JP2023006350A - antiviral composition - Google Patents

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

The present invention relates to antiviral compositions.

Oseltamivir, Zanamivir, etc. are known as antiviral drugs used against viruses such as influenza virus.
For example, as disclosed in Patent Document 1, inhibition of neuraminidase activity is an anti-influenza virus effect of oseltamivir, zanamivir, and the like. Neuraminidase is an enzyme that hydrolyzes terminal sialic acid residues on polysaccharide chains. Inhibition of the neuraminidase activity suppresses the release of the replicated virus from the host cell, thereby suppressing the spread of infection to other host cells. On the other hand, Patent Document 1 describes that antiviral drugs such as oseltamivir and zanamivir have side effects. In addition, Non-Patent Document 1 describes viruses that have acquired oseltamivir resistance or zanamivir resistance.

Patent Document 1 proposes an antiviral agent containing a watermelon extract as an active ingredient as an antiviral agent other than oseltamivir and zanamivir.

JP 2017-178913 A

"Influenza neuraminidase: a druggable target for natural products.", Natural product reports, 2012, 29(1), p.11-36

There is a demand for antiviral compositions that exhibit antiviral effects other than antiviral drugs such as oseltamivir and zanamivir.

An antiviral composition for solving the above problems is characterized by containing an extract from a plant or seaweed as an active ingredient.
In the above antiviral composition, the plant or seaweed is at least one selected from fucus, saxifrage, tea tree, evening primrose, eucalyptus, St. John's wort, burnet, Houttuynia cordata, cordata, cassia, botan, European grape, and wild thyme. preferably included.

The antiviral composition of the present invention can exert an antiviral action.

An embodiment of the antiviral composition is described below.
The antiviral composition of this embodiment contains a plant or seaweed extract as an active ingredient. Antiviral compositions exert their antiviral effects by inhibiting the activity of neuraminidase.

<Extract>
Extracts from plants or seaweed are described. In the present embodiment, an effective extract was defined as an extract that inhibited neuraminidase activity at a concentration of 0.01 w/v % during the reaction. In the following, the rate of inhibition of neuraminidase activity is also referred to as NA inhibition rate. As an example, the NA inhibition rate was evaluated as the rate of inhibition of activity against influenza A virus H1N1 subtype neuraminidase and influenza A virus H3N2 subtype neuraminidase. More specifically, an effective extract is one in which either the NA inhibition rate for influenza A virus H1N1 subtype or the NA inhibition rate for influenza A virus H3N2 subtype is 1% or more at the extract concentration described above. bottom. In the following, the influenza A virus H1N1 subtype is also referred to as "H1N1". The H3N2 subtype of influenza A virus is also referred to as “H3N2”. A method for calculating the NA inhibition rate will be described later.

The NA inhibition rate is preferably 50% or higher, more preferably 80% or higher, and even more preferably 90% or higher. Also, it is more preferable that both the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are high.

Although the extract from plants or seaweed is not particularly limited, it is preferably 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 alcohol and water may be used.

The extract from plants or seaweed may be purified as necessary. One of the following extracts may be used alone, or two or more may be used in combination.
Plants used in the antiviral composition of the present embodiment are not particularly limited, but for example, Saxifrageaceae, Onagraceae, Rosaceae, Theaceae, Myrtaceae, Hypericaceae, Lindenaceae, Lauraceae, Houttuyaceae, and Grapeaceae. , Peonies, Labiatae, and the like.

As a plant of the Saxifrageaceae family, specifically, the genus Saxifraga is exemplified, and more specifically, saxifrage is exemplified. For example, an extract extracted from the whole plant of Saxifrage can be used as the extract.

Examples of plants of the family Onagraceae specifically include evening primrose, more specifically evening primrose. For example, an extract extracted from evening primrose seeds can be used as the extract.

As a plant of the family Rosaceae, specific examples include the genus Burne, and more specifically, Burn this plant. For example, an extract extracted from the root and rhizome of burnet can be used as the extract.

