Neuraminidase inhibitors

Abstract

To exhibit an antiviral action.SOLUTION: An antiviral composition contains extract from plants or seaweeds as an active ingredient. The antiviral composition can be expected to exhibit an antiviral action by inhibiting an activity of neuraminidase.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to Neuraminidase inhibitors .

Background Art

[0002] As antiviral drugs used against viruses such as influenza virus, oseltamivir, zanamivir, etc. are known. For example, as disclosed in Patent Document 1, as the anti-influenza virus action of oseltamivir, zanamivir, etc., there is inhibition of the activity of neuraminidase. Neuraminidase is an enzyme that hydrolyzes terminal sialic acid residues on a polysaccharide chain. By inhibiting the activity of neuraminidase, the release of replicated virus from host cells is suppressed, so the spread of infection to other host cells can be suppressed. On the other hand, Patent Document 1 describes that antiviral drugs such as oseltamivir and zanamivir have side effects. Also, Non-Patent Document 1 describes viruses that have acquired oseltamivir resistance or zanamivir resistance.

[0003] In Patent Document 1, as an antiviral agent other than oseltamivir and zanamivir, an antiviral agent containing an extract of watermelon as an active ingredient has been proposed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

[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] To solve the above problems Neuraminidase inhibitors contain an extract from Fucus vesiculosus as their active ingredient. One example of the neuraminidase inhibitors mentioned above is for oral use. [Effects of the Invention]

[0008] Honpatsu Clearly Therefore, it can exert antiviral effects. [Modes for carrying out the invention]

[0009] The following describes one embodiment of an antiviral composition. The antiviral composition of this embodiment contains an extract from a plant or seaweed as an active ingredient. The antiviral composition exerts its antiviral effect by inhibiting the activity of neuraminidase.

[0010] <Extract> Extracts from plants or seaweed are described below. In this embodiment, an effective extract was defined as one in which the concentration of the extract inhibited neuraminidase activity at 0.01 w / v% during the reaction. Hereafter, the inhibition rate of neuraminidase activity will also be referred to as the NA inhibition rate. The NA inhibition rate was evaluated as an example of the inhibition rate of neuraminidase activity against influenza A virus H1N1 subtype and influenza A virus H3N2 subtype. More specifically, an effective extract was defined as one in which the NA inhibition rate for either influenza A virus H1N1 subtype or influenza A virus H3N2 subtype was 1% or higher at the above extract concentration. Hereafter, influenza A virus H1N1 subtype will also be referred to as "H1N1". Influenza A virus H3N2 subtype will also be referred to as "H3N2". The method for calculating the NA inhibition rate will be described later.

[0011] The above NA inhibition rate is preferably 50% or higher, more preferably 80% or higher, and even more preferably 90% or higher. Furthermore, 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 plants or seaweed is not particularly limited, but it is preferable that it has been 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 mixture of the above alcohols and water may also be used as the solvent.

[0013] Extracts from plants or seaweed may be purified as needed. The following extracts may be used individually or in combination of two or more. The plants used in the antiviral composition of this embodiment are not particularly limited, but examples include plants from the Saxifragaceae, Onagraceae, Rosaceae, Theaceae, Myrtaceae, Hypericaceae, Tiliaceae, Lauraceae, Saururaceae, Vitaceae, Paeoniaceae, and Lamiaceae families.

[0014] As a plant of the Saxifragaceae family, specifically, the genus Saxifraga can be mentioned, and more specifically, Saxifraga can be mentioned. For example, an extract obtained from the whole herb of Saxifraga can be used as an extract.

[0015] As a plant of the Asteraceae family, specifically, the genus Leptinella can be mentioned, and more specifically, Leptinella squalida can be mentioned. For example, an extract obtained from the seeds of Leptinella squalida can be used as an extract.

[0016] As a plant of the Rosaceae family, specifically, the genus Rubus can be mentioned, and more specifically, Rubus parvifolius can be mentioned. For example, an extract obtained from the roots and rhizomes of Rubus parvifolius can be used as an extract.

