Neuraminidase activity inhibitor and antiviral composition

Abstract

To inhibit activity against neuraminidase derived from bacteria.SOLUTION: A neuraminidase activity inhibitor comprises at least one selected from licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an inhibitor of neuraminidase activity and an antiviral composition. [Background technology]

[0002] Oseltamivir, zanamivir, and the like are known as antiviral drugs used against viruses such as influenza viruses. For example, Patent Document 1 discloses that oseltamivir, zanamivir, and the like have an anti-influenza virus effect of inhibiting the activity of neuraminidase. Neuraminidase is an enzyme that exists on the surface of the virus and hydrolyzes terminal sialic acid residues on polysaccharide chains. By inhibiting the activity of neuraminidase, the release of replicated viruses from host cells is suppressed, and the spread of infection to other host cells can be suppressed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-178913 A Summary of the Invention [Problem to be solved by the invention]

[0004] Meanwhile, neuraminidase exists not only on the surface of viruses but also in bacteria. Bacterial neuraminidase exists, for example, in the oral cavity of humans and animals. There is a risk that bacterial neuraminidase may also promote influenza virus infection. Therefore, there is a demand for a neuraminidase activity inhibitor that can inhibit the activity of bacterial neuraminidase. [Means for solving the problem]

[0005] A neuraminidase activity inhibitor for solving the above problems contains at least one selected from the group consisting of licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin.

[0006] The antiviral composition preferably contains the above-mentioned neuraminidase activity inhibitor. Effect of the Invention

[0007] The neuraminidase activity inhibitor of the present invention can inhibit the activity of bacterial neuraminidase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] An embodiment of the neuraminidase activity inhibitor of the present invention will be described. The neuraminidase activity inhibitor contains at least one selected from the group consisting of licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin.

[0009] Each component constituting the neuraminidase activity inhibitor will be described below. <Licorice extract> Licorice extract is an extract of Glycyrrhiza L., a perennial plant of the genus Licorice in the family Fabaceae. Specific examples of Glycyrrhiza L. include Russian licorice (Glycyrrhiza echinata), Spanish licorice (also called licorice) (Glycyrrhiza glabra), American licorice (Glycyrrhiza lepidota), Ural licorice (Glycyrrhiza uralensis), and Xinjiang licorice (Glycyrrhiza inflate).

[0010] When extracting the extract from the above plant, the flower, stem, leaf, rhizome, root bark, root, or whole plant of the above plant can be used, but rhizome is particularly preferred. Examples of the extracting solvent that can be used include alcohols such as ethanol, glycerin, propylene glycol, and 1,3-butylene glycol, and water. A mixed solvent of the above alcohols and water may also be used. Purification may be performed after extraction as necessary. A liquid licorice extract is also called a licorice soft extract. The licorice extract may also be a licorice dry extract obtained by concentrating the extracted licorice extract by drying it.

[0011] Ingredients contained in licorice extract include β-glycyrrhetinic acid, monoammonium glycyrrhizinate, dipotassium glycyrrhizinate, etc. Commercially available licorice extracts can be used.

[0012] <Dipotassium glycyrrhizinate> Dipotassium glycyrrhizinate is known as an anti-inflammatory agent. Dipotassium glycyrrhizinate is contained in licorice extract. Dipotassium glycyrrhizinate may be isolated from licorice extract or may be obtained by synthesis. Dipotassium glycyrrhizinate may be a commercially available product.

[0013] <Sodium copper chlorophyllin> Copper chlorophyllin sodium is known as a coloring agent approved for use under the Food Sanitation Act. Commercially available copper chlorophyllin sodium products can be used.

[0014] The neuraminidase activity inhibitor may contain only one of licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin, or may contain a suitable combination of two or more of them.