Specific examples of the plants of the Camellia family include the genus Camellia, and more specifically, the tea tree. The extracts extracted from tea leaves include green tea extract, black tea extract and the like. An example of black tea is Assam tea.

As a plant of Myrtaceae, specifically Eucalyptus can be mentioned. For example, an extract extracted from eucalyptus leaves can be used as the extract.
As a plant belonging to the family Hypericum, specifically, the genus Hypericum is mentioned, and more specifically, St. John's wort is mentioned. For example, an extract extracted from the aerial parts of St. John's wort can be used as the extract.

Specific examples of the plant of the linden family include the genus Linden, and more specifically, the linden tree. For example, an extract extracted from flowers of Corianthus can be used as the extract.

Specific examples of plants of the Lauraceae family include the genus Cinnamomum, and more specifically cassia. For example, an extract extracted from cassia bark can be used as the extract.

Examples of plants belonging to the family Houttuynia include specifically the genus Houttuynia, and more specifically Houttuynia cordata. For example, an extract extracted from the aerial parts of Houttuynia cordata can be used as the extract.

Specific examples of the plants of the family Grapeaceae include the genus Grapes, and more specifically European grapes, which are a type of red grape. For example, an extract extracted from European grape leaves can be used as the extract.

As a plant belonging to the Peonaceae family, Peonies are specifically exemplified, and Peonies are more specifically exemplified. For example, an extract extracted from the root bark of button can be used as the extract.

Specific examples of plants of the Labiatae family include Thymus genus, and more specific examples include wild thyme (Thymus serpyllum). For example, an extract extracted from wild thyme can be used as the extract.

The seaweed used in the antiviral composition of the present embodiment is not particularly limited, and examples thereof include brown algae. Specifically, fucus is mentioned. For example, an extract extracted from whole algae of fucus can be used as the extract.

Plants and seaweeds used in the antiviral composition of the present embodiment are preferably ingredients that can be incorporated into quasi-drugs. By being a component that can be blended into quasi-drugs, it is possible to suppress side effects and use it more safely.

<Applicable form>
The application form of the antiviral composition is not particularly limited, and for example, it can be used as foods, cosmetics, pharmaceuticals, and quasi-drugs.

When the antiviral composition is used as an oral composition such as food, it can be applied to, for example, candy, troche, tablet, gum, gummies, granules, powder, jelly, syrup, beverage and the like.

When the antiviral composition is used as an external composition such as cosmetics, it can be applied to, for example, face packs, pastes, ointments, creams, gels, lotions, milky lotions, serums, lotions, and the like. In addition, the antiviral composition can be applied not only to products used on the human body, but also to liquid agents that are sprayed or applied to products that come into contact with the skin, such as bedding, clothing, furniture, fixtures, and articles. can also Examples of materials for target products include natural fibers, synthetic fibers, natural leathers, artificial leathers, synthetic leathers, woods, synthetic resins, metals, and painted surfaces.

The antiviral composition can be used to cleanse the skin or skin, cleanse the mouth, keep the skin or skin healthy. The antiviral composition can be used to suppress or alleviate sore throat, swelling in the throat, discomfort in the throat, sore throat, or hoarseness due to inflammation of the throat. The antiviral composition can be used to disinfect, disinfect, or cleanse the skin, skin, fingers, oral cavity, or throat (pharynx). The antiviral composition can also be used for virus barrier, virus blocking, virus shutting, antivirus, and virus elimination.

When the antiviral composition is used as an oral composition such as a quasi-drug or a throat composition, for example, sprays, toothpastes, liquid dentifrices, mouthwashes, rinses, etc. can be applied. Sprays include, for example, nasal sprays, throat sprays, oral sprays and the like.

<Action and effect>
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 an extract as an active ingredient has the effect of inhibiting the activity of neuraminidase. That is, the composition containing the active ingredient extract acts as a neuraminidase inhibitor. In the following, neuraminidase inhibitors are also referred to as NA inhibitors. Neuraminidase is an enzyme present on the surface of, for example, influenza virus. Influenza viruses are released from host cells by neuraminidase activity to spread infection to other cells. According to the antiviral composition of the present embodiment, inhibition of neuraminidase activity suppresses release of influenza virus from host cells.

