JP2016044155A - Neuraminidase inhibitor, anti-influenza agent, food product and agent comprising the same, and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a neuraminidase inhibitor derived from a Ganoderma lingzhi extract, food products, cosmetics and pharmaceuticals (anti-influenza agents) comprising the neuraminidase inhibitor, and a method for producing the neuraminidase inhibitor.SOLUTION: A neuraminidase inhibitor has the effect of inhibiting the activity of neuraminidase (NA), the neuraminidase inhibitor comprising an organic solvent extract from Ganoderma lingzhi or a triterpenoid contained therein, or an aqueous solvent extract from Ganoderma lingzhi. There are also provided food products, cosmetics and pharmaceuticals (anti-influenza agents) comprising the same.SELECTED DRAWING: None

Description

The present invention relates to a neuraminidase inhibitor containing an extract of Ganoderma lingzhi , particularly fruiting bodies thereof, an anti-influenza agent containing the same, foods and drugs, and methods for producing them.

Ganoderma lingzhi has long been used for health promotion and treatment of various diseases in East Asian countries, especially China and Japan.
Research has revealed that triterpenoids are included as one of the main bioactive components in Ganoderma lingzhi . Over the last few years, more than 150 new triterpenoids have been discovered in Ganoderma lingzhi due to advances in isolation technology and the use of the latest analytical instruments. Of these, 50 triterpenoids were found to be unique to Ganoderma lingzhi . The physiological activities of these triterpenoids are usually analyzed individually with a focus on antitumor activity and immunomodulatory activity. Patent Document 1 discloses that a mixture of Sendai leaf extract powder, Ganoderma extract powder (hot water extract) and Ganoderma black baked powder at a predetermined weight ratio is useful as an influenza drug. However, Patent Document 1 does not mention a chemical substance contained in ganoderma, particularly a component extracted with an organic solvent. Therefore, the antiviral activity, especially the anti-influenza virus activity of triterpenoids in Ganoderma has been little investigated.

Furthermore, Patent Document 2 discloses an antiviral preparation containing, as an active ingredient, a pentameric procyanidin flavonoid obtained from Ganoderma or the like.
Every year, it is said that 5-10% of adults and 20-30% of children in the world are infected with seasonal influenza. Type A influenza virus has two coat proteins, hemagglutinin (HA) and neuraminidase (NA), of which HA binds to sialic acid present on the surface of cells such as the human respiratory tract. Due to this binding, the influenza virus is surrounded by the cell membrane and taken into the cell, and a vesicle (endosome) encapsulating the influenza virus is formed in the cell. When the pH inside the vesicle encapsulating the influenza virus is lowered, the coat protein portion of the influenza virus in the vesicle is fused with the inner wall of the vesicle, and the genome portion of the influenza virus is released into the cell. Thereafter, a replication function of the cell works based on the genome, and influenza virus is produced in large quantities in the cell. Next, the mass-produced influenza virus is released from the cells, where the neuraminidase (NA) portion of the influenza virus binds the HA portion of the influenza virus and the sialic acids of the glycoprotein of the cell. Disconnect. Since the influenza virus is released from the cells by this cleavage, neuraminidase (NA) on the surface of the influenza virus is an essential enzyme for releasing the influenza virus from cells such as the human respiratory tract. Therefore, the neuraminidase (NA) portion of influenza virus is regarded as a special target molecule for anti-influenza drugs.

  “Relenza” (registered trademark) and “Tamiflu” (registered trademark), which are generally known as anti-influenza agents, inhibit the activity of neuraminidase (NA), thereby causing influenza virus to escape from (host) cells such as the human respiratory tract. By inhibiting the release, it is prevented from moving to other cells or other humans and spreading and proliferating.

  If a new substance (neuraminidase inhibitor) that can inhibit the activity of neuraminidase is found in the same manner as “Relenza” and “Tamiflu”, influenza that is resistant to existing anti-influenza drugs (eg, Tamiflu resistance) Can also be used for humans and other animals of idiosyncratic nature such as side effects caused by known anti-influenza drugs such as Tamiflu. It is also beneficial from the viewpoint of expanding the options for anti-influenza drugs and contributing to diversification of anti-influenza drugs. Therefore, various anti-influenza agents are desired.

JP 2011-1321 A Special table 2011-530609 gazette

The present inventors include an inhibition model in which the neuraminidase (NA) activity of influenza virus is inhibited using Relenza (registered trademark) or Tamiflu (registered trademark), and is included in Ganoderma lingzhi 33 As a result of examining whether various types of triterpenoids inhibit neuraminidase (NA) of influenza virus, it has been found that some triterpenoids have NA inhibitory ability.

Therefore, the present invention contains a triterpenoid contained in Ganoderma lingzhi as an active ingredient, and a neuraminidase inhibitor capable of inhibiting or suppressing influenza virus neuraminidase (NA) activity, An object is to provide anti-influenza drugs, foods and drugs, and methods for producing them.

The present invention provides the following neuraminidase inhibitors and the like.
[1] A neuraminidase inhibitor comprising tripenoids obtained by extraction from Ganoderma lingzhi with an organic solvent or an aqueous solvent as an active ingredient.

[2] One or two selected from the group consisting of the following compounds (1) to (33), including a tripenoid contained in the extract of the organic solvent as an active ingredient of the neuraminidase inhibitor. The neuraminidase inhibitor according to [1], wherein the neuraminidase inhibitor is a compound of more than one species (note that the numbers (1) to (33) are given for the convenience of explanation of the present invention).
(1) Ganoderic acid Z (Ganoderic acid Z),
(3) Ganoderic acid S (Ganoderic acid S),
(4) Ganoderic acid F,
(5) Ganoderic acid SZ (Ganoderic acid SZ),
(6) Ganoderic acid LM2 (Ganoderic acid LM2),
(7) Ganoderic acid DM (Ganoderic acid DM),
(8) Ganoderic acid A,
(9) Ganoderic acid AM1 (Ganoderic acid AM1),
(10) Ganoderic acid B (Ganoderic acid B),
(12) Ganoderic acid Y,
(13) Ganoderic acid TN (Ganoderic acid TN),
(14) Ganoderic acid TQ (Ganoderic acid TQ),
(15) Ganolucidic acid A,
(16) Ganodermanontriol,
(17) Ganodermanondiol,
(18) Ganoderol A (Ganoderol A),
(19) Ganoderol B,
(20) Ganoderiol F,
(21) Lucialdehyde A,
(22) Lucialdehyde B,
(23) Ganoderic acid C1 (Ganoderic acid C1),
(24) Ganoderic acid C2 (Ganoderic acid C2),
(25) Ganoderic acid C6,
(26) Ganoderic acid H,
(27) Ganoderic acid K,
(28) Ganoderic acid TR (Ganoderic acid TR),
(29) Ganoderenic acid A,
(30) Ganoderenic acid C,
(31) Ganoderenic acid D,
(32) Ganoderenic acid F,
(33) Ganoderenic acid H.

  [3] (1) Ganoderic acid Z, (2) Ganoderic acid N, (3) Ganoderic acid S, (4) Ganodermic acid F ( Ganoderic acid F), (5) Ganoderic acid SZ (Ganoderic acid SZ), (6) Ganoderic acid LM2 (Ganoderic acid LM2), (7) Ganoderic acid DM (Ganoderic acid DM), (8) Ganoderic acid DM A (Ganoderic acid A), (9) Ganoderic acid AM1, (10) Ganoderic acid B, (11) Ganoderic acid D, (12) Ganodermic acid D Derrick acid Y (Ganoderic acid Y), (13) Ganoderic acid TN (Ganoderic acid TN), (14) Ganoderic acid TQ (Ganoderic acid TQ), (1 ) Ganolucidic acid A, (16) Ganodermanontriol, (17) Ganodermanondiol, (18) Ganoderol A, (19) Ganoderol B ), (20) Ganoderiol F, (21) Lucialdehyde A, (22) Lucialdehyde B, (23) Ganoderic acid C1, (24) Ganoderic acid C2 (Ganoderic acid C2), (25) Ganoderic acid C6 (Ganoderic acid C6), (26) Ganoderic acid H (Ganoderic acid H), (27) Ganoderic acid K (Ganoderic acid K), ( 28) Ganoderic acid TR, (29) Ganoderenic acid A, (30) Ganoderin 1 selected from the group consisting of C (Ganoderenic acid C), (31) Ganoderenic acid D, (32) Ganoderenic acid F, and (33) Ganoderenic acid H. A neuraminidase inhibitor comprising a species or two or more of the tripenoids. (In [3], it does not matter whether the tripenoids are derived from ganoderma or synthesized.)

  [4] The neuraminidase inhibitor according to [1], wherein the aqueous solvent is hot water and the extraction component is a hot water extract.

[5] Extract (extract) extracted from Ganoderma lingzhi with an organic solvent or an aqueous solvent in an amount equivalent to an organic solvent extract (extract) of 400 μg / mL or more. The neuraminidase inhibitor according to [1], which is contained in an amount of 1600 μg / mL or more in terms of a solvent extract (extract).

  [6] A food (health supplement) or an anti-influenza agent for preventing influenza infection or suppressing or reducing the progression of symptoms, comprising the neuraminidase inhibitor according to any one of [1] to [5].

  [7] The anti-influenza agent according to [6], wherein the symptom is pneumonia, acute encephalopathy or Reye syndrome, cardiac complications, acute myositis, otitis media, conjunctivitis, sinusitis, myocarditis).

  [8] A bittering agent containing the neuraminidase inhibitor according to any one of [1] to [5].

[9] A method for producing an anti-influenza agent having an anti-influenza action, comprising a step of extracting a component having a neuraminidase inhibitory action from Ganoderma lingzhi using an organic solvent or an aqueous solvent. .

  [10] The method for producing an anti-influenza agent according to [9], wherein the aqueous solvent is hot water.

