Neuraminidase inhibitors and antiviral compositions

Abstract

To provide a neuraminidase inhibitor and an antiviral composition exhibiting an excellent antiviral action.SOLUTION: The neuraminidase inhibitor contains at least one kind selected from a Platycodon root extract and cinnamon bark powder. The antiviral composition contains an extract from a plant or a plant powder as an active ingredient. The antiviral composition can be expected to exhibit an antiviral action by inhibiting the activity of neuraminidase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a neuraminidase inhibitor 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, as disclosed in Patent Document 1, the anti-influenza virus effect of oseltamivir, zanamivir, and the like is inhibition of neuraminidase activity. Neuraminidase is an enzyme that hydrolyzes terminal sialic acid residues on polysaccharide chains. Inhibition of neuraminidase activity suppresses the release of replicated viruses from host cells, thereby suppressing the spread of infection to other host cells. However, Patent Document 1 describes that antiviral drugs such as oseltamivir and zanamivir have side effects. Furthermore, Non-Patent Document 1 describes viruses that have acquired resistance to oseltamivir or zanamivir.

[0003] Patent Document 1 proposes an antiviral agent containing a watermelon extract as an active ingredient, as an antiviral agent other than oseltamivir or zanamivir. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2017-178913 [Non-patent literature]

[0005] [Non-Patent Document 1] "Influenza neuraminidase: a druggable target for natural products.", Natural product reports, 2012, 29(1), p.11-36 Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for antiviral compositions that exhibit antiviral activity other than antiviral drugs such as oseltamivir and zanamivir. [Means for solving the problem]

[0007] The neuraminidase inhibitor for solving the above problems is characterized by containing at least one selected from the group consisting of Platycodon grandiflorum extract and cinnamon bark powder. Further, the antiviral composition for solving the above problems is characterized by containing at least one selected from the group consisting of a platycodon extract and a cinnamon bark powder as an active ingredient. [Effects of the Invention]

[0008] The antiviral composition of the present invention can exert an antiviral effect. DETAILED DESCRIPTION OF THE INVENTION

[0009] One embodiment of the antiviral composition will be described below. The antiviral composition of this embodiment contains at least one active ingredient selected from licorice extract, platycodon grandiflorum extract, and cinnamon powder. The antiviral composition exerts its antiviral effect by inhibiting the activity of neuraminidase. Note that licorice extract is referred to as a reference example below.

[0010] <Extract> Plant extracts and plant powders will now be described. In this embodiment, an extract that inhibits neuraminidase activity at a concentration of 0.5 w / w% during reaction and a powder that inhibits neuraminidase activity at a concentration of 0.025 w / v% were defined as effective extracts. Hereinafter, the inhibition rate of neuraminidase activity is also referred to as NA inhibition rate. As an example, the NA inhibition rate was evaluated as the inhibition rate of activity against neuraminidase of influenza A virus subtype H1N1 and neuraminidase of influenza A virus subtype H3N2. More specifically, an effective extract or powder was defined as one that exhibited an NA inhibition rate of 20% or higher against either influenza A virus subtype H1N1 or influenza A virus subtype H3N2 at the above concentrations. Hereinafter, influenza A virus subtype H1N1 will also be referred to as "H1N1." Influenza A virus subtype H3N2 will also be referred to as "H3N2." The method for calculating the NA inhibition rate will be described later.

[0011] The NA inhibition rate is preferably 30% or more, more preferably 50% or more, and even more preferably 70% or more. It is more preferable that both the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are high.

[0012] The plant extract is not particularly limited, but is preferably one extracted using a known extraction solvent. Examples of known extraction solvents include alcohols such as ethanol, glycerin, propylene glycol, and 1,3-butylene glycol, as well as water. A mixed solvent of the above alcohols and water may also be used. The plant extract may be purified as necessary. The following extracts may be used alone or in combination of two or more.

[0013] The plant powder is not particularly limited, but is preferably one obtained by known means. Known methods include, for example, drying the whole plant or a specific part (seed, root, stem, leaf, bark, etc.) at a temperature preferably below 60°C, and then pulverizing it to obtain a powder of a specific size. The following powders may be used singly or in combination of two or more.

[0014] The plant used in the antiviral composition of this embodiment is not particularly limited, but examples thereof include plants of the Fabaceae, Campanulaceae, and Lauraceae families. Specific examples of legume plants include the genus Glycyrrhiza, more specifically Glycyrrhiza uralensis and Glycyrrhiza glabra. For example, an extract extracted from the roots and stolons of licorice can be used as the extract.

[0015] Specific examples of plants of the Campanulaceae family include the genus Platycodon, and more specifically Platycodon grandiflorum. For example, an extract extracted from the roots of Platycodon grandiflorum can be used as the extract.