Effects of the present embodiment will be described.
(1) Antiviral compositions contain plant or seaweed extracts as active ingredients and exhibit antiviral effects.

The higher the rate of inhibition of neuraminidase activity, the more preferably the release of influenza virus grown in host cells from the host cells can be suppressed. That is, the higher the NA inhibition rate, the better the antiviral effect can be exhibited.

(2) It is possible to provide oral compositions, external compositions, oral compositions, and throat compositions that exhibit antiviral effects.
(3) The antiviral composition may be useful not only against influenza virus but also against viruses whose infection involves neuraminidase activity. For example, it can be expected to exhibit an antiviral effect against viruses such as human parainfluenza virus, mumps virus, Sendai virus, and Newcastle disease virus.

(4) According to the antiviral composition, prevention of viral infection, treatment of viral infection, inhibition of viral proliferation, suppression of viral infection, suppression of viral proliferation, inactivation of viral enzyme activity, suppression of viral enzyme activity, viral enzyme Activation and inactivation can be expected.

The antiviral composition will be described in more detail based on the following examples. In addition, the antiviral composition is not limited to the configuration described in the Examples section.
This test was based on 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. .

<<Calculation principle of NA inhibition rate>>
The principle of the method for calculating the NA inhibition rate will be explained. A kit for measuring neuraminidase activity (NA-Fluor Influenza Neuraminidase Assay Kit, Thermo Fisher Scientific) was used to calculate the NA inhibition rate.

The above kit contains the fluorescent substrate MUNANA (4-(methylumbelliferyl)-N-acetylneuraminic acid). MUNANA is degraded by neuraminidase activity. When MUNANA is decomposed, N-acetylneuraminic acid and a fluorescent substance 4-MU (4-Methylumbelliferone) are liberated. 4-MU can be detected by fluorescence measurement with 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 4-MU produced.

Addition of an NA inhibitor to the system in addition to MUNANA and neuraminidase inhibits neuraminidase activity depending on the inhibitory potency of the added NA inhibitor. That is, the amount of 4-MU produced decreases according to the inhibitory ability of the NA inhibitor. Therefore, the ability of each NA inhibitor to inhibit neuraminidase can be calculated based on the fluorescence intensity. Specifically, the NA inhibition rate can be calculated by the following calculation formula (Formula 1).

算出式（式１）において、ＲＦＵｃは、ノイラミニダーゼ及び基質を添加した場合のＲＦＵ値（relative fluorescence unit）である。ＢＧ２は、バックグラウンドノイズ測定のために、ノイラミニダーゼを添加せず基質のみを添加した場合の蛍光強度である。ＲＦＵｓは、各ＮＡ阻害剤、ノイラミニダーゼ及び基質を添加した場合のＲＦＵ値である。ＢＧ１は、バックグラウンドノイズ測定のために、ノイラミニダーゼを添加せずＮＡ阻害剤と基質とを添加した場合の蛍光強度である。算出式（式１）によれば、ＮＡ阻害剤の阻害能によって蛍光強度が低下するほどＮＡ阻害率が高く算出される。

In the calculation formula (Formula 1), RFUc is the RFU value (relative fluorescence unit) when neuraminidase and substrate are added. BG2 is the fluorescence intensity when no neuraminidase was added and only substrate was added for background noise measurement. RFUs is the RFU value when each NA inhibitor, neuraminidase and substrate were added. BG1 is the fluorescence intensity when no neuraminidase was added and NA inhibitor and substrate were added for background noise measurement. According to the calculation formula (Equation 1), the NA inhibition rate is calculated to be higher as the fluorescence intensity decreases due to the inhibition ability of the NA inhibitor.