According to the present invention, a neuraminidase inhibitor which contains specific triterpenoids (derived from or synthesized from Ganoderma lingzhi ) as an active ingredient and can inhibit or suppress the activity of neuraminidase (NA) of influenza virus, Provided are anti-influenza agents, foods and medicaments, and methods for their efficient production.

FIG. 1 is a diagram illustrating the principle of a method for measuring the inhibitory activity of a neuraminidase inhibitor using the principle of measuring neuraminidase activity. In FIG. 1, MUNANA (4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid) is converted to 4-MU (4-methylumbelliferone), which is a phosphor, by the action of neuraminidase. 4-MU is a substance that emits pale blue fluorescence having a peak near a wavelength of 450 nm when irradiated with ultraviolet light near a wavelength of 360 nm. The fluorescence intensity (F) at a wavelength of 450 nm of the produced 4-MU can be quantified, and the neuraminidase (NA) activity can be quantified from the fluorescence intensity. Since the activity of neuraminidase is inhibited by adding an inhibitor to the reaction system of FIG. 1 according to the NA inhibitory ability of the added inhibitor, the amount of 4-MU produced is reduced. The ability to inhibit neuraminidase can be quantified. FIGS. 2A and 2B are graphs of inhibitor concentration (nM) -inhibition rate (IR (%)) against neuraminidase (H1N1) for Tamiflu known as an anti-influenza agent (NA inhibitor). FIG. From the approximate expression in FIG. 2B, it can be seen that the IC _{50 of} Tamiflu (the concentration of inhibitor necessary to obtain half the maximum inhibition rate (50%)) is 260 nM. 2A and 2B are diagrams for comparing the NA inhibitory ability of the neuraminidase inhibitor according to the present invention with the NA inhibitory ability of Tamiflu. FIG. 3 shows (3) ganoderic acid S, (9) ganoderic acid AM1, (13) ganoderic acid TN, (14) against neuraminidase (H1N1) derived from influenza virus as the inhibitor of FIG. ) Ganoderlic acid TQ, (19) Ganoderol B, (21) Rusylaldehyde A, (22) Rusylaldehyde B, (27) Ganodermic acid K, (28) Ganoderic acid TR, (29) Ganoderenic acid A And (30) Fluorescence intensity of 4-MU (fluorescence intensity after subtracting blank value, unit: RFU) produced when ganoderenic acid C is used at each concentration (0 μM, 25 μM, 50 μM, 100 μM, 200 μM) FIG. According to FIG. 3, it can be said that each inhibitor concentration from 25 μM to at least 200 μM has a positive correlation between the concentration of each inhibitor and the inhibitory ability (compound derived from Ganoderma (3 ) Etc., the NA inhibitory ability increases. In FIG. 3, when the concentration of the compound derived from Ganoderma is 0 μM, it means that the compound is not added. FIG. 4 divides each triterpenoid (each compound (1) to (33)) examined for the presence or absence of NA inhibitory ability in Test Example 1 into three groups based on the skeleton structure, and explains the skeleton structure of each group FIG. In FIG. 4, for example, Δ in “Δ ^7,8 ” means a carbon-carbon double bond (C═C), and the subscript “7, 8” is the position of carbon (C) where the double bond exists. Means a number. Further, “+ COOH”, “+ OH” and the like indicate the types of (main) functional groups. FIG. 5-1 (A) is a diagram showing the binding state of Tamiflu to neuraminidase (NA) using a ribbon model. In FIG. 5-1 (A), the host cell and the influenza virus are bound to a spherical space having a radius of 10 angstroms including a pocket in which the sialic acid site of the cell and the binding site of the neuraminidase (NA) of the virus reside. It is shown that Tamiflu has entered. FIG. 5-1 (B) is a diagram showing a binding state of Tamiflu to neuraminidase (NA) in (A) by a space filling model. FIG. 5-2 (A) is a diagram showing a state in which ganodermic acid TR is bound to neuraminidase (H1N1) by a ribbon model. It has been shown that neuraminidase (H1N1) has a pocket in which a binding site to which a substrate binds is present, and that ganoderic acid TR enters a spherical space having a radius of 10 angstrom including the pocket. FIG. 5-2 (B) is a diagram showing the binding state of ganodermic acid TR to neuraminidase (H1N1) shown in FIG. 5-2 (A) with a space-filling model. FIG. 6 shows NA inhibition with respect to neuraminidase (H1N1, N295S) with compounds (1), (3) to (33) as inhibitors of FIG. 1 and a final concentration of 200 μM (final concentration of 100 μM only for ganoderic acid DM). It is a figure which shows the result of having analyzed ability. FIG. 6 shows that any compound has an inhibitory ability against NA (H3N1, N295S). “N295S” indicates that the 295th asparagine of neuraminidase is substituted with serine. FIG. 7 shows each compound against neuraminidase (H5N1) when each compound (1), (3) to (33) is used as an inhibitor of FIG. 1 at a final concentration of 200 μM (final concentration of 100 μM only for ganoderic acid DM). It is a figure which shows the result of having analyzed NA inhibitory ability. FIG. 7 shows that each compound (1), (3) to (33) has an inhibitory ability against neuraminidase (H5N1). FIG. 8 shows a comparison with neuraminidase (H3N2, E119V) when each compound (1), (3) to (33) was used as an inhibitor of FIG. 1 at a final concentration of 200 μM (final concentration of 100 μM only for ganoderic acid DM). It is a figure which shows the result of having analyzed the NA inhibitory ability of each compound. FIG. 8 shows that each compound (1), (3) to (33) has an inhibitory ability against NA (H3N2, E119V). FIG. 9 shows each compound against neuraminidase (H7N9) when each compound (1), (3) to (33) is used as an inhibitor in FIG. 1 at a final concentration of 200 μM (final concentration of 100 μM only for ganoderic acid DM). It is a figure which shows the result of having analyzed NA inhibitory ability. FIG. 9 shows that each compound (1), (3) to (33) has an inhibitory ability against neuraminidase (H7N9). FIG. 10 (A) shows a chloroform extract of Ganoderma lingzhi (purified by immersing the ganoderma in chloroform and extracting it by shaking for 3 days at room temperature and removing the solvent from the oil phase) and a hot water extract. (The reishi was extracted with hot water in an autoclave (121 ° C, 20 minutes) and the extract was concentrated and dried with an evaporator desiccator), ethanol extract (Ethanol-Ex .; Reishi was 99% ethanol After 48 hours of shaking extraction with suction, the filtrate was concentrated and dried with an evaporator desiccator), 30% ethanol extract (ganoderma powder was shaken with 30% ethanol for 48 hours, suction filtered, filtrate 1 was dissolved in DMSO and dissolved in DMSO to inhibit the inhibition of FIG. 1 at each final concentration (0 μg / mL, 400 μg / mL, 800 μg / mL, 1600 μg / mL). Used as, against influenza virus-derived neuraminidase (H1N1, N295S), it is a graph showing the results of measuring the activity of neuraminidase. FIG. 10 (B) is a graph showing the inhibition rate against neuraminidase (H1N1, N295S) at each concentration of each extract calculated from FIG. 10 (A). FIG. 11 (A) shows how much the activity of neuraminidase (H3N2, E119V) is inhibited when neuraminidase (H3N2, E119V) is used instead of neuraminidase (H3N2, N295S) in FIG. 10 (A). It is a graph which shows the result investigated. FIG. 11 (B) is a graph showing the inhibition rate for the neuraminidase (H3N2, E119V) at each concentration of each extract calculated from the result of FIG. 11 (A). FIG. 12 (A) shows the result of examining how much the activity of neuraminidase (H5N1) is inhibited when neuraminidase (H5N1) is used instead of neuraminidase (H1N1, N295S) in FIG. 10 (A). It is a graph to show. FIG. 12 (B) is a graph showing the inhibition rate for neuraminidase (H5N1) at each concentration of each extract calculated from the results of FIG. 12 (A). FIG. 13A shows the results of examining how much the activity of neuraminidase (H7N9) is inhibited when neuraminidase (H7N9) is used instead of neuraminidase (H3N2, N295S) in FIG. 10A. It is a graph. FIG. 13 (B) is a graph showing the inhibition rate for neuraminidase (H7N9) at each concentration of each extract calculated from the results of FIG. 13 (A). 12A to 12D are conceptual diagrams illustrating the interaction between the active site pocket of neuraminidase (NA) and each site of the chemical structure of a known drug. In any case, it can be seen that the carboxyl group or guanidine group of a known drug interacts with the neuraminidase site facing the pocket. FIG. 15 shows a chloroform extract of Ganoderma lingzhi (the Ganoderma lingzhi soaked in chloroform and extracted by shaking for 3 days at room temperature and solvent removed from the oil phase) and a hot water extract (the spirit The turf was extracted with hot water by autoclaving (121 ° C., 20 minutes) and the extract was dried) and dissolved in DMSO and used at the final concentrations (0 μg / mL and 1600 μg / mL) as the inhibitors in FIG. It is a figure which shows the result of having measured NA activity using neuraminidase (H1N1) derived from influenza virus.

  The neuraminidase inhibitor, the anti-influenza agent containing the neuraminidase inhibitor, foods and drugs, methods for producing them, and the ganoderma tripenoid compound as an active ingredient of the neuraminidase inhibitor described above, its physical properties, effects and uses, etc. .

《Neuraminidase inhibitor (NA inhibitor)》
The neuraminidase inhibitors (NA inhibitors) according to the present invention include those containing an extraction component obtained by extraction from Ganoderma lingzhi with an organic solvent or an aqueous solvent. Further, since the extract component contains triterpenoids (compounds (1) to (33) (described later)) having an action of inhibiting neuraminidase, the neuraminidase inhibitor (NA inhibitor) according to the present invention includes Those containing any one or more of these compounds (1) to (33) are also included.

(Extraction)
Ganoderma lingzhi can be used as a raw material, but the collected natural product (ganoderma) can be processed by drying, crushing, etc. in terms of extraction efficiency. Those prepared in the above are preferred. When the extracted material is pulverized, pulverization by a roll-type pulverizer, a food processor, a ball mill pulverizer, a hammer-type pulverizer, or the like can be exemplified.