[0015] <Content of each ingredient> The contents of licorice extract, dipotassium glycyrrhizinate, and copper chlorophyllin sodium in the neuraminidase activity inhibitor are not particularly limited. The contents of licorice extract, dipotassium glycyrrhizinate, and copper chlorophyllin sodium can be appropriately set within the range that exhibits the effect as a neuraminidase activity inhibitor. In the following, the contents of each component in the neuraminidase activity inhibitor are expressed by abbreviating "mass %" to "%".

[0016] When the total mass of the neuraminidase activity inhibitor is taken as 100%, the content of the licorice extract is preferably 0.1% or more and 3% or less, more preferably 0.5% or more, and more preferably 2% or less.

[0017] When the total mass of the neuraminidase activity inhibitor is taken as 100%, the content of dipotassium glycyrrhizinate is preferably 0.0001% or more and 3% or less, more preferably 1% or more, and more preferably 2% or less.

[0018] The content of copper chlorophyllin sodium is preferably 0.1 μg / mL or more and 1500 μg / mL or less per total volume (mL) of the neuraminidase activity inhibitor, more preferably 10 μg / mL or more, and more preferably 1000 μg / mL or less of the licorice extract.

[0019] <Other ingredients> The neuraminidase activity inhibitor may contain other components in addition to the above-mentioned components depending on the application purpose, form, use, etc. Examples of the other components include surfactants, flavoring agents, sweeteners, wetting agents, binders, preservatives, colorants, pH adjusters, chelating agents, medicinal ingredients, bases, abrasives, stabilizers, etc. The other components may be any known components that are blended into neuraminidase activity inhibitors. The neuraminidase activity inhibitor may contain only one of the above other components alone, or may contain two or more of them in combination.

[0020] <Application form, use, and dosage form> The application form of the neuraminidase activity inhibitor is not particularly limited, and it can be used, for example, as food, cosmetics, medicines, quasi-drugs, etc. Also, it can be used as an antiviral composition containing the neuraminidase activity inhibitor.

[0021] When the antiviral composition is used as an oral composition such as a food, it can be applied to, for example, candies, troches, tablets, gum, granules, powders, jellies, syrups, beverages, and the like.

[0022] When the antiviral composition is used as a composition for external use such as cosmetics, it can be applied, for example, to face packs, pastes, ointments, creams, gels, lotions, milky lotions, beauty essences, skin lotions, etc. In addition, the antiviral composition is not limited to products for use on the human body, and can also be applied as a liquid agent to be sprayed or applied to products that come into contact with the skin, such as bedding, clothing, furniture, fittings, and articles. Examples of materials of target products include natural fibers, synthetic fibers, natural leather, artificial leather, synthetic leather, wood, synthetic resins, metals, painted surfaces, etc.

[0023] The antiviral composition can be used to cleanse the skin or skin, purify the mouth, and keep the skin or skin healthy. The antiviral composition can be used to suppress or reduce sore throat, swollen throat, throat discomfort, sore throat, or hoarseness caused by throat inflammation. The antiviral composition can be used to sterilize, disinfect, or cleanse the skin, hands, mouth, or throat (pharynx). The antiviral composition can also be used for virus barrier, virus blocking, virus shutting, antivirus, and virus elimination.

[0024] When the antiviral composition is used as an oral cavity composition or throat composition such as a quasi-drug, it can be applied, for example, to a spray, toothpaste, liquid toothpaste, mouthwash, rinse, etc. Examples of the spray include a nasal spray, throat spray, and oral spray.

[0025] Neuraminidase activity inhibitors are also called neuraminidase inhibitors, NA activity inhibitors, or NA inhibitors. <Bacterial neuraminidase> The neuraminidase derived from bacteria is not particularly limited. Examples of the neuraminidase derived from bacteria include neuraminidase derived from streptococci, salmonella, cholera, and Clostridium bacteria (Clostridium tetanus, Clostridium botulinum, and Clostridium perfringens). Specific examples of streptococci include bacteria belonging to the genus Streptococcus.