<<Evaluation test>>
The NA inhibition rate was evaluated using the extracts of Examples 1 to 41 shown in Table 1 as NA inhibitors. NA inhibition rate was assessed by studies with H1N1 neuraminidase and studies with H3N2 neuraminidase. The concentration of each NA inhibitor during reaction was 0.01 w/v%. Details of the NA inhibitor in each example are as follows.

(Example 1)
As an extract extracted from whole algae of fucus, Maruzen Pharmaceutical Co., Ltd.'s "Kaiso extract BG-J" was used.

(Example 2)
As an extract extracted from the whole plant of Saxifrage, Maruzen Pharmaceutical Co., Ltd.'s "Saxifrage extract BG" was used.

(Example 3)
As an extract extracted from Assam tea, "Black Tea Liquid" manufactured by Ichimaru Farcos Co., Ltd. was used.

(Example 4)
As the extract extracted from the seeds of evening primrose, "Luna White B" manufactured by Ichimaru Farcos Co., Ltd. was used.

(Example 5)
As an extract extracted from eucalyptus leaves, “Eucalyptus Extract BG” manufactured by Maruzen Pharmaceutical Co., Ltd. was used.

(Example 6)
As an extract extracted from the aerial part of St. John's wort, "Hypericum perforatum B" of Maruzen Pharmaceutical Co., Ltd. was used.

(Example 7)
As an extract extracted from the roots and rhizomes of burnet, Maruzen Pharmaceutical Co., Ltd.'s "Jiyu Extract Liquid BG-R" was used.

(Example 8)
As an extract extracted from the aerial part of Houttuynia cordata, “Falcorex Dokudami B” manufactured by Ichimaru Falcos Co., Ltd. was used.

(Example 9)
Maruzen Pharmaceutical Co., Ltd.'s "linden extract BG-J" was used as the extract extracted from the flowers of Tricholoma chinensis.

(Example 10)
As an extract extracted from cassia bark, Maruzen Pharmaceutical Co., Ltd.'s "Keihi Extract W-LA" was used.

(Example 11)
As an extract extracted from the root bark of the button, "Falcorex buttonpi B" manufactured by Ichimaru Falcos Co., Ltd. was used.

(Example 12)
As the extract extracted from the European grape leaves, "red grape extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd. was used.

(Example 13)
As an extract extracted from wild thyme, "Falcorex Wild Thyme B" manufactured by Ichimaru Falcos Co., Ltd. was used.

(Example 14)
As an extract extracted from green tea, “waism <green tea>” manufactured by Maruzen Pharmaceutical Co., Ltd. was used.

(Examples 15-41)
Table 2 shows Examples 15-41. Each example contains extracts extracted from plants or seaweed. Table 2 also shows the product name and manufacturer of the extract used in each example.

<Preparation of reagent>
(1x Assay Buffer)
2x Assay Buffer (66.6 mM MES, 8 mM CaCl2, pH 6.5) included in the above kit was diluted _twice with distilled water to prepare 1x Assay Buffer.

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

(NA inhibitor)
Distilled water was added to dilute the extract so that the concentration of the extract in the NA inhibitor was 0.04 w/v%.
(NA solution)
A neuraminidase solution of influenza A virus subtype H1N1 was prepared. As the H1N1 subtype neuraminidase, "Influenza A H1N1 (A/California/04/2009) Neuraminidase/NA (Active)" from Sino Biological Inc. was used. It was diluted with 1×Assay Buffer so that the neuraminidase concentration would be 0.1 U/mL when mixed with the substrate.

A neuraminidase solution of influenza A virus subtype H3N2 was prepared. As the H3N2 subtype neuraminidase, "Influenza A H3N2 Neuraminidase/NA (Active)" from Sino Biological Inc. was used. It was diluted with 1×Assay Buffer so that the concentration of neuraminidase when mixed with the substrate would be 0.03 U/mL.

<Test method>
Using a black 96-well plate, 25 μL of prepared NA inhibitor was added to each well. Instead of the NA inhibitor, 25 μL of 1×Assay Buffer was added to the wells for measuring BG2 and the wells for measuring RFUc.