(Extraction solvent)
From the viewpoint of efficiently extracting components such as ganoderma triterpenoids having an action of inhibiting neuraminidase, the organic solvent for extraction is preferably a solvent containing 10% or more of an organic solvent as the solvent. A solvent containing 50% or more (organic solvent) is more preferable. In addition, it is desirable that the amount of the organic solvent contained in the organic solvent is usually 10% or more, more preferably about 30 to 99% with respect to the total amount of the solvent.

As the examples of the organic solvent, halogenated alkyl (e.g. chloroform), alcohols (e.g. methanol, ethanol, isopropyl alcohol or the like, a monovalent aliphatic alcohols of about C ₁ ~ ₁₀ or ethylene glycol or glycerin, polyhydric alcohol), hydrocarbons (e.g., such as: n-hexane, etc., C ₁ ~ about ₁₀ aliphatic hydrocarbons), ether (e.g. dimethyl ether, ethyl methyl ether, formula such as diethyl ether, "R ¹ -O- R ² (R ¹ and R ² are alkyl groups of about C ₁ to ₅ ) ”; aliphatic or aromatic cyclic ethers such as furan, dibenzofuran, and tetrahydrofuran; esters (eg, ethyl acetate, etc.) , An organic solvent of a monovalent to polyvalent carboxylic acid having a C1-5 alkyl ester), or a mixture of water and these organic solvents. I can make it.

  In particular, methanol, ethanol, and isopropyl alcohol are preferable as the alcohols, but ethanol or hydrous ethanol is particularly preferable from the viewpoint that even if it remains in the extract component, there is little adverse effect on the human body. As the water-containing ethanol, for example, water-containing ethanol having an ethanol concentration of 5 to 99.5% can be used.

  From the viewpoint of suitably obtaining an extract capable of inhibiting the activity of neuraminidase as an aqueous solvent for extraction, for example, water, buffer solution (for example, PBS, Tris-HCl), 30% -50% ethanol aqueous solution, etc. Can be mentioned.

  As a suitable extraction solvent for preparing an inhibitor of neuraminidase (H1N1, N295S), 99% to 50% ethanol, hot water {for example, from the viewpoint of obtaining a crude purified product with high NA inhibitory ability {for example, Heat and pressurize with steam and / or hot water over a pressure of 0.1 to 10 atmospheres, a temperature of 90 ° C. to 200 ° C., and a time of 1 minute to 100) minutes. The same applies below. }, 50% to 1% ethanol, chloroform and the like.

  As a suitable extraction solvent for preparing an inhibitor of neuraminidase (H3N2, E119V), 99% to 50% ethanol, hot water (the above heat The same as the water conditions.), Chloroform, 50% to 1% ethanol, and the like.

  As a suitable extraction solvent for preparing an inhibitor of neuraminidase (H5N1), chloroform and hot water (the same as the above hot water conditions) are preferred in order from the viewpoint of obtaining a crude purified product having a high NA inhibitory ability. 99% to 50% ethanol, 50% to 1% ethanol, and the like.

  As a suitable extraction solvent for preparing an inhibitor of neuraminidase (H7N9), 99% to 50% ethanol, 50% to 1% ethanol, Hot water (the same as the above hot water conditions), chloroform and the like can be mentioned.

(Extraction process)
Extraction processing is not particularly limited, but, for example, a natural product collected from Yamano or artificially cultivated ganoderma is dried or pulverized as necessary, and then immersed in an extraction solvent and allowed to stand to extract desired components. Known extraction methods such as an immersion method (cold immersion method, digestion method) and a Soxhlet extraction method by heating reflux using a Soxhlet extractor can be applied. The extracted liquid thus extracted may be concentrated or dried by evaporating the solvent by, for example, a spray drying method or the like, and can be prepared in the form of a solid or a powder thereof.

  The extraction temperature is not particularly limited as long as the active ingredient of the NA inhibitor can be extracted from Ganoderma, and the extraction solvent used is not frozen or solidified. In the case of extraction with an organic solvent, for example, 0 to 100 ° C. is there. In the case of an aqueous solvent, it is 30 degreeC-125 degreeC, for example, Preferably it is 80 degreeC-125 degreeC, Most preferably, it is around 121 degreeC (115 degreeC-125 degreeC).

  Further, the extraction time is not particularly limited as long as a desired component (said tripenoids) can be extracted, and is, for example, 1 hour to 3 days. The pressure of the extraction treatment is not particularly limited, and is, for example, normal pressure to 5 atmospheres.

  In addition, the extract of organic solvent or aqueous solvent is subjected to various chromatography such as silica gel column chromatography, reverse phase preparative liquid chromatography, thin layer chromatography (TLC), etc. The amount and purity may be increased.

(Extracted components)
The following tripenoids (compounds (1) to (33)) are included as components (reishi organic solvent extract) obtained by extraction from Ganoderma lingzhi with the above organic solvent or aqueous solvent. The presence of these triterpenoids in the extract can be confirmed by LCMS analysis (liquid chromatograph mass spectrometry) using, for example, LCMS-IT-TOF (Shimadzu Corporation "LCMS-2020").

  Although the present inventors have high or low inhibitory ability for these tripenoid compounds (1) to (33), at least various neuraminidases (H1N1, H1N1 (N295S), H3N2 (E119V), H5N1 types) , H7N9 type).

  Therefore, the neuraminidase inhibitor (NA inhibitor) according to the present invention includes the above aqueous solvent (hot water, less than 50% to 0.1% ethanol or more) or an organic solvent (99% to 50% ethanol, chloroform, etc.). And those containing any one or more selected from the group consisting of compounds (1) to (33).

Here, since the NA inhibitory ability of each extract or each compound differs depending on the type of neuraminidase (type such as H1N1), the crude extract or compound as an active ingredient suitable for each type of neuraminidase also differs ( (See Table 4 and FIGS. 6 to 13: A compound having a lower IC ₅₀ value and RFU value indicates higher NA inhibitory ability).

<< NA (H1N1 type)
Influenza (H1N1 type) is known as influenza that infects humans or pigs. Examples of NA inhibitors that inhibit the influenza-derived neuraminidase (H1N1 type) include those containing 1600 μg / mL or less of either a hot water extract or a chloroform extract.

  Moreover, as a suitable NA inhibitor or anti-influenza agent against this influenza-derived neuraminidase (H1N1 type), it is preferable as an active ingredient from the viewpoint of having NA inhibitory activity (see FIG. 3, Table 3 to Table 5). Ganoderic acid S (3), ganoderic acid AM1 (9), ganoderic acid B (10), ganoderic acid TN (13), ganoderic acid TQ (14), ganoderol B (19) ), Lucyl aldehyde A (21), lucyl aldehyde B (22), ganoderic acid C1 (23), ganoderic acid C6 (25), ganoderic acid H (26), ganoderic acid K (27), ganoderic Acid TR (28), Ganoderic acid C (30), Ganoderic acid D (31), Ganode NA inhibitors or anti-influenza agent containing one or two kinds selected from the group consisting of phosphate F (32) and Ganoderen acid H (33) can be mentioned.

  More preferably, NA inhibitors or anti-influenza agents containing one or two compounds selected from the group consisting of compounds (14) and (27) having an IC50 value of 100 μM or less are mentioned.

<< NA (H1N1 (N295S) type >>
Influenza (H1N1 (N295S) type) is known to be a Tamiflu-resistant influenza virus that infects humans or pigs. As an NA inhibitor that inhibits this neuraminidase derived from influenza (H1N1 (N295S) type), the ethanol concentration of the solution used for extracting the extract from Ganoderma in the order of preference is 99% to 50%. (Hereinafter also referred to simply as “99% or less to 50% or more ethanol extract”. The same applies to other concentrations), the concentration of ethanol used in the extract extraction from ganoderma is less than 50% to 30% Examples of the ethanol extract, hot water extract, and chloroform extract include one or more of these, preferably 400 μg / mL or more to 1600 μg / mL or less, more preferably 800 μg / mL or more. Examples include those containing ˜1600 μg / mL or less (see FIG. 10).

  As a suitable NA inhibitor or anti-influenza agent for neuraminidase derived from this influenza virus (H1N1 (N295S)), from the viewpoint of the high inhibition rate (200%) (described later) of each compound, As an active ingredient, one or more selected from the group consisting of Ganodermic Acid S (3), Ganodermic Acid TQ (14), and Ganodermic Acid TR (28) having an inhibition rate of 30% or more NA inhibitor or anti-influenza agent, and more preferably one or two selected from the group consisting of ganoderic acid TQ (14) and ganoderic acid TR (28), wherein the inhibition rate is 40% or more Include species-containing NA inhibitors or anti-influenza agents (see Table 10).

<< NA (H3N2 (E119V) type >>
The influenza virus (H3N2 (E119V) type) is known as a Tamiflu-resistant influenza virus that mainly infects humans and pigs.

  As an NA inhibitor that inhibits this neuraminidase derived from influenza (H3N2 (E119V) type), the ethanol concentration of the solution used for extracting the extract from Ganoderma in order of preference is 99% to 50%. , Hot water extract, ethanol extract of liquid used for extraction from Ganoderma extract, ethanol extract of less than 50% to 30% or more, chloroform extract, one or more, preferably 400 μg Examples include those containing not less than / mL and not more than 1600 μg / mL, more preferably not less than 800 μg / mL and not more than 1600 μg / mL (see FIG. 11).