[0017] As a plant of the Theaceae family, specifically, the genus Camellia can be mentioned, and more specifically, Camellia sasanqua can be mentioned. Examples of extracts obtained from the leaves of Camellia sasanqua include green tea extract, black tea extract, etc. An example of black tea is Assam tea.

[0018] As a plant of the Myrtaceae family, specifically, the genus Eucalyptus can be mentioned. For example, an extract obtained from the leaves of Eucalyptus can be used as an extract. As a plant of the Caprifoliaceae family, specifically, the genus Lonicera can be mentioned, and more specifically, Lonicera periclymenum can be mentioned. For example, an extract obtained from the aerial parts of Lonicera periclymenum can be used as an extract.

[0019] As a plant of the Betulaceae family, specifically, the genus Betula can be mentioned, and more specifically, Betula platyphylla var. japonica can be mentioned. For example, an extract obtained from the flowers of Betula platyphylla var. japonica can be used as an extract.

[0020] As a plant of the Burseraceae family, specifically, the genus Commiphora can be mentioned, and more specifically, Commiphora myrrha can be mentioned. For example, an extract obtained from the bark of Commiphora myrrha can be used as an extract.

[0021] As a plant of the Saururaceae family, specifically, the genus Houttuynia can be mentioned, and more specifically, Houttuynia cordata can be mentioned. For example, an extract obtained from the above-ground part of Houttuynia cordata can be used as the extract.

[0022] As a plant of the Vitaceae family, specifically, the genus Vitis can be mentioned, and more specifically, Vitis vinifera, which is a type of red grape, can be mentioned. For example, an extract obtained from the leaves of Vitis vinifera can be used as the extract.

[0023] As a plant of the Caryophyllaceae family, specifically, the genus Dianthus can be mentioned, and more specifically, Dianthus caryophyllus can be mentioned. For example, an extract obtained from the root bark of Dianthus caryophyllus can be used as the extract.

[0024] As a plant of the Lamiaceae family, specifically, the genus Thymus can be mentioned, and more specifically, Thymus serpyllum can be mentioned. For example, an extract obtained from Thymus serpyllum can be used as the extract.

[0025] The seaweed used in the antiviral composition of this embodiment is not particularly limited, and for example, brown algae can be mentioned. Specifically, Ecklonia cava can be mentioned. For example, an extract obtained from the whole alga of Ecklonia cava 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 formulated into quasi-drugs. By being ingredients that can be formulated into quasi-drugs, side effects can be suppressed and it becomes possible to use them more safely.

[0027] 〈Application form〉 The application form of the antiviral composition is not particularly limited, and for example, it can be used as food, cosmetics, pharmaceuticals, or quasi-drugs.

[0028] When an antiviral composition is used as an oral composition in food products, for example, it can be applied to candies, lozenges, tablets, gum, gummies, granules, powders, jellies, syrups, beverages, etc.

[0029] When antiviral compositions are used as external compositions such as cosmetics, they can be applied to, for example, face packs, pastes, ointments, creams, gels, lotions, emulsions, serums, and toners. In addition, antiviral compositions are not limited to products used on the human body, but can also be applied to liquids that are sprayed or applied to products that come into contact with the skin, such as bedding, clothing, furniture, fixtures, and articles. Examples of materials used in these products include natural fibers, synthetic fibers, natural leather, artificial leather, synthetic leather, wood, synthetic resins, metals, and painted surfaces.

[0030] Antiviral compositions can be used to cleanse the skin, purify the mouth, and maintain healthy skin. Antiviral compositions can be used to suppress or alleviate sore throat, swelling, discomfort, irritation, or hoarseness caused by throat inflammation. Antiviral compositions can be used to sterilize, disinfect, or cleanse the skin, hands, mouth, or throat (pharynx). Antiviral compositions can also be used as virus barriers, virus blocks, virus shutouts, antivirals, and virus-out agents.

