Neuraminidase inhibitor

Abstract

To provide a neuraminidase inhibitor exhibiting antiviral action.SOLUTION: The neuraminidase inhibitor contains an extract from at least one kind selected from Saxifraga stolonifera, Camellia sinensis, evening primrose, Eucaligus cuneata, Hypericum perforatum, Sanguisorba officinalis, Houttuynia cordata, Tilia cordata, Cassia, Peony root, Vitis vinifera, and wild thyme.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to neuraminidase inhibitors. [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 demand 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 contains an extract from at least one selected from saxifrage, tea plant, evening primrose, eucalyptus, St. John's wort, burnet, houttuynia cordata, Tilia cordata, cassia, peony, European grape, and wild thyme. [Effects of the Invention]

[0008] According to the present invention, an antiviral effect can be exerted. DETAILED DESCRIPTION OF THE INVENTION

[0009] One embodiment of the antiviral composition will be described below. The antiviral composition of this embodiment contains an extract from a plant or seaweed as an active ingredient, and exerts its antiviral effect by inhibiting the activity of neuraminidase.

[0010] <Extract> Extracts from plants or seaweed are described below. In this embodiment, an extract that inhibits neuraminidase activity at a concentration of 0.01 w / v% during reaction is defined as an effective extract. 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 is defined as one that exhibits an NA inhibition rate of 1% or higher for either influenza A virus subtype H1N1 or influenza A virus subtype H3N2 at the above extract concentration. Hereinafter, influenza A virus subtype H1N1 is also referred to as "H1N1." Influenza A virus subtype H3N2 is also referred to as "H3N2." The method for calculating the NA inhibition rate will be described later.

[0011] The NA inhibition rate is preferably 50% or more, more preferably 80% or more, and even more preferably 90% 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 extract from the plant or seaweed 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, and water. A mixed solvent of the above alcohols and water may also be used.

[0013] The extracts from plants or seaweeds may be purified as necessary. The following extracts may be used singly or in combination of two or more. The plant used in the antiviral composition of the present embodiment is not particularly limited, and examples thereof include plants of the Saxifragaceae family, Onagraceae family, Rosaceae family, Theaceae family, Myrtaceae family, Hypericaceae family, Tiliaceae family, Lauraceae family, Houttuyniaceae family, Vitaceae family, Paeoniaceae family, and Lamiaceae family.

[0014] Specific examples of plants of the Saxifragaceae family include the genus Saxifragaceae, and more specifically Saxifraga saxifragaceae. For example, an extract extracted from the whole plant of Saxifragaceae can be used as the extract.

[0015] Specific examples of plants of the Onagraceae family include the genus Oenothera, and more specifically, Oenothera biennis. For example, an extract extracted from the seeds of Oenothera biennis can be used as the extract.

[0016] The plant of the Rosaceae family specifically includes the genus Sanguisorba, more specifically Sanguisorba officinalis. For example, an extract extracted from the roots and rhizomes of Sanguisorba officinalis can be used as the extract.

[0017] The plant of the Theaceae family specifically includes the genus Camellia, more specifically the tea plant. Extracts extracted from tea plant leaves include green tea extract and black tea extract. An example of black tea is Assam tea.

[0018] Specific examples of plants in the Myrtaceae family include Eucalyptus. For example, an extract extracted from eucalyptus leaves can be used as the extract. Specific examples of plants of the Hypericaceae family include the genus Hypericum, and more specifically, St. John's wort. For example, an extract extracted from the aerial parts of St. John's wort can be used as the extract.

[0019] Specific examples of plants of the Tilia family include Tilia genus, more specifically Tilia cordata. For example, an extract extracted from Tilia cordata flowers can be used as the extract.

[0020] The plant of the Lauraceae family specifically includes the genus Cinnamomum, more specifically cassia, and for example, an extract extracted from the bark of cassia can be used as the extract.

[0021] Specific examples of plants of the Houttuynia family include the genus Houttuynia, and more specifically, Houttuynia cordata. For example, an extract extracted from the above-ground parts of Houttuynia cordata can be used as the extract.

[0022] The Vitaceae plant specifically includes the genus Vitis, more specifically Vitis vinifera, a type of red grape. For example, an extract extracted from Vitis vinifera leaves can be used as the extract.