  The NA inhibitor or anti-influenza agent for the neuraminidase (H3N2 (E119V) type) is preferably an active ingredient having an inhibition rate of 200 μM from the viewpoint of the high inhibition rate (%) of each compound. 30% or more of Ganodermic Acid S (3), Ganodermic Acid A (8), Ganoderic Acid AM1 (9), Ganodermic Acid B (10), Ganodermic Acid TN (13), Ganodermic Acid TQ ( 14), ganoderic manon diol (17), lucyl aldehyde B (22), ganoderic acid C1 (23), ganoderic acid C2 (24), ganoderic acid K (27), ganoderic acid TR (28), Ganoderic acid C (30), Ganoderic acid D (31), and Ganoderin NA inhibitors or anti-influenza agent containing one or more compounds selected from the group consisting of H (33) can be mentioned. More preferably, each of the inhibition rates is 40% or more, ganodermic acid S (3), ganodermic acid TN (13), ganodermic acid TQ (14), lucylaldehyde B (22), NA inhibitor or anti-influenza agent containing 1 type, or 2 or more types selected from the group which consists of ganoderic acid C2 (24) and ganoderic acid TR (28) is mentioned. Particularly preferably, the NA inhibitor comprising one or more selected from the group consisting of ganoderic acid TQ (14) and ganoderic acid TR (28), each having an inhibition rate of 50% or more, or Anti-influenza agents are mentioned (see Table 10).

《Neuraminidase (H5N1 type)》
Influenza virus (H5N1) causes influenza in birds, humans and many other animals.

  As an NA inhibitor that inhibits this neuraminidase derived from influenza (H5N1 type), an ethanol extract having an ethanol concentration of 99% or less to 50% or more in an extract extraction from Ganoderma in order of preference, Examples include ethanol extract, hot water extract, chloroform extract whose ethanol concentration of the liquid used for extraction from Ganoderma lucidum is less than 50% to 30% or more, and any one or more of these. Is preferably 400 μg / mL to 1600 μg / mL, more preferably 800 μg / mL to 1600 μg / mL (see FIG. 12).

  The NA inhibitor or anti-influenza agent against neuraminidase (H5N1 type) derived from this influenza virus is preferably 30% or more from the viewpoint of the high inhibition rate (200%) of each compound. Ganoderic Acid S (3), Ganoderic Acid F (4), Ganoderic Acid LM2 (6), Ganoderic Acid DM (7), Ganoderic Acid A (8), Ganoderic Acid AM1 (9) , Ganodermic Acid Y (12), Ganodermic Acid TN (13), Ganoderic Acid TQ (14), Ganoside Acid A (15), Ganodermanone Triol (16), Ganodermanone Diol (17), Ganoderol A (18), Ganoderol B (19), Ganodeliol F (20) Lucidaldehyde A (21), Lucidaldehyde B (22), Ganoderic Acid C1 (23), Ganoderic Acid C2 (24), Ganoderic Acid H (26), Ganoderic Acid K (27), Ganoderic Acid TR (28), one or two selected from the group consisting of ganoderenic acid A (29), ganodellenic acid C (30), ganodellenic acid D (31), ganodellenic acid F (32), and ganodellenic acid H (33) NA inhibitors and anti-influenza agents containing the above can be mentioned. More preferably, the inhibition rate is 40% or more for all the compounds (3), (4), (6), (7), (9), (12), (13) to (19), ( 21)-(24), (26)-(28), NA inhibitor containing one or more selected from the group consisting of (31)-(33) includes anti-influenza agents. More preferably, the above compounds (3), (6), (7), (9), (12) to (19), (21) to (24), (26) each having an inhibition rate of 50% or more. )-(28), (31)-(33) NA inhibitors and anti-influenza agents containing one or more selected from the group consisting of (33) as active ingredients may be mentioned (see Table 10).

  Similarly, NA inhibitors and anti-influenza agents containing one or more compounds included in each group having an inhibition rate of 60% or more, 70% or more, or 80% or more as active ingredients are preferred, but the inhibition rate is particularly high. 90% or more of a compound having a very potent NA inhibitory ability (one selected from the group consisting of ganoderic acid TN (13), ganoderic acid TQ (14), and ganoderic acid TR (28)) It can be seen that NA inhibitors and anti-influenza agents containing two or more active ingredients are effective and desirable against influenza viruses (H5N1 type) to the extent comparable to known anti-influenza agents (Tamiflu) (Table 10). reference).

(4) Inhibitory ability against neuraminidase (H7N9 type) Influenza virus (H7N9 type) is a virus that has been reported to mainly infect birds and infect humans in China. Examples of NA inhibitors that inhibit this influenza-derived neuraminidase (H7N9 type) include ethanol extracts having an ethanol concentration of 99% or less to 50% or more in the order of extract extraction from reishi, Examples include ethanol extract, hot water extract, and chloroform extract with an ethanol concentration of less than 50% to 30% or more for extraction of turf extract. Preferably, it includes those containing 400 μg / mL to 1600 μg / mL, more preferably 800 μg / mL to 1600 μg / mL (see FIG. 13).

  The NA inhibitor or anti-influenza agent against the neuraminidase (H7N9 type) is preferably 20% or more from the viewpoint of the high inhibition rate (%) of 200 μM of each compound. Ganoderic Acid S (3), Ganoderic Acid DM (7), Ganoderic Acid TN (13), Ganoderic Acid TQ (14), Ganodermanone Diol (17), Ganoderol B (19), Ganoderol F (20), Lucylaldehyde A (21), Lucylaldehyde B (22), Ganodermic Acid C1 (23), Ganoderic Acid C2 (24), Ganodermic Acid C6 (25), Ganodermic Acid H (26), Ganoderic Acid K (27), Ganoderic Acid TR (28), Ganoderen (29), NA containing at least one selected from the group consisting of ganoderenic acid C (30), ganoderenic acid D (31), ganoderenic acid F (32) and ganoderenic acid H (33) Inhibitors and anti-influenza agents are mentioned. More preferably, as an active ingredient in which the inhibition rate (%) exceeds 30%, ganodermic acid S (3), lucylaldehyde B (22), ganoderene acid D (31), ganoderene acid F (32) and NA inhibitors and anti-influenza agents containing one or more selected from the group consisting of ganoderenic acid (33) can be mentioned.

  These triterpenoid compounds are commercially available from various manufacturers (Quality Phytochemicals LLC, American Custom Chemicals Corporation, AK scientific, BOC Sciences, Cheminstock, Chromadex, ChemFaces, BioBioPha, etc.) as compounds derived from Ganoderma lucidum. You may purchase and use. Further, these triterpenoid compounds are not limited to Ganoderma lucidum, and may be synthesized.

(1) Ganoderic acid Z (Ganoderic acid Z),

(2) Ganoderic acid N (Ganoderic acid N),

(3) Ganoderic acid S

(4) Ganoderic acid F

(5) Ganoderic acid SZ (Ganoderic acid SZ),

(6) Ganoderic acid LM2

(7) Ganoderic acid DM

(8) Ganoderic acid A

(9) Ganoderic acid AM1

(10) Ganoderic acid B

(11) Ganoderic acid D

(12) Ganoderic acid Y

(13) Ganoderic acid TN

(14) Ganoderic acid TQ

(15) Ganolucidic acid A

(16) Ganodermanontriol

(17) Ganodermanondiol

(18) Ganoderol A

(19) Ganoderol B

(20) Ganoderiol F

(21) Lucialdehyde A

(22) Lucialdehyde B

(23) Ganoderic acid C1

(24) Ganoderic acid C2

(25) Ganoderic acid C6

(26) Ganoderic acid H

(27) Ganoderic acid K

(28) Ganoderic acid TR

(29) Ganoderenic acid A

(30) Ganoderenic acid C

(31) Ganoderenic acid D

(32) Ganoderenic acid F

(33) Ganoderenic acid H

(Salt, ester)
Compounds (1) to (33) (extracted from Ganoderma or synthesized) may be esterified by reacting with alcohol, or may be reacted with a base to form a salt. Examples of esters of these compounds (1) to (33) include linear or branched alkyl esters having 1 to 11 carbon atoms such as methyl esters. Examples of salts of these compounds (1) to (33) include alkali metal salts such as sodium and potassium, ammonium salts and the like.

  The concentration of the active ingredient contained in the neuraminidase inhibitor according to the present invention is such that NA inhibition occurs when the neuraminidase inhibitor reaches the mucosal epithelial cells of the throat (upper respiratory tract) where the influenza virus is infected (affected site). The concentration of the active ingredient in the agent is not particularly limited as long as it is a concentration capable of inhibiting various neuraminidases. For example, the concentration of the active ingredient in the epithelial cells such as the throat is 0.1 to 200 μM. In addition, in 100% by weight of the neuraminidase inhibitor, it can be appropriately set within the range of 0.1 to 100% by weight according to the dosage form, usage, and dose.

For example, various extracts derived from Ganoderma lingzhi (ethanol extract with 99% ethanol concentration, extract from hot water, hot water extract, 30% ethanol extract, chloroform extract) In the case of NA inhibitors containing) as an active ingredient, NA inhibitors and anti-influenza agents prepared so as to have a concentration of 1 to 1600 μg / mL in mucosal cells of the throat where influenza virus can be infected shall be exemplified. Can do. Here, for example, when the active ingredient concentration is diluted with saliva or the like, the concentration after dilution may be adjusted to 1 to 1600 μg / mL. Specifically, when the concentration of the active ingredient is halved when it reaches the mucous membrane with saliva or the like, for example, when it is prepared to have the concentration in the epithelial cells, the concentration is 2 to 3200 μg / mL. Examples thereof include NA inhibitors and anti-influenza agents containing any one or more of the above extracts.

(Evaluation of neuraminidase inhibitors)
Conventionally, there are various neuraminidase inhibitors of Tamiflu (registered trademark) (oseltamivir), Relenza (registered trademark, also known as: zanamivir), Rapiacta (peramivir), and laninamivir. Tamiflu has the following chemical structure (A1). It has the following chemical structure (A2), zanamivir has the following chemical structure (A3), and laninamivir has the following chemical structure (A4).