[0026] <Action and Effects> The operation of this embodiment will be described. Influenza viruses spread their infection to other cells by being released from host cells by the activity of neuraminidase. Neuraminidase is an enzyme present on the surface of influenza viruses, for example. Neuraminidase is also present in bacteria, for example, in the oral cavity. Therefore, there is a risk that bacterial neuraminidase may also promote influenza virus infection.

[0027] According to the neuraminidase activity inhibitor of the present embodiment, by containing at least one selected from licorice extract, dipotassium glycyrrhizinate, and copper chlorophyllin sodium, the activity of bacterial neuraminidase can be inhibited. Furthermore, an antiviral composition containing the neuraminidase activity inhibitor of the present embodiment exerts the same action as the neuraminidase activity inhibitor of the present embodiment.

[0028] The effects of this embodiment will be described. (1) The neuraminidase activity inhibitor contains at least one selected from the group consisting of licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin, and is therefore capable of inhibiting the activity of bacterial neuraminidase.

[0029] (2) The antiviral composition may be useful not only against influenza viruses but also against viruses whose infection is related to neuraminidase activity, and is expected to exert an antiviral effect against, for example, human parainfluenza virus, mumps virus, Sendai virus, Newcastle disease virus, etc.

[0030] (3) Neuraminidase activity inhibitors are expected to inhibit not only neuraminidase derived from bacteria but also neuraminidase present on the surface of viruses. (4) According to the antiviral composition, prevention of viral infections, treatment of viral infections, inhibition of virus growth, suppression of virus infection, suppression of virus growth, inactivation of viral enzyme activity, inhibition of viral enzyme activity, and deactivation of viral enzyme activity can be expected.

Examples

[0031] The neuraminidase activity inhibitor will be further described in more detail based on the following examples. Note that the neuraminidase activity inhibitor is not limited to the configuration described in the examples section.

[0032] This test referred to the methods described in JP-A-2019-163292 and "A microplate-based screening assay for neuraminidase inhibitors", Drug Discoveries & Therapeutics, 2009, 3(6), p260-265.

[0033] <Principle of calculating NA inhibition rate> The principle of the method for calculating the NA inhibition rate will be explained. To calculate the NA inhibition rate, a kit for measuring the activity of neuraminidase (NA-Fluor Influenza Neuraminidase Assay Kit, manufactured by Thermo Fisher Scientific) was used.

[0034] The above kit contains MUNANA (4-(methylumbelliferyl)-N-acetylneuraminic acid), which is a fluorescent substrate. MUNANA is decomposed by the activity of neuraminidase. When MUNANA is decomposed, N-acetylneuraminic acid and the fluorescent substance 4-MU (4-Methylumbelliferone) are released. 4-MU can be detected by fluorescence measurement with an excitation wavelength of 350 - 365 nm and an emission wavelength of 440 - 460 nm. The activity of neuraminidase can be measured based on the fluorescence intensity of the generated 4-MU.

[0035] When a neuraminidase activity inhibitor is added to the system in addition to MUNANA and neuraminidase, the activity of neuraminidase is inhibited according to the inhibitory ability of the added neuraminidase activity inhibitor. That is, the amount of 4-MU produced is reduced according to the inhibitory ability of the neuraminidase activity inhibitor. Therefore, the inhibitory ability of each neuraminidase activity inhibitor against neuraminidase can be calculated based on the fluorescence intensity. Specifically, the NA inhibition rate can be calculated by the following calculation formula (Formula 1).

[0036]

number

[0037] RFUbackground2 is the fluorescence intensity when only the substrate was added without adding neuraminidase in order to measure background noise. RFU サンプル indicates the RFU value when each neuraminidase activity inhibitor, neuraminidase, and substrate were added.

[0038] RFUbackground1 is the fluorescence intensity when neuraminidase was not added but a neuraminidase activity inhibitor and a substrate were added in order to measure background noise.

[0039] According to the calculation formula (Formula 1), the more the fluorescence intensity decreases due to the inhibitory ability of the neuraminidase activity inhibitor, the higher the NA inhibition rate is calculated. <Evaluation test> The NA inhibition rate was evaluated using the neuraminidase activity inhibitors of Examples 1 to 17 shown in Tables 1 and 2. The NA inhibition rate was evaluated by a test using neuraminidase derived from bacteria.