[0031] When an antiviral composition is used as an oral or throat composition in a quasi-drug or the like, it can be applied to, for example, a spray, toothpaste, liquid toothpaste, mouthwash, or rinsing solution. Examples of sprays include nasal sprays, throat sprays, and oral sprays.

[0032] <Mechanism of Action and Effects> The operation of this embodiment will now be described. In this embodiment, an extract from a plant or seaweed is used as the active ingredient. The composition containing the extract as the active ingredient has the effect of inhibiting the activity of neuraminidase. That is, the composition containing the extract as the active ingredient acts as a neuraminidase inhibitor. Hereinafter, a neuraminidase inhibitor will also be referred to as an NA inhibitor. Neuraminidase is an enzyme present on the surface of, for example, the influenza virus. The influenza virus spreads 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 the influenza virus from host cells is suppressed by inhibiting the activity of neuraminidase.

[0033] The effects of this embodiment will now be explained. (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 inhibition rate of neuraminidase activity, the more effectively the release of influenza viruses that have proliferated within host cells from the host cells can be suppressed. In other words, the higher the NA inhibition rate, the better the antiviral effect can be exhibited.

[0035] (2) We can provide oral compositions, topical 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 in which neuraminidase activity is involved in infection. For example, it can be expected to exert antiviral activity against viruses such as human parainfluenza virus, mumps virus, Sendai virus, and Newcastle disease virus.

[0036] (4) The antiviral composition can be expected to prevent viral infections, treat viral infections, inhibit viral replication, suppress viral infections, suppress viral replication, inactivate viral enzyme activity, inhibit viral enzyme activity, and inactivate viral enzyme activity. 〈Technical thought〉 The technical concepts that can be understood from the above embodiments are described below. [Note 1] An antiviral composition characterized by containing an extract from a plant or seaweed as an active ingredient. [Note 2] The antiviral composition according to Appendix 1, wherein the plant or seaweed comprises at least one selected from Fucus vesiculosus, Saxifraga stolonifera, Camellia sinensis, Evening primrose, Eucalyptus, Hypericum erectum, Sanguisorba officinalis, Houttuynia cordata, Tilia cordata, Cassia, Peony, Vitis coignetiae, and wild thyme. [Examples]

[0037] The antiviral composition will be described in more detail based on the following examples. However, the antiviral composition is not limited to the configurations described in the Examples section. Examples 2 to 41 below should be replaced by Reference Examples 2 to 41, respectively. This study was conducted based on the methods described in Japanese Patent Publication No. 2019-163292 and "A microplate-based screening assay for neuraminidase inhibitors," Drug Discoveries & Therapeutics, 2009, 3(6), pp. 260-265.

[0038] <<Principle for calculating NA inhibition rate>> This section explains the principle behind calculating the NA inhibition rate. A kit for measuring neuraminidase activity (NA-Fluor Influenza Neuraminidase Assay Kit, Thermo Fisher Scientific) was used to calculate the NA inhibition rate.

[0039] The above 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 excitation wavelengths of 350-365 nm and fluorescence wavelengths of 440-460 nm. The activity of neuraminidase can be measured based on the fluorescence intensity of the generated 4-MU.

[0040] When an NA inhibitor is added to the system in addition to MUNANA and neuraminidase, the activity of neuraminidase is inhibited in proportion to the inhibitory activity of the added NA inhibitor. In other words, the amount of 4-MU produced decreases in proportion 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).

[0041]

number

[0042] <<Evaluation Test>> The NA inhibition rates were evaluated using extracts from Examples 1 to 41 shown in Table 1 as NA inhibitors. NA inhibition rates were evaluated by tests using H1N1 neuraminidase and H3N2 neuraminidase. The reaction concentration of each NA inhibitor was set to 0.01 w / v%. Details of the NA inhibitors in each example are as follows.

[0043] (Example 1) We used Maruzen Pharmaceutical Co., Ltd.'s "Kaisou Extract BG-J" as an extract derived from the entire vegetative algae of Fucus vesiculosus.