[0023] The plant of the Paeoniaceae family includes the genus Paeonia, and more specifically, the peony. For example, an extract extracted from the root bark of the peony can be used as the extract.

[0024] Specific examples of plants in the Lamiaceae family include Thymus genus, and more specifically, wild thyme (Thymus serpyllum). For example, an extract extracted from wild thyme can be used as the extract.

[0025] The seaweed used in the antiviral composition of the present embodiment is not particularly limited, but examples thereof include brown algae. Specifically, Fucus vesiculosus is exemplified. For example, an extract extracted from the whole Fucus vesiculosus can be used as the extract.

[0026] The plants and seaweed used in the antiviral composition of this embodiment are preferably ingredients that can be incorporated into quasi-drugs, which can reduce side effects and enable safer use.

[0027] <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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] <Action and Effects> The operation of this embodiment will be described. In this embodiment, an extract from a plant or seaweed is used as an active ingredient. A composition containing the extract 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 are also 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. According to the antiviral composition of this embodiment, the release of influenza viruses from host cells is suppressed by inhibiting the activity of neuraminidase.

[0033] The effects of this embodiment will be described. (1) The antiviral composition contains an extract from a plant or seaweed as an active ingredient and exerts an antiviral effect.

[0034] 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.

[0035] (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.

[0036] (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.

[0037] 〈Technical thought〉 The technical concept that can be understood from the above embodiment will be described. [Appendix 1] An antiviral composition characterized by containing an extract from a plant or seaweed as an active ingredient.

[0038] [Appendix 2] The antiviral composition according to Appendix 1, wherein the plant or seaweed comprises at least one selected from fucus, saxifrage, tea plant, evening primrose, eucalyptus, St. John's wort, burnet, houttuynia cordata, tilia cordata, cassia, peony, European grape, and wild thyme. [Example]

[0039] The antiviral composition will be described in more detail based on the following examples. The antiviral composition is not limited to the configurations described in the Examples section. Examples 1 and 15 to 41 below should be replaced with Reference Examples 1 and 15 to 41, respectively.

[0040] 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.

[0041] <<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).

[0042] 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.

[0043] 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):

[0044]

number

[0045] 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.

[0046] <<Evaluation Test>> The extracts of Examples 1 to 41 shown in Table 1 were used as NA inhibitors to evaluate the NA inhibition rate. The NA inhibition rate was evaluated by a test using H1N1 neuraminidase and a test using H3N2 neuraminidase. The concentration of each NA inhibitor during the reaction was 0.01 w / v%. Details of the NA inhibitor in each Example are as follows.

[0047] Example 1 Maruzen Pharmaceutical Co., Ltd.'s "Kaisou Extract BG-J" was used as an extract extracted from the whole algae of Fucus vesiculosus.

[0048] Example 2 Maruzen Pharmaceutical Co., Ltd.'s "Saxifrage Extract BG" was used as an extract extracted from the whole plant of saxifrage.

[0049] Example 3 The extract extracted from Assam tea was "Black Tea Liquid" from Ichimaru Falcos Co., Ltd.

[0050] Example 4 The extract used was "Luna White B" from Ichimaru Falcos Co., Ltd., extracted from evening primrose seeds.

[0051] Example 5 Maruzen Pharmaceutical Co., Ltd.'s "Eucalyptus Extract BG" was used as the extract extracted from eucalyptus leaves.

[0052] Example 6 "Hypericum B" manufactured by Maruzen Pharmaceutical Co., Ltd. was used as an extract extracted from the above-ground parts of St. John's wort.

[0053] Example 7 Maruzen Pharmaceutical Co., Ltd.'s "Jiyu Extract BG-R" was used as an extract extracted from the roots and rhizomes of Sanguinea chinensis.

[0054] Example 8 The extract used was "Falcorex Houttuynia Cordata B" from Ichimaru Falcos Co., Ltd., extracted from the above-ground parts of Houttuynia cordata.

[0055] Example 9 Maruzen Pharmaceutical Co., Ltd.'s "Linnish Extract BG-J" was used as an extract extracted from Tilia cordata flowers.

[0056] Example 10 The extract extracted from cassia bark was "Cinnamon Extract W-LA" by Maruzen Pharmaceutical Co., Ltd.

[0057] Example 11 The extract used was Falcorex Botanpi B from Ichimaru Falcos Co., Ltd., extracted from the root bark of the peony.