Tamiflu (registered trademark) binds to the active site of neuraminidase (NA) to form a new hydrophobic pocket, and when the neuraminidase (NA) tries to bind to a substrate, Inhibits neuraminidase activity by binding with affinity. On the other hand, Relenza, veramivir or laninamivir has not only a carboxyl group and glycerol in its chemical structure but also a guanidine group [H ₂ NC (═NH) —NH—]. Tamiflu and the like have a guanidine group, but this guanidine group is electrically bound to neuraminidase (NA) possessed by influenza virus and inhibits enzyme activity without causing a structural change of NA. In this electrical coupling, the Tamiflu guanidine group is in the active site at the bottom of the NA pocket and is electrically coupled to a negatively charged amino acid residue (FIG. 14).

Therefore, the inhibitory performance of neuraminidase is (i) whether the inhibitor molecule has a chemical structure that can interact with each site of the active center (such as the energy value of the docking score value), or (ii) the IC ₅₀ value. Judgment can be made.

(Structure of triterpenoid)
Many of the triterpenoids (compounds (1) to (33)) having NA inhibitory ability as an active ingredient of the NA inhibitor according to the present invention have a carboxyl group (described later), and this carboxyl group is publicly known. There is a high possibility that it functions as a functional group corresponding to the carboxyl group of the NA inhibitor. In addition, the hydrophobic chemical structure portion of the steroid skeleton of the compounds (1) to (33) may function as a chemical structure portion corresponding to the hydrophobic group of Tamiflu.

  In addition, compounds (1) to (33) are molecules having a steroid skeleton as a basic skeleton, but have a molecular size similar to that of known NA inhibitors, and structure-activity relationship analysis (SAR). ) As a result, for ganoderic acid TQ (14) and ganoderic acid TR (28), neuraminidase docking scores (Kcal / mol) of various influenza viruses (H1N1, H3N2, H5N1, H7H9, H1N1 (N295S)) ) Has a negative value as in the case of known NA inhibitors such as Tamiflu, at least ganodermic acid TQ (14) and ganodermic acid TR (28) are active centers of any type of neuraminidase. It is possible to enter the pocket without colliding with the pocket (see Table 12).

  Ganodermic Acid TQ (14) and Ganodermic Acid TR (28) have NA inhibitory ability that can be used as a substitute for known NA inhibitors (such as Tamiflu). Compared to Tamiflu, which is a known NA inhibitor, the IC50 value, which is an index of NA inhibition ability, is about 120 times that of Tamiflu for neuraminidase (H1N1), neuraminidase (H1N1, N295S) About 20 times, 14.5 times for neuraminidase (H3N2, E119V) and 420 times or more for neuraminidase (H7N9), which is slightly inferior to Tamiflu. Surprisingly, for neuraminidase (H5N1), the IC50 value of ganoderic acid T-Q is 0.51 times that of Tamiflu, and for neuraminidase (H5N1), ganoderic acid T-Q. Q has a NA inhibitory capacity that exceeds Tamiflu.

  On the other hand, the IC50 value is about 177 times that of Tamiflu for neuraminidase (H1N1) and Tamiflu for neuraminidase (H1N1, N295S) when comparing Ganodermic Acid TR with the known NA inhibitor Tamiflu. About 12 times as much as Tamiflu for neuraminidase (H3N2, E119V), about 6 times that for Tamiflu for neuraminidase (H5N1), and 425 for Tamiflu for neuraminidase (H7N9). More than double, slightly inferior to Tamiflu.

Therefore, ganodermic acid TQ (14) and ganodermic acid T-R (28) have NA inhibitory ability to the extent that they can be used as alternatives such as Tamiflu.
Moreover, as shown to FIGS. 6-9, other triterpenoids (compound (1)-(13), (15)-(27) other than ganoderic acid TQ (14) and ganoderic acid TR (28). ) And (29) to (33)) are triterpenoids that can be used as substitutes for known NA inhibitors (such as Tamiflu), although NA inhibition ability is high or low.

  Therefore, the NA inhibitor according to the present invention containing such a triterpenoid as an active ingredient has a neuraminidase (NA) inhibitory activity, like Tamiflu, and is very useful as an anti-influenza agent.

  As described above, since the NA inhibitor according to the present invention is useful as an anti-influenza agent, it contributes to diversification of known anti-influenza agents. This diversification of anti-influenza agents is very advantageous when influenza viruses resistant to known anti-influenza agents spread. In fact, influenza viruses that have drug resistance against known anti-influenza drugs (NA inhibitors) such as osemitamiflu and veramivir have been reported (see the table below, NIID; “Anti-influenza drug resistant strain surveillance, July 11, 2014 Quoted from ").

  If a resistant influenza virus appears in all or many of the known anti-influenza drugs in Table 1 below, the known anti-influenza drugs cannot be qualitatively and / or quantitatively dealt with. Moreover, it can also contribute to the expansion of the choice of anti-influenza agents that can be used for humans of other idiosyncrasies such as constitutions in which side effects are remarkably caused by known anti-influenza agents and other animals. In that sense, the NA inhibitor according to the present invention is very significant in that it contributes to diversification of options for influenza drugs.

(Inhibition rate)
The inhibition rate of the anti-influenza agent (NA inhibitor) according to the present invention against neuraminidase can be measured by a conventionally known method. The inhibition rate can be expressed by how much the activity of the original neuraminidase is reduced by adding an inhibitor to the reaction system for measuring the activity of neuraminidase.

  As the neuraminidase activity measurement method itself, for example, the measurement method performed by removing the inhibitor in FIG. 1 can be exemplified as the NA activity measurement method. According to this exemplary method, neuraminidase hydrolyzes 4-MUNANA (methylumbelliferic-α-DN-acetylneuraminic acid) as a substrate to produce 4-methylumbelliferone (4-MU). . Here, since the produced 4-methylumbelliferone (4-MU) is a substance that is excited at a wavelength of 365 nm and emits fluorescence at a wavelength of 450 nm, neuraminidase is obtained by optically quantifying the amount of 4-MU produced. The activity of (NA) can be quantified. The amount of activity at this time can be expressed as the relative fluorescence intensity (RFU) of the total amount of 4-MU produced. Here, other compounds included in the reaction system of FIG. 1 other than 4-MU may emit light having a wavelength of 450 nm slightly, and this may be included as noise, so the relative fluorescence intensity ( It is desirable to subtract the amount of background noise fluorescence from (RFU).

  Here, when an NA inhibitor is added to the reaction system for measuring the neuraminidase (NA) activity, the amount of 4-MU produced decreases, so the relative fluorescence intensity (RFU) derived from 4-MU produced also decreases. Therefore, by determining the amount of neuraminidase activity reduced by the addition of NA inhibitor relative to the amount of original neuraminidase activity in which no NA inhibitor is present, how much the activity of the original neuraminidase is due to the addition of inhibitor. It can be expressed as a percentage (%) of decrease, and this becomes an inhibition rate (IR (%)). In the following formula [I], each RFU is expressed as a value obtained by subtracting the amount of fluorescence of background noise.

For the above-described neuraminidase (NA) activity measurement, for example, a commercially available kit for measuring neuraminidase activity (eg, “NA-Fluor Influenza Neurminidase Assay Kit” (Life Technologies)) can be used.

(IC ₅₀ )
The concentration of inhibitor (IC ₅₀ ) required to inhibit neuraminidase activity by 50% (= 50% inhibition rate) is determined by the chromophore produced as the final product when no NA inhibitor is present (above) In the example, when the amount of 4-MU) is 100%, the concentration of the NA inhibitor when the amount of the chromophore produced is 50% when the NA inhibitor is added.

IC ₅₀ can be determined by the following method. For example, in the activity measurement of neuraminidase (NA) in FIG. 1 described above, compounds (1) to (33) are used as inhibitors at different final concentrations (eg, 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15 125 μM), and the inhibition rate against neuraminidase is determined according to the concentration of NA inhibitor. Next, the value of each inhibition rate determined is plotted as a graph of NA inhibitor concentration (horizontal axis) -inhibition rate (%) (vertical axis), and an approximate line of the plot is created. Finally, by obtaining the concentration of NA inhibitor with an inhibition rate of 50% from the approximate line, the concentration can be specified as IC ₅₀ .

(Chemical structure of triterpenoid contributing to NA inhibitory activity)
(Skeletal structure A)
Preferable active ingredients of the NA inhibitor according to the present invention are triterpenoids having the following skeletal structure A or skeletal structure B, but other triterpenoids have skeletal structure C. In the following description, for example, “Δ” in “Δ ^7,8 ” means a double bond, and the subscript “7, 8” on the right shoulder indicates the position of carbon (C).

(Skeletal structure B)

(Skeletal structure C)

(Skeletal structure A)
The skeletal structure A has two double bonds (Δ ^7,8 , Δ ^9,11 ) in the tetracyclic ring and has a branched chain having a carboxyl group. Triterpenoids having this skeletal structure A include (3) ganoderic acid S (5), (5) ganoderic acid SZ, (12) ganoderic acid Y, (13) ) Ganodermic acid TN (Ganoderic acid TN), (14) Ganoderic acid TQ (Ganoderic acid TQ), (28) Ganoderic acid TR (Ganoderic acid TR).

(Skeletal structure B)
The skeleton structure B has one double bond (Δ ^8,9 ) in the tetracyclic ring and has a branched chain having a carboxyl group. Triterpenoids having this skeletal structure B include (8) ganoderic acid A, (29) ganoderic acid A, (24) ganoderic acid C2, (9) ganoderic acid AM1, (27) ganoderic acid K, ( 33) Ganoderic acid H, (26) Ganoderic acid H, (10) Ganoderic acid B, (32) Ganoderic acid F, (30) Ganoderic acid C, (31) Ganoderic acid D, (25) Ganoderic acid C6 (23) Ganodermic acid C1, (2) Ganodermic acid N, (7) Ganodermic acid DM, (15) Ganodic acid A, (11) Ganodermic acid D, (1) Ganodermic acid Z, (6 ) Ganodermic Acid LM2, (4) Ganodermic Acid F, (22) Lucidaldehyde B And the like.