25 μL of the prepared NA solution was added to each well. Instead of the NA solution, 25 μL of 1×Assay Buffer was added to the wells for measuring BG1 and the wells for measuring BG2.

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

Plates were read at an excitation wavelength of 360 nm and an emission wavelength of 450 nm using a plate reader (Cytation5, BioTek Instruments, Inc.). Based on the measurement results, the NA inhibition rate in each example was calculated. Furthermore, evaluation was performed according to the following criteria. Tables 1 and 2 show the results.

· Evaluation criteria for antiviral action ○○○ (excellent): When both the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 are 90% or more.
○○ (Good): NA inhibition rate for H1N1 and NA inhibition rate for H3N2 are both 50% or more, and at least one is less than 90%.
○ (fairly good): when either one of the NA inhibition rate for H1N1 and the NA inhibition rate for H3N2 is 50% or more and the other is less than 50%.
△ (Possible): Either NA inhibition rate for H1N1 or NA inhibition rate for H3N2 is 1% or more, and both NA inhibition rate for H1N1 and NA inhibition rate for H3N2 are less than 50% If it is.

<Test results>
As shown in Table 1, in Examples 1 to 14, the NA inhibition rate of H1N1 and NA inhibition rate of H3N2 were 50% or more. That is, in Examples 1 to 14, the evaluation of the antiviral action was good. In particular, in Examples 1 to 7, the NA inhibition rate of H1N1 and the NA inhibition rate of H3N2 were 80% or more, showing better results. Furthermore, in Examples 2 and 4, the H1N1 NA inhibition rate and the H3N2 NA inhibition rate were 90% or more, and a more excellent antiviral effect can be expected.

As shown in Table 2, Examples 15 to 17 exhibited an NA inhibition rate of 50% or more for H1N1, indicating that H1N1 neuraminidase activity was inhibited. On the other hand, the NA inhibition rate for H3N2 was less than 50%. That is, Example 15, which contains the bark extract of Amur cork, Example 16, which contains the root extract of Scutellaria baicalensis, and Example 17, which contains the extract of gardenia fruit, are considered to have slightly weak antiviral effects. Yellowfin is a plant belonging to the genus Amurinus of the Rutaceae family. Scutellaria baicalensis is a plant belonging to the genus Scutellariae of the Labiatae family. Gardenia is a plant belonging to the genus Gardenia of the family Rubiaceae.

Examples 28, 29, 31 and 34 were shown to inhibit the neuraminidase activity of H3N2 with a NA inhibition rate of 50% or greater for H3N2. On the other hand, the NA inhibition rate for H1N1 was less than 50%. That is, Example 28 containing horsetail whole plant extract, Example 29 containing mandarin orange pericarp extract, Example 31 containing Calendula officinalis flower extract, and Magwa root bark extract are contained. In Example 34, the antiviral action is considered to be slightly weak. Horsetail is a plant belonging to the genus Horsetail of the family Horsetail family. Unshu mikan is a plant belonging to the genus Citrus of the family Rutaceae. Calendula officinalis is a plant belonging to the genus Calendula of the Asteraceae family. Mugwort is a plant belonging to the genus Mulberry of the family Moraceae.

As a positive control, a test was performed using oseltamivir phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) and Zanamivir Hydrate (Tokyo Chemical Industry Co., Ltd.). As a result of testing using oseltamivir phosphate at a concentration of 25 μM at the time of reaction, 100% NA inhibition rate could be confirmed for H1N1. As a result of testing using Zanamivir Hydrate at a concentration of 25 μM at the time of reaction, a 96% NA inhibition rate was confirmed for H1N1.

Claims (2)

An antiviral composition comprising a plant or seaweed extract as an active ingredient. 2. The plant or seaweed includes at least one selected from fucus, saxifrage, tea tree, evening primrose, eucalyptus, St. John's wort, burnet, houttuynia cordata, cassia, botan, European grape, and wild thyme. A composition for viruses.