dry. Equipped with, (1) and (2) are combined into one. Tear gas generating kit.

2. (1) The tear gas component generating kit according to claim 1, further comprising sugars.

3. The tear gas component generating kit according to claim 2, comprising 5 parts by mass or more of sugars per 1 part by mass of PRENCSO.

4. (1) A dried PRENCSO composition comprising PRENCSO and a porous carrier supporting it, and which is dry, and (2) A dry enzyme composition comprising alliinase, lacrimal enzyme (LFS), and a porous carrier on which they are supported, and which is dry. This includes bringing it into contact with water. A method for generating tear gas components, which combines (1) and (2).

5. The method according to claim 4, wherein (1) further comprises sugars.

6. The method according to claim 5, comprising 5 parts by mass or more of sugars per 1 part by mass of PRENCSO.

7. The method according to any one of claims 4 to 6, wherein the water is an aqueous solution containing a surfactant.

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