(Skeletal structure C)
The skeleton structure C has two double bonds (Δ ^7,8 , Δ ^9,11 ) in the tetracyclic ring and does not have a carboxyl group in the branched chain. Triterpenoids having this skeletal structure B include (16) Ganodermanontriol, (17) Ganodermanondiol, (18) Ganoderol A, and (19) Ganoderol B. ), (20) Ganoderiol F (Ganoderiol F), and (21) Lucialdehyde A.

  Of the triterpenoids (1) to (33), many of the triterpenoids having the ability to inhibit neuraminidase activity have a skeletal structure A or a skeletal structure B, and these skeletal structures A and B have a carboxyl group. . Here, a known NA inhibitor such as Tamiflu has a carboxyl group (COOH), and as shown in FIG. 14, the known NA inhibitor enters the pocket of the active center of NA. It is thought to interact with the pocket of the active center. Therefore, it is considered that the carboxyl group in the chemical structure of the various triterpenoids derived from Ganoderma (that is, the compounds (1) to (33)) performs the same function as the carboxyl group of a known NA inhibitor. It is done. When searching for a compound that can be an active ingredient of an NA inhibitor from triterpenoids, screening for selecting one having a carboxyl group may be performed.

(Docking simulation)
By performing a docking simulation of triterpenoids against neuraminidase, the triterpenoids enter the neuraminidase active center pocket and become the neuraminidase's original substrate (eg, on the surface of the human throat mucosa and on the host cell surface where influenza is infected) The inhibition mode, such as whether or not to inhibit neuraminidase by competing with the sialic acid sugar chain), and the ease of binding of triterpenoids to neuraminidase can be investigated. At this time, if the docking score indicating the binding energy between triterpenoids and neuraminidase is negative, it means that the triterpenoids have entered the pocket of the active center of neuraminidase. It means that the class does not enter the pocket of the active center of the neuraminidase, and the magnitude of the absolute value means a binding strength when it is negative, and a magnitude of repulsion when it is positive.

  Ganodermic acid TQ (14) and Ganodermic acid TR (28) were combined into five neuraminidases derived from different influenza virus subtypes (H1N1 / H1N1 (N295S) / H3N2 (E119V) / H5N1, H7N9). Since the docking score obtained when docked with a three-dimensional structure is a negative value similar to that of known NA inhibitors (Tamiflu, etc.) (see Table 13), as with Tamiflu, etc. It competes with the substrate to enter the active site pocket of neuraminidase and inhibit neuraminidase (NA). Therefore, the neuraminidase according to the present invention can be used as an anti-influenza agent that competitively inhibits NA similarly to known NA inhibitors such as Tamiflu.

  The docking score for various neuraminidases of triterpenoids (H1N1 / H1N1 (N295S) / H3N2 (E119V) / H5N1, H7N9) indicates how easily these triterpenoids bind to various neuraminidases. When determining whether a triterpenoid is useful as an NA inhibitor, a docking simulation is performed on various neuraminidases using the chemical structure of the triterpenoid, and a docking score close to that of a known inhibitor is obtained based on the docking score. It is good also as selecting only the triterpenoid which has as an active ingredient of NA inhibitor.

(Cell delivery of NA inhibitors)
Including the types of influenza viruses that infect humans, pigs, birds, etc. As of July 2014, 16 types of surface antigens of hemagglutinin of influenza virus (H1-H16) and 9 types of neuraminidase (N1-N9) ) Has been confirmed. Influenza virus types (eg, H7N9, etc.) are classified according to the combination of hemagglutinin and neuraminidase, and influenza virus antigens differ depending on the type.

  In addition, influenza virus binds to the viral receptors of epithelial cells, especially in the trachea, bronchi, and bronchioles during human infection, but galactose binds to the α2-6 position of epithelial cells in human influenza viruses. The avian influenza virus recognizes and binds to sialic acid bound to galactose at the α2-3 position of the epithelial cell as a viral receptor. Therefore, if NA inhibitors can be delivered specifically targeting specific antigens of influenza viruses and epithelial cells, NA inhibition will be more efficiently suppressed, and thus the progression of influenza virus symptoms will be prevented, and the spread of influenza infection will be prevented. Can be connected.

  Specifically, at least one of the above compounds (1) to (33), preferably any one of the first to third compounds among the compounds selected in descending order of the inhibitory ability against various neuraminidases, for example. An antibody (A) against the above, the virus receptor or the antibody (B) as an antigen, and a complex of each compound and the antibody (hereinafter referred to as “antibody drug complex”), It is conceivable to use this as a DDS preparation containing a NA inhibitor according to the present invention (a drug configured to deliver a predetermined drug only to specific cells using a drug delivery system). Two or more of the above compounds may be used simultaneously by mixing DDS lumber.

  Here, the antibody (A) has a function of specifically retaining each of the compounds (1) to (33), and the antibody (B) is a specific cell (epithelial cell or virus up to trachea, bronchi, bronchiole) A function of specifically binding to a specific protein expressed only in the virus (such as the HA part of a viral receptor or a viral coat protein), and a complex of antibodies (A) and (B) (the above antibody Since the drug complex)) has both these functions, each compound (1) to (33) can be specifically delivered to the influenza virus or a host cell infected with the influenza virus.

  In this case, between each of the compounds (1) to (33) and the antibody, a linker that can be selectively cleaved (for example, a linker such as Seattle Genetics, which contains a protein sequence that can be cleaved selectively with a specific protease). Etc.). Since this antibody-drug conjugate is stable in blood, it selectively acts on epithelial cells up to the trachea, bronchi, and bronchioles infected with influenza virus, and after being taken up into vascular epithelial cells, Each of the compounds (1) to (33) can be released by cleaving the linker with a proteolytic enzyme. As a result, each compound (1) to (33) is delivered to a specific cell, and neuraminidase (NA) is specifically inhibited in the cell (vascular epithelial cell or the like).

<< Use of NA inhibitor >>
The NA inhibitor according to the present invention can be used as food (supplements, food additives, etc.), cosmetics, pharmaceuticals (anti-influenza agents), research reagents and the like. These foods and the like have an excellent neuraminidase (NA) inhibitory activity ability of any one or more of the organic solvent extract, aqueous solvent extract, and tripenoid compounds (1) to (33), (human and For prevention and treatment of influenza in animals).

[Food]
As described above, the food according to the present invention comprises the above organic solvent extract (eg, 99% to 50% ethanol extract of chloroform, extract of chloroform), aqueous solvent extract (ex: extract from ganoderma). The above compound (1) as an active ingredient of an NA inhibitor contained in a high concentration in an ethanol extract or hot water extract having an ethanol concentration of 50% to 1% in the liquid used for extraction, or in the organic solvent extract ) To (33) any one or two or more of them.

  Furthermore, since the NA inhibitory ability of each compound (1) to (33) is different from various neuraminidases (H1N1, H1N1 (N295S) type, H3N2 (E119V) type, H5N1 type and H7N9 type), the type of neuraminidase The compound having a high NA inhibitory capacity is selected according to (for example, among the aforementioned compound groups formed by sorting various neuraminidases on the basis of NA inhibition rate (%) at 200 μM or IC50 (μM)). One or two or more compounds may be selected from a group having a high NA inhibitory ability) and a food containing the compound may be produced. Moreover, you may manufacture the foodstuff containing any 1 type, or 2 or more types of compounds selected from the group with 2nd and 3rd highest NA inhibitory ability.

  The blood concentration in a human or the like who ingested the food, and the amount of any one or more of the compounds (1) to (33) is 0.01 to 100 μM. It is preferable to make it contain. For example, an example in which one or more of compounds (1) to (33) or the above extract is ingested in an aspect of 0.1 mg / kg / day to 500 mg / kg / day for an adult (body weight 60 kg). Can be mentioned. In addition, for example, the amount of the active ingredient (each compound) contained in a single intake of food (drink, jelly, supplement, etc.) is equal to or greater than the amount equivalent to Tamiflu Tablets (75 mg) as described below for the anti-influenza agent. You may adjust so that it may become the range of minimum poisoning amount, or less than Tamiflu tablet (75 mg) equivalent amount-0.1 mg or more.

  As the dosage form of the food according to the present invention, any one or more of the organic solvent extract, the aqueous solvent extract, or the compounds (1) to (33) were directly prepared as food. Illustrate any food form normally used, such as foods, foods added to other foods, or jelly (eg sticks), drinks, capsules, tablets, etc. in the form of food or health food Can do.

  Further, when the NA inhibitor according to the present invention (active ingredient thereof) is blended in food and taken by humans or other animals, excipients, extenders, binders, thickeners, emulsifiers are contained in the food. Colorants, fragrances, food additives, seasonings and the like can be appropriately added together with the NA inhibitor. In that case, the NA inhibitor according to the present invention can be mixed with food and these optional components, and the dosage form can be formed into a powder, granule, tablet or the like according to the application. In particular, since triterpenoids are very bitter, it is desirable to make them easy to ingest, including sweetening ingredients. Furthermore, foodstuffs can be prepared by mixing NA inhibitors into food ingredients and commercialized as functional foods.

(drink)
When producing the food according to the present invention as a drink, since the drink itself is ingested and swallowed in a short time and the active ingredient contained in the drink is not easily diluted with saliva etc., the above various extracts (99% ethanol extract, (Hot water extract, chloroform extract, 30% ethanol extract) is adjusted to a concentration range in which the inhibitory effect is reliably obtained, for example, 400 to 250,000 μg / 30 cc bottle (13.3 μg to 10,000 μg / mL). Can be manufactured.

(Jelly (stick shape))
When producing the food according to the present invention as a jelly, because the jelly is chewed in the mouth and thinned by saliva before reaching the mucous membrane of the throat such as the esophagus, considering the amount diluted with the amount of saliva, You may determine the density | concentration of the active ingredient in a foodstuff so that it may become a density | concentration which can inhibit various neuraminidases effectively as NA inhibitor when reaching the mucous membrane of the throat.