[0044] (Example 2) As an extract derived from the whole plant of Saxifraga stolonifera, we used "Saxifraga stolonifera Extract BG" from Maruzen Pharmaceutical Co., Ltd.

[0045] (Example 3) For the extract derived from Assam tea, we used "Black Tea Liquid" from Ichimaru Falcos Co., Ltd.

[0046] (Example 4) We used "Luna White B" from Ichimaru Falcos Co., Ltd. as an extract derived from the seeds of the evening primrose.

[0047] (Example 5) We used "Eucalyptus Extract BG" from Maruzen Pharmaceutical Co., Ltd. as the extract derived from eucalyptus leaves.

[0048] (Example 6) We used "Otogirisou B" from Maruzen Pharmaceutical Co., Ltd., an extract derived from the above-ground parts of St. John's wort.

[0049] (Example 7) Maruzen Pharmaceutical Co., Ltd.'s "Jiyu Extract BG-R" was used as an extract derived from the roots and rhizomes of Sanguisorba officinalis.

[0050] (Example 8) For the extract derived from the above-ground parts of Houttuynia cordata, we used "Falcorex Houttuynia B" from Ichimaru Falcos Co., Ltd.

[0051] (Example 9) We used "Tilia Cordata Extract BG-J" from Maruzen Pharmaceutical Co., Ltd., which is an extract derived from the flowers of the winter linden tree.

[0052] (Example 10) For the extract derived from cassia bark, we used "Cinnamon Extract W-LA" from Maruzen Pharmaceutical Co., Ltd.

[0053] (Example 11) For the extract derived from the root bark of the peony, we used "Falcorex Peony Bark B" from Ichimaru Falcos Co., Ltd.

[0054] (Example 12) As an extract derived from European grape leaves, we used "Red Grape Extract BG" from Maruzen Pharmaceutical Co., Ltd.

[0055] (Example 13) For the extract derived from wild thyme, we used "Falcorex Wild Thyme B" from Ichimaru Falcos Co., Ltd.

[0056] (Example 14) We used Maruzen Pharmaceutical Co., Ltd.'s "Waism <Green Tea>" as the extract derived from green tea.

[0057] (Examples 15-41) Table 2 shows Examples 15-41. Each example contains an extract derived from a plant or seaweed. Table 2 also shows the product name and manufacturer of the extract used in each example.

[0058] <Preparation of Reagents> (1 × Assay Buffer) 1× Assay Buffer was prepared by diluting the 2× Assay Buffer (66.6 mM MES, 8 mM CaCl2, pH 6.5) included in the above kit 2-fold with distilled water.

[0059] (200μM substrate) A 2.5 mM substrate was prepared by dissolving the MUNANA contained in the above kit in distilled water. A 200 μM substrate was prepared by diluting the 2.5 mM substrate with 1 × Assay Buffer.

[0060] (NA inhibitors) The NA inhibitor was diluted with distilled water to a concentration of 0.04 w / v% of the extract. (NA solution) A neuraminidase solution of influenza A virus H1N1 subtype was prepared. The H1N1 subtype neuraminidase used was "Influenza A H1N1 (A / California / 04 / 2009) Neuraminidase / NA (Active)" from Sino Biological Inc. The solution was diluted with 1× Assay Buffer to a neuraminidase concentration of 0.1 U / mL when mixed with the substrate.

[0061] A neuraminidase solution of influenza A virus H3N2 subtype was prepared. The H3N2 subtype neuraminidase used was "Influenza A H3N2 Neuraminidase / NA (Active)" from Sino Biological Inc. The solution was diluted with 1× Assay Buffer to a neuraminidase concentration of 0.03 U / mL when mixed with the substrate.

[0062] <Testing Method> Using a black 96-well plate, 25 μL of the prepared NA inhibitor was added to each well. In the wells for measuring BG2 and RFUc, 25 μL of 1× Assay Buffer was added instead of the NA inhibitor.