[0058] Example 12 Maruzen Pharmaceutical Co., Ltd.'s "Red Grape Extract BG" was used as an extract extracted from European grape leaves.

[0059] Example 13 The extract extracted from wild thyme used was "Falcorex Wild Thyme B" from Ichimaru Falcos Co., Ltd.

[0060] Example 14 Maruzen Pharmaceutical Co., Ltd.'s "Waism <Green Tea>" was used as the extract extracted from green tea.

[0061] (Examples 15 to 41) Table 2 shows Examples 15 to 41. Each Example contains an extract extracted from a plant or seaweed. Table 2 also lists the product name and manufacturer of the extract used in each Example.

[0062] 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.

[0063] (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.

[0064] (NA inhibitor) The extract was diluted with distilled water so that the concentration of the extract in the NA inhibitor was 0.04 w / v%. (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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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. Based on the measurement results, the NA inhibition rate for each example was calculated. Furthermore, evaluation was performed according to the following criteria. The results are shown in Tables 1 and 2.

[0070] Evaluation criteria for antiviral activity ○○○(Excellent): When the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are both 90% or higher. 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 90%. ○ (fairly good): When either the NA inhibition rate for H1N1 or the NA inhibition rate for H3N2 is 50% or more, and the other is less than 50%. △ (Acceptable): Either the NA inhibition rate for H1N1 or the NA inhibition rate for H3N2 is 1% or more, and both the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are less than 50%.

[0071] <Test Results> As shown in Table 1, in Examples 1 to 14, the NA inhibition rates of H1N1 and H3N2 were 50% or higher. That is, in Examples 1 to 14, the evaluation of the antiviral activity was favorable. In particular, in Examples 1 to 7, the NA inhibition rates of H1N1 and H3N2 were 80% or higher, which were even better results. Furthermore, in Examples 2 and 4, the NA inhibition rates of H1N1 and H3N2 were 90% or higher, which means that even better antiviral activity can be expected.

[0072] As shown in Table 2, Examples 15 to 17 showed NA inhibition rates of 50% or more for H1N1, indicating that they inhibit the activity of H1N1 neuraminidase. On the other hand, the NA inhibition rates for H3N2 were less than 50%. That is, Example 15, which contains an extract of Phellodendron amurense bark, Example 16, which contains an extract of Scutellaria baicalensis root, and Example 17, which contains an extract of gardenia fruit, are considered to have somewhat weak antiviral effects. Phellodendron amurense is a plant of the genus Phellodendron amurense in the family Rutaceae. Scutellaria baicalensis is a plant of the genus Scutellaria in the family Lamiaceae. Gardenia is a plant of the genus Gardenia in the family Rubiaceae.

[0073] Examples 28, 29, 31, and 34 showed NA inhibition rates of 50% or more against H3N2, indicating their ability to inhibit H3N2 neuraminidase activity. On the other hand, the NA inhibition rates against H1N1 were less than 50%. That is, Example 28, which contains an extract of the whole plant of horsetail, Example 29, which contains an extract of Satsuma mandarin peel, Example 31, which contains an extract of calendula flower, and Example 34, which contains an extract of morus alba root bark, are considered to have somewhat weak antiviral activity. Horsetail is a plant of the Equisetaceae family, Equisetum genus. Satsuma mandarin is a plant of the Rutaceae family, Citrus genus. Calendula is a plant of the Asteraceae family, Calendula genus. Morus alba is a plant of the Moraceae family, Mulberry genus.

[0074] As positive controls, tests were conducted using oseltamivir phosphate (FUJIFILM Wako Pure Chemical Industries, Ltd.) and zanamivir hydrate (Tokyo Chemical Industry Co., Ltd.). Tests using oseltamivir phosphate at a reaction concentration of 25 μM confirmed 100% NA inhibition for H1N1. Tests using zanamivir hydrate at a reaction concentration of 25 μM confirmed 96% NA inhibition for H1N1.

[0075] [Table 1]

[0076] [Table 2]

Claims

[Claim 1] The neuraminidase inhibitor contains an extract from at least one plant selected from saxifrage, tea plant, evening primrose, eucalyptus, St. John's wort, burnet, houttuynia cordata, Tilia cordata, cassia, peony, European grape, and wild thyme.

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