(supplement)
The NA inhibitor according to the present invention can be used as a supplement. In the case of supplements, for example, the above-mentioned organic solvent extract, aqueous solvent extract, or any one or more of compounds (1) to (33), or preferably neuraminidase as described above One or two or more compounds from a group of suitable compounds (eg, a group of compounds having the highest NA inhibitory ability) that differ depending on the type, and are granulated in a conventional manner using known excipients. It can be used in the form of powder, gel, capsule, paste, or tablet. Among these, when making into a paste, the above compounds (1) to (33) and the like may be encapsulated in the cyclodextrin pores as an active ingredient of the NA inhibitor.

  The total content of any one or more of Ganoderma tripenoids (compounds (1) to (33)) in the supplement is not particularly limited, but about 200 mg of ganoderic acid is contained in 100 g of Ganoderma In general, since 25 to 30 g of Ganoderma is used in a single intake, for example, one supplement or more of compounds (1) to (33) in one supplement (1 g), or It is preferable that any one or more of compound (3) and the like is contained in a total amount of about 5 mg to 60 mg.

(Prey for domestic animals and poultry)
Infectious influenza viruses differ among host animals such as humans, birds, and pigs, so the active ingredients of NA inhibitors and anti-influenza drugs are selected according to the type of host animal (among compounds (1) to (33)). It is good also as preparing the foodstuff containing this. For example, as described later, when preparing poultry food having an effect of preventing infection of birds such as influenza (H7N9 type and H5N1 type) mainly infecting birds, the NA (H7N9 type) described above is prepared. It is desirable to select one or more compounds from the group of compounds suitable for inhibiting (H5N1) and to prepare poultry feed containing them. As for pig food, the preferred triterpenoids group (inhibition rate (inhibition rate) described above as an effective component for inhibiting the above-described influenza (H1N1, H1N1 (N295S), H3N2 (E119V), N5N1)) that can infect pigs. %) Or a group of the above-mentioned compounds based on IC50) to produce a livestock feed according to the present invention containing one or more compounds selected from known feeds (pigs, etc.) May be.

  Suitable concentrations of the active ingredients contained in poultry and livestock feed are as described above. For example, in the state where the feed reaches the throat mucosal epithelial cells of poultry and livestock, it is contained in the feed. In the case of an aqueous solvent extract or organic solvent extract, the concentration of the active ingredient is adjusted to 400 μg / mL to 1600 μg / mL, and in the case of a compound, the concentration is adjusted to 1 to 200 μM. . In addition, for example, the amount of the active ingredient (each compound) as a single intake amount is in a range from a Tamiflu tablet (75 mg) equivalent amount to a minimum poisoning amount or a Tamiflu tablet ( 75 mg) It may be adjusted to be less than the equivalent amount to 0.1 mg or more. In the latter case, an infection prevention effect can be expected.

  For avian influenza neuraminidase (H5N1 type, H7N9 type), foods (food) containing NA inhibitors and anti-influenza agents containing the above-mentioned compounds suitable for inhibiting these neuraminidases are very effective. If the poultry is raised by feeding the poultry, for example, it is possible to prevent the influenza (H7N9 type or H5N1 type) from infecting humans from birds. Therefore, foods (food) containing the NA inhibitor and the like can be used for poultry (chicken, ducks, emu, geese, Indian peafowl, mute swan, ostrich, turkey, guinea fowl, pheasant pheasant, goldfish, rare, quail, etc.) and pets (pets). ) Bird (such as parakeets (budgerigars, etc.), ducks, parrots, bunchos, nine-birds, canary, juvenile pine, long-tailed dragonfly, chabo, etc.).

(Food additive)
Since compounds (1) to (33) exhibit a strong bitter taste, they can be used as soft drinks, alcoholic beverages and the like that allow bitterness as food additives (bitterings). Compounds (1) to (33) can be handled basically in the same manner as known bittering agents such as sesquiterpenes.

  Conversely, it is also possible to suppress the bitterness of the compounds (1) to (33). In this case, after masking the bitterness with a bitterness masking agent (Kao “Benecoat BMI-40” etc.) Expected influenza prevention based on NA inhibition, pneumonia caused by influenza infection, acute encephalopathy and Reye syndrome, cardiac complications, acute myositis, otitis media, conjunctivitis, sinusitis, myocarditis) It may be used as a food additive.

  When the compounds (1) to (33) are used as the food additive, the following other food additives permitted in terms of safety may be used in combination to make them water-soluble or oil-soluble. Other food additives include monohydric alcohols (such as ethanol), polyhydric alcohols (eg, ethylene glycol, glycerin, etc.), animal and vegetable oils (eg, glycerin mono, di, tri-fatty acid esters). A known solvent such as a fatty acid ester of a monohydric alcohol), a known surfactant or a known emulsifier {eg sugar ester, sorbitan fatty acid ester (sorbitan ester), propylene glycol fatty acid ester (PG ester), lecithin)}, etc. Can be mentioned.

  In addition, the compounds (1) to (33) are emulsified using a common solvent or emulsifier together with a polymer substance such as water / natural gums and polysaccharides to obtain oily food (mayonnaise, etc.), aqueous food (soft drink, etc.) ).

  The total content of any one or more of compounds (1) to (33) in the food additive is not particularly limited. However, assuming a 60 kg adult, the minimum poisoning amount of ganoderic acid per day: Since it is 30000 mg, for example, it is preferably contained in the food additive (for 1 day) in a total amount of 0.1 mg to less than 20000 mg. In the case of this example, it may be produced as a supplement of mg tablet containing 0.1 mg to 100 mg, or it may be produced so as to contain 100 mg to 20000 mg in total in the form of the compound (3) of the active ingredient. In addition, for example, the amount of the active ingredient (each compound) as a single intake amount is in a range from a Tamiflu tablet (75 mg) equivalent amount to a minimum poisoning amount or a Tamiflu tablet ( 75 mg) It may be adjusted to be less than the equivalent amount to 0.1 mg or more.

[Cosmetics]
The cosmetics (medicinal cosmetics) according to the present invention contain one or more of the organic solvent extract or aqueous solvent extract and ganoderma tripenoid compounds (1) to (33) described above. is there. Since Ganoderma tripenoid compounds (1) to (33) have a basic steroid skeleton, they are easily taken into cells from the skin due to the interaction between lipids of cells and compounds (1) to (33). It is preferably used as a cosmetic. Examples of cosmetic dosage forms include solution systems, solubilization systems, emulsification systems, powder dispersion systems, water-oil two-layer systems, water-oil-powder three-layer systems, gels, mists, sprays, mousses, roll-ons, sticks, etc. In addition, a sheet impregnated or coated on a sheet such as a non-woven fabric can be used, and spray or mist that can be easily applied in the throat or nasal cavity (particularly in the back) is particularly preferable.

  The cosmetic preparation is applied to the mucous membrane of the nasal cavity, palatine tonsil, throat, trachea, bronchi, bronchiole, and esophagus by a method such as spraying, so that the NA inhibitor according to the present invention in the epithelial cells of the mucosa is targeted Therefore, it is useful in these applications because it can prevent influenza A and reduce its symptoms. When applying by spraying, for example, the amount of active ingredient (amount of each compound) delivered from the spray nozzle with a single press can be adjusted to the amount indicated by an anti-influenza agent as described below. Good. For example, the amount of the active ingredient (each compound) delivered from the nozzle by a single press of the spray is in the range from the equivalent of Tamiflu Tablets (75 mg) to the minimum poisoning amount, or less than the equivalent of Tamiflu Tablets (75 mg) to 0.00. You may adjust so that it may become 1 mg or more.

(Concentration of active ingredients in cosmetics)
Unlike cosmetic supplements and anti-influenza drugs that are administered by oral intake, cosmetics can be applied to epithelial cells such as the nasal cavity and throat infected by influenza, so they are included in cosmetics. The total dose of any one or more of the above compounds (1) to (33), particularly any one or more of the compound (3), etc. is very small compared to the case of ingestion, and higher. It can be contained in a concentration.

  Therefore, in the case of cosmetics, the inhibition rate of NA is a total of any one or more of the compounds (1) to (33), preferably compound (3), which are active ingredients of NA inhibitors in cosmetics. Is highly preferable because it can be applied to the above nasal cavity, tonsil, throat, trachea, bronchi, bronchiole, and esophageal mucosa at a high concentration (eg, 40 μM to 300 μM). Not limited to this, the NA inhibition rate is, for example, 0.1% to 100% in total in one or more of the compounds (1) to (33) or any one or more of the compound (3) in the cosmetics. In a concentration (for example, 0.1 μM to 300 μM). In addition, for example, the amount of the active ingredient (each compound) as a single intake amount is in a range from a Tamiflu tablet (75 mg) equivalent amount to a minimum poisoning amount or a Tamiflu tablet ( 75 mg) It may be adjusted to be less than the equivalent amount to 0.1 mg or more.

  In this case, dimethyl sulfoxide (DMSO), ion activator, ethanol, propylene glycol, fatty acid, fatty acid ester, etc. are used in order to efficiently penetrate the extracted components applied to the skin (especially mucosal epithelial cells) into the cells. A penetration enhancer (transdermal absorption enhancer) may be included as a cosmetic ingredient. Among these, when using DMSO, it is preferable to contain 10 weight% or less with respect to the whole cosmetics. When the above-described extraction is performed using ethanol, ethanol is included again as a cosmetic component, which is advantageous in that it is not necessary to remove the ethanol used for extraction.

  In the present invention, any one or more of the compounds (1) to (33), or any one or more of the compound (3), oils and fats such as vegetable oil, waxes such as lanolin and beeswax, and hydrocarbons , Fatty acids, higher alcohols, esters, various surfactants, pigments, fragrances, vitamins, plant and animal extract components, UV absorbers, antioxidants, antiseptics, bactericides, etc. May be manufactured.

[Pharmaceuticals (anti-influenza)]
The anti-influenza agent according to the present invention is any one of the aforementioned organic solvent extract or aqueous solvent extract, any one or more of the aforementioned triterpenoid compounds (1) to (33), or the compound (3), etc. One or more types are contained.