[0063] 25 μL of the prepared NA solution was added to each well. For the wells used to measure BG1 and BG2, 25 μL of 1×Assay Buffer was added instead of the NA solution.

[0064] 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 in the dark. The reaction was stopped by adding 100 μL of the reaction stop solution (0.2 M Na2CO3 solution) included in the above kit to each well.

[0065] A plate reader (Cytation5, BioTek Instruments, Inc.) was used to measure the plate at an excitation wavelength of 360 nm and an 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.

[0066] • Criteria for evaluating antiviral activity ○○○ (Excellent): When the NA inhibition rate for both H1N1 and H3N2 is 90% or higher. ○○ (Good): When the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are both 50% or higher, and at least one of them is less than 90%. ○ (Slightly Good): When either the NA inhibition rate for H1N1 or the NA inhibition rate for H3N2 is 50% or higher, and the other is less than 50%. △ (Acceptable): If either the NA inhibition rate for H1N1 or the NA inhibition rate for H3N2 is 1% or higher, and both the NA inhibition rates for H1N1 and H3N2 are less than 50%.

[0067] <Test Results> As shown in Table 1, in Examples 1 to 14, the NA inhibition rates for H1N1 and H3N2 were 50% or higher. In other words, the antiviral activity was well evaluated in Examples 1 to 14. In particular, in Examples 1 to 7, the NA inhibition rates for H1N1 and H3N2 were 80% or higher, showing even better results. Furthermore, in Examples 2 and 4, the NA inhibition rates for H1N1 and H3N2 were 90% or higher, suggesting even better antiviral activity.

[0068] As shown in Table 2, Examples 15-17 demonstrated that the NA inhibition rate for H1N1 was 50% or higher, indicating inhibition of H1N1 neuraminidase activity. On the other hand, the NA inhibition rate for H3N2 was less than 50%. In other words, 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 jasminoides fruit, are considered to have somewhat weak antiviral activity. Phellodendron amurense is a plant belonging to the genus Phellodendron in the family Rutaceae. Scutellaria baicalensis is a plant belonging to the genus Scutellaria in the family Lamiaceae. Gardenia jasminoides is a plant belonging to the genus Gardenia in the family Rubiaceae.

[0069] Examples 28, 29, 31, and 34 showed that the NA inhibition rate for H3N2 was 50% or higher, indicating inhibition of H3N2 neuraminidase activity. On the other hand, the NA inhibition rate for H1N1 was less than 50%. In other words, Example 28, which contains an extract of the whole horsetail plant; Example 29, which contains an extract of the peel of Satsuma mandarin; Example 31, which contains an extract of the flower of calendula; and Example 34, which contains an extract of the root bark of mulberry, are considered to have somewhat weak antiviral activity. Horsetail is a plant of the genus Equisetum in the family Equisetaceae. Satsuma mandarin is a plant of the genus Citrus in the family Rutaceae. Calendula is a plant of the genus Calendula in the family Asteraceae. Mulberry is a plant of the genus Morus in the family Moraceae.

[0070] As positive controls, tests were conducted using oseltamivir phosphate from Fujifilm Wako Pure Chemical Corporation and zanamivir hydrate from Tokyo Chemical Industry Co., Ltd. Tests using oseltamivir phosphate at a reaction concentration of 25 μM showed a 100% NA inhibition rate for H1N1. Tests using zanamivir hydrate at a reaction concentration of 25 μM showed a 96% NA inhibition rate for H1N1.

[0071] [Table 1]

[0072] Table 2

Claims

1. A neuraminidase inhibitor containing an extract from Fucus vesiculosus as an active ingredient, which inhibits the activity of influenza virus neuraminidase, In tests using neuraminidase of influenza A virus H1N1 subtype and tests using neuraminidase of influenza A virus H3N2 subtype, the inhibition rate of neuraminidase activity, calculated at a neuraminidase inhibitor concentration of 0.01 w / v%, was 80% or higher in both cases. Neuraminidase inhibitors.

2. The neuraminidase inhibitor according to claim 1, for oral use.

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