[0040] (Examples 1 to 7) As the licorice extract, "Licorice Extract-L2", a licorice soft extract from Japan Powder Pharmaceutical Co., Ltd., was used.

[0041] (Examples 8 to 12) As dipotassium glycyrrhizinate, Maruzen Pharmaceutical Co., Ltd.'s "dipotassium glycyrrhizinate (GK2)" was used.

[0042] (Examples 13 to 17) As the copper chlorophyllin sodium, "copper chlorophyllin sodium" manufactured by Tama Biochemical Co., Ltd. was used.

[0043] Each of the components shown in Examples 1 to 17 was diluted with ultrapure water to the concentrations shown in Tables 1 and 2. [Preparation of Reagents] (1x Assay Buffer) The 2x Assay Buffer (66.6 mM MES, 8 mM CaCl2, pH 6.5) included in the above kit was diluted two-fold with distilled water to prepare 1x Assay Buffer.

[0044] (200μM substrate) MUNANA included in the above kit was dissolved in distilled water to prepare 2.5 mM substrate, which was then diluted with 1× Assay Buffer to prepare 200 μM substrate.

[0045] (NA solution) Bacterial neuraminidase manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used. This neuraminidase is derived from bacteria belonging to the genus Streptococcus. This neuraminidase was diluted to 0.18 U / mL using 2x Assay Buffer.

[0046] (Test Method) A black 96-well plate was used, and 25 μL of the neuraminidase activity inhibitor of Examples 1 to 17 was added to each well. 25 μL of 1× Assay Buffer was added instead of the neuraminidase activity inhibitor to the wells for measuring RFUbackground2 and the wells for measuring RFUcontrol.

[0047] The prepared NA solution was added to each well at 25 μL. Instead of the NA solution, 25 μL of 1× Assay Buffer was added to the wells for measuring RFUbackground1 and RFUbackground2.

[0048] 50 μL of the prepared 200 μM substrate was added to each well. The plate was covered and incubated at 37° C. for 60 minutes, protected from light. The reaction was stopped by adding 100 μL of the reaction stop solution (0.2 M Na 2 CO 3 solution) included in the kit to each well.

[0049] The plate was measured using a plate reader (Cytation5, BioTek Instruments, Inc.) at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. In Examples 1 to 17, the test was performed with an n number of 3. Based on the measurement results, the average value of the NA inhibition rate in each Example was calculated. Furthermore, evaluation was performed according to the following criteria. The results of Examples 1 to 12 are shown in Table 1. The results of Examples 13 to 17 are shown in Table 2.

[0050] Evaluation criteria for NA inhibition rate ◎◎(particularly excellent): NA inhibition rate is 60% or more ◎ (Excellent): NA inhibition rate is 40% or more and less than 60% ○○(Good): NA inhibition rate is 20% or more and less than 40% ○(Acceptable): NA inhibition rate is 1% or more but less than 20% × (Not acceptable): NA inhibition rate is less than 1% (Test Results) As shown in Tables 1 and 2, all of Examples 1 to 17 had an NA inhibition rate of 1% or more, and it was confirmed that the activity of bacterial neuraminidase could be inhibited. Furthermore, Examples 4 and 16 had an NA inhibition rate of 20% or more, Example 9 had an NA inhibition rate of 40% or more, and Examples 1 to 3, 8, and 13 to 15 had an NA inhibition rate of 60% or more. It was confirmed that the activity of bacterial neuraminidase could be inhibited more suitably.

[0051] [Table 1]

[0052] [Table 2]

Claims

1. A neuraminidase activity inhibitor comprising at least one member selected from the group consisting of licorice extract, dipotassium glycyrrhizinate, and sodium copper chlorophyllin.

2. 2. An antiviral composition comprising the neuraminidase activity inhibitor according to claim 1.

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