  The dosage forms of the anti-influenza agent include liquids, extracts, elixirs, capsules, granules, pills, powders, spirits, tablets, syrups, soaking agents, decoctions, injections, lozenges, limonades, From the viewpoint of being applicable to the nasal cavity, palatine tonsil, throat, trachea, bronchus, bronchiole, and esophagus, liquids, syrups, troches, and fluid extracts are particularly preferable.

[dose]
(Anti-influenza agent for H5N1)
The dose of Tamiflu is 75 mg per day, and in the case of an adult (body weight 60 kg), the dose is 1.25 mg Tamiflu / kg / day. Based on this, the amount of the above compound in the anti-influenza drug can be determined. Good.

The amount of triterpenoids (compounds (1) to (33)) corresponding to the daily Tamiflu dose is calculated from the IC ₅₀ value for influenza (H5N1 type) described above (see Table 6 below). . Here, the minimum daily poisoning amount of ganoderic acid is 500 mg / kg / day.

  Therefore, the content of any one of the compounds (1) to (33) in the anti-influenza agent (for H5N1 type; 1 day) according to the present invention is determined based on the following table and the above minimum poisoning amount. Daily dose (mg / kg / day) of any compound (1) to (33) × 60 kg × 1 day (unit: mg) ”or more to“ minimum poisoning amount 500 mg / kg / day × 60 kg × It is preferable to set within a range of “1 day (unit: mg)” or less. For example, if it is Ganoderic acid TQ (Ganoderic acid TQ (14), the content of Ganodermic acid TQ in anti-influenza drugs (for H5N1 type: 1 day) is 66.0 mg to less than 30000 mg It is preferable to set within the range.

  However, since it is thought that the same level of effect as Tamiflu should be obtained, the amount of the compound as an active ingredient in the anti-influenza drug (H5N1 type: 1 day) It is preferable to set the amount to be an amount close to the amount of active ingredient corresponding to 75 mg) (eg, ± 0% to ± 5%, 10%, 15%, 20%, 30%). For example, in the case of ganodermic acid TQ, 66.0 mg of ganodermic acid TQ corresponds to 75 mg of Tamiflu, and therefore, for example, an amount of ± 10% thereof, specifically, an anti-influenza agent (for H5N1 type; 1 As a preferred example, a composition containing 59.4 mg to 72.6 mg of ganoderic acid TQ in (day).

  Here, in the case of aiming at prevention of influenza (H5N1 type), as a preferred example, any one of compounds (1) to (33) in the anti-influenza agent (for H5N1 type; for 1 day) according to the present invention The example which sets content of 1 type to less than Tamiflu tablet (75 mg) equivalent is mentioned. For example, an example of an anti-influenza agent (H5N1; for prevention) containing 1 mg to 66.0 mg of ganoderic acid TQ can be given (the same applies to other compounds).

  Although compounds not shown in the following table such as Ganoderic Acid D are being analyzed and no data are available, the amount corresponding to the daily dose of Tamiflu is calculated from the IC50 value, and this amount is reduced to the minimum poisoning. An amount in the range of the amount (500 mg) may be contained in the anti-influenza agent (H5N1 type). In addition, IC50 values are not strictly determined for the compounds with inequality signs in the following table. For example, the ganoderic acid S (3) means “IC50 (μM) <100”, which means that it has been found to be less.

(Anti-influenza agent for H1N1 type)
As in the case of H5N1, the daily dose of each compound corresponding to the daily dose of Tamiflu (1.25 mg / kg / day) is shown in Table 7 below. As shown in Table 7 below, the content of each compound contained in the anti-influenza agent (for H1N1; for 1 day) according to the present invention is “one day of any of the compounds (1) to (33)”. The dose (mg / kg / day) × 60 kg × 1 day (unit: mg) ”to“ minimum poisoning 500 mg / kg / day × 60 kg × 1 day (unit: mg) ”or less. Is preferred. For example, in the case of Ganoderic acid TQ (Ganoderic acid TQ (14)), the content of ganodermic acid TQ in the anti-influenza drug (for H1N1 type: 1 day) is 14.6 g to 30. It is preferable to set in a range of less than 0 g.

  However, since it is considered that the same effect as Tamiflu can be obtained, the amount of the compound as an active ingredient in the anti-influenza drug (H1N1 type; 1 day) It is preferable to set the amount to be an amount close to the amount of active ingredient corresponding to 75 mg) (eg, ± 0% to ± 5%, 10%, 15%, 20%, 30%). For example, in the case of ganodermic acid TQ, 14.56 g of ganodermic acid T-Q corresponds to 75 mg of Tamiflu, and therefore, for example, an amount of ± 10% thereof, specifically, an anti-influenza agent (for H1N1 type; 1 As a suitable example, a composition containing 13.1 g to 16.0 g of ganoderic acid TQ in (day portion) can be shown.

  Here, in the case of aiming at prevention of influenza (H1N1 type), as a preferred example, any one of compounds (1) to (33) in the anti-influenza agent (for H1N1 type; for 1 day) according to the present invention The example which sets content of 1 type to less than Tamiflu tablet (75 mg) equivalent is mentioned. For example, an example of an anti-influenza agent (H1N1; for prevention) containing 1 mg to 13.1 g of ganoderic acid TQ can be given (the same applies to other compounds).

  Although compounds not shown in the following table such as Ganoderic Acid D are being analyzed and no data are available, the amount corresponding to the daily dose of Tamiflu is calculated from the IC50 value, and this amount is reduced to the minimum poisoning. An amount in the range of the amount (500 mg) may be contained in the anti-influenza agent (H1N1 type). In addition, IC50 values are not strictly determined for the compounds with inequality signs in the following table. For example, the ganoderic acid S (3) means “IC50 (nM) <100”, which means that it has been found to be less.

(Other anti-influenza agents for NA)
As in the case of H5N1, the daily dose of each compound corresponding to the daily dose of Tamiflu (1.25 mg / kg / day) is shown in Table 7A below. As shown in Table 7A below, each of the anti-influenza agents according to the present invention (for H1N1 (N295S); for 1 day, for H3N2 (E119V); for 1 day, NA (H7N9); for 1 day)) The content of the compound is “a daily dose (mg / kg / day) of each compound (14), (28) × 60 kg × 1 day (unit: mg)” or more to “minimum poisoning amount” It is preferable to set within a range of “500 mg / kg / day × 60 kg × 1 day (unit: mg)” or less.

  For example, if it is Ganoderic acid TQ (14), the content of Ganodermic acid TQ in the anti-influenza agent (for 1 day) is anti-neuraminidase (H1N1 (N295S)). It is preferable to set in the range of 2.45 g or more to less than 30.0 g for influenza drugs, and in the range of 1.78 g or more to less than 30.0 g for anti-influenza drugs for neuraminidase (H3N2 (E119V)) Is preferred.

  The same applies to ganoderic acid TR (28), and the content of ganoderic acid TR (28) in the anti-influenza agent (for 1 day) is 2. for the anti-influenza agent for neuraminidase (H1N1 (N295S)). It is preferable to set in the range of 25 g or more to less than 30 g, and in the anti-influenza agent for neuraminidase (H3N2 (E119V)), it is preferable to set in the range of 1.28 g or more to less than 30.0 g.

  However, since it is considered that the same effect as Tamiflu can be obtained, the amount of the compound as an active ingredient in the anti-influenza drug (H1N1 type; 1 day) It is preferable to set the amount to be an amount close to the amount of active ingredient corresponding to 75 mg) (eg, ± 0% to ± 5%, 10%, 15%, 20%, 30%).

  In the case of Ganodermic Acid TQ, for example, in the anti-influenza agent for neuraminidase (H3N2 (E119V)), Ganodermic Acid T-Q 1.782 g is equivalent to 75 mg of Tamiflu. Specifically, an anti-influenza drug (for H3N2 (E119V) type; one day) containing 1.60 g to 1.96 g of ganoderic acid TQ can be shown as a suitable example.

  Here, in the case of aiming at prevention of influenza (H1N1 type), as a preferred example, any one of compounds (1) to (33) in the anti-influenza agent (for H1N1 type; for 1 day) according to the present invention The example which sets content of 1 type to less than Tamiflu tablet (75 mg) equivalent is mentioned. For example, an example of an anti-influenza drug (H1N1; for prevention) containing 1 mg or more and less than 1.60 g of ganoderic acid TQ can be given (the same applies to other compounds). The same applies to Ganodermic Acid TR.

[Research reagents]
Ganoderma-derived tripenoid compounds (1) to (33) have different NA inhibitory activities due to differences in their chemical structures, and thus each compound (1) to (33) has a mode of binding to NA and NA. Although the mechanism of expression of inhibitory activity is thought to be different, the details are currently unknown. Therefore, compounds (1) to (33) having different levels of NA inhibitory activity can be used as research reagents for elucidating the mechanism by which triterpenoids derived from Ganoderma lingzhi inhibit NA.

  Moreover, since each compound (1)-(33) has a different chemical structure, as will be described later, the mode of binding to neuraminidase (NA) and NA inhibitory activity are different. Therefore, based on these compounds (1) to (33), it is possible to predict and develop NA inhibitors having higher NA inhibitory activity, and they can also be used as research reagents for this purpose.

Hereinafter, the acquisition method etc. are demonstrated about the reagent etc. which are commonly used by each test example.
《Various neuraminidases》
The following five subtypes of representative recombinant neuraminidase were purchased from Sino Biological Inc.
・ NA (A / California / 04/2009 / (H1N1)) (neuraminidase (H1N1))
・ NA (A / California / 04/2009 (H1N1) (N295S) (neuraminidase (N295S)
・ NA (A / Babol / 36/2005 (H3N2) (E119V) (neuraminidase (E119V))
・ NA (A / Hubei / 1/2011 (H5N1) (neuraminidase (N5N1))
・ NA (A / Hangzhou / 1/2013 (H7N9)) (neuraminidase (H7N9))
《Neuraminidase activity assay kit》
"Neuraminidase (NA) Recombinant Viral Protein, N