[0016] Examples of plants of the Lauraceae family include the genus Cinnamomum, and more specifically Cinnamomum chinensis. For example, powder of the bark or bark from which part of the periderm has been removed of Cinnamomum chinensis can be used as the plant powder.

[0017] <Application form> The application form of the antiviral composition is not particularly limited, and it can be used as, for example, food, cosmetics, pharmaceuticals, or quasi-drugs.

[0018] When the antiviral composition is used as an oral composition such as a food, it can be applied to, for example, candy, troches, tablets, gum, granules, powder, jelly, syrup, beverages, etc.

[0019] When the antiviral composition is used as a topical composition such as a cosmetic, it can be applied, for example, to face packs, pastes, ointments, creams, gels, lotions, emulsions, beauty serums, and skin lotions. Furthermore, the antiviral composition is not limited to products used on the human body, and can also be applied as a liquid to be sprayed or applied to products that come into contact with the skin, such as bedding, clothing, furniture, fixtures, and other articles. Examples of materials for the target products include natural fibers, synthetic fibers, natural leather, artificial leather, synthetic leather, wood, synthetic resins, metals, and painted surfaces.

[0020] The antiviral composition can be used to cleanse the skin or the mouth, and to maintain healthy skin or the mouth. The antiviral composition can be used to suppress or alleviate 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, the oral cavity, or the throat (pharynx). The antiviral composition can also be used for virus barrier, virus blocking, virus shutting, antivirus, and virus elimination.

[0021] 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 sprays, toothpastes, liquid toothpastes, mouthwashes, rinses, etc. Examples of sprays include nasal sprays, throat sprays, and oral sprays.

[0022] <Action and Effects> The action of this embodiment will be described. In this embodiment, a plant extract or powder is used as an active ingredient. A composition containing the extract or powder as an active ingredient has the effect of inhibiting the activity of neuraminidase. In other words, a composition containing the extract as an active ingredient acts as a neuraminidase inhibitor. Hereinafter, neuraminidase inhibitors will also be referred to as NA inhibitors. Neuraminidase is an enzyme present on the surface of, for example, influenza viruses. Influenza viruses spread their infection to other cells by being released from host cells by the activity of neuraminidase. The antiviral composition of this embodiment inhibits the activity of neuraminidase, thereby suppressing the release of influenza viruses from host cells.

[0023] The effects of this embodiment will be described. (1) The antiviral composition contains an extract from a plant as an active ingredient and exerts an antiviral effect.

[0024] The higher the rate of inhibition of neuraminidase activity, the more effectively influenza viruses that have multiplied within host cells can be prevented from being released from the host cells. In other words, the higher the rate of NA inhibition, the more effective the antiviral effect.

[0025] (2) It is possible to provide oral compositions, external compositions, oral compositions, and throat compositions that exhibit antiviral activity. (3) The antiviral composition may be useful not only against influenza viruses but also against viruses whose infection is related to neuraminidase activity, such as human parainfluenza virus, mumps virus, Sendai virus, and Newcastle disease virus.

[0026] (4) The antiviral composition is expected to prevent viral infections, treat viral infections, inhibit viral proliferation, suppress viral infection, suppress viral proliferation, inactivate viral enzyme activity, suppress viral enzyme activity, and inactivate viral enzyme activity. [Example]

[0027] The antiviral composition will be described in more detail based on the following examples. Note that the antiviral composition is not limited to the configurations described in the examples. This test was performed based on the methods described in JP 2019-163292 A and “A microplate-based screening assay for neuraminidase inhibitors,” Drug Discoveries & Therapeutics, 2009, 3(6), pp. 260-265.

[0028] <<Calculation principle of NA inhibition rate>> The principle of the method for calculating the NA inhibition rate will be explained below. The NA inhibition rate was calculated using a kit for measuring neuraminidase activity (NA-Fluor Influenza Neuraminidase Assay Kit, manufactured by Thermo Fisher Scientific).

[0029] The kit contains the fluorescent substrate MUNANA (4-(methylumbelliferyl)-N-acetylneuraminic acid). MUNANA is degraded by the activity of neuraminidase. When MUNANA is degraded, N-acetylneuraminic acid and the fluorescent substance 4-MU (4-methylumbelliferone) are released. 4-MU can be detected by fluorescence measurement at an excitation wavelength of 350-365 nm and an emission wavelength of 440-460 nm. Neuraminidase activity can be measured based on the fluorescence intensity of the generated 4-MU.

[0030] When an NA inhibitor is added to the system in addition to MUNANA and neuraminidase, the activity of neuraminidase is inhibited according to the inhibitory activity of the added NA inhibitor. In other words, the amount of 4-MU produced is reduced according to the inhibitory activity of the NA inhibitor. Therefore, the inhibitory activity of each NA inhibitor against neuraminidase can be calculated based on the fluorescence intensity. Specifically, the NA inhibition rate can be calculated using the following formula (Equation 1):

[0031]

number

[0032] In the calculation formula (Equation 1), RFUc is the RFU value (relative fluorescence unit) when neuraminidase and substrate are added. BG2 is the fluorescence intensity when only substrate is added without adding neuraminidase for background noise measurement. RFUs is the RFU value when each NA inhibitor, neuraminidase, and substrate are added. BG1 is the fluorescence intensity when NA inhibitor and substrate are added without adding neuraminidase for background noise measurement. According to the calculation formula (Equation 1), the NA inhibition rate is calculated higher as the fluorescence intensity decreases due to the inhibitory ability of the NA inhibitor.

[0033] <<Evaluation Test>> The extracts or powders of Reference Example 1 and Examples 2 and 3 shown in Table 1 were used as NA inhibitors to evaluate the NA inhibition rate. The NA inhibition rate was evaluated by tests using H1N1 neuraminidase and H3N2 neuraminidase. The concentrations of each NA inhibitor during the reaction were 0.5 w / w% for the extract and 0.025 w / v% for the powder. Details of the NA inhibitors in Reference Example 1 and each Example are as follows.

[0034] (Reference example 1) The "Licorice Flow Extract" manufactured by Alps Pharmaceutical Co., Ltd. was used as an extract extracted from the whole plant of licorice.

[0035] Example 2 The extract extracted from platycodon root was "Japanese Pharmacopoeia Platycodon Root Extract" manufactured by Alps Pharmaceutical Co., Ltd.

[0036] Example 3 As the Ceylon cinnamon powder, "Japanese Pharmacopoeia Cinnamon Powder" manufactured by Nippon Powder Pharmaceutical Co., Ltd. was used.

[0037] Preparation of Reagents (1x Assay Buffer) The 2x Assay Buffer (66.6 mM MES, 8 mM CaCl2, pH 6.5) included in the kit was diluted two-fold with distilled water to prepare 1x Assay Buffer.

[0038] (200μM substrate) MUNANA included in the kit was dissolved in distilled water to prepare a 2.5 mM substrate, which was then diluted with 1x Assay Buffer to prepare a 200 µM substrate.

[0039] (NA inhibitor) The NA inhibitor was diluted with 1x Assay Buffer so that the extract concentration in the NA inhibitor was 0.5 w / w% and the powder concentration was 0.025 w / v%.

[0040] (NA solution) A neuraminidase solution containing influenza A virus subtype H1N1 was prepared. The H1N1 neuraminidase used was "Influenza A H1N1 (A / California / 04 / 2009) Neuraminidase / NA (Active)" from Sino Biological Inc. The neuraminidase was diluted with 1x Assay Buffer to a concentration of 0.1 U / mL when mixed with the substrate.

[0041] A neuraminidase solution containing influenza A virus subtype H3N2 was prepared. The H3N2 neuraminidase used was "Influenza A H3N2 Neuraminidase / NA (Active)" from Sino Biological Inc. The neuraminidase was diluted with 1x Assay Buffer to a concentration of 0.03 U / mL when mixed with the substrate.

[0042] Test Method A black 96-well plate was used, and 25 μL of the prepared NA inhibitor was added to each well. 25 μL of 1× Assay Buffer was added instead of the NA inhibitor to the wells for measuring BG2 and RFUc.

[0043] 25 μL of the prepared NA solution was added to each well. 25 μL of 1× Assay Buffer was added instead of the NA solution to the wells for measuring BG1 and BG2.

[0044] 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.

[0045] The plate was measured using a plate reader (Cytation5, manufactured by BioTek Instruments, Inc.) at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. Based on the measurement results, the NA inhibition rate for the Reference Example and each Example was calculated. Furthermore, evaluation was performed according to the following criteria. The results are shown in Table 1.

[0046] Evaluation criteria for antiviral activity ○○○(Excellent): When the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are both 70% or higher.

[0047] XX (Good): The NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are both 50% or higher, and at least one is less than 70%. ○ (fairly good): When either the NA inhibition rate for H1N1 or the NA inhibition rate for H3N2 is 20% or more, and both the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are less than 50%.

[0048] <Test Results> As shown in Table 1, in Example 3, the NA inhibition rates of H1N1 and H3N2 were 70% or more, indicating very good antiviral activity. Furthermore, in Reference Example 1, the NA inhibition rate of H1N1 was 50% or more, and the NA inhibition rate of H3N2 was 70% or more, indicating good antiviral activity. Furthermore, in Example 2, the NA inhibition rate of H1N1 was 20% or more, and the NA inhibition rate of H3N2 was 20% or more, indicating good antiviral activity.

[0049] [Table 1]

Claims

1. A neuraminidase inhibitor comprising at least one selected from a platycodon extract and a cinnamon bark powder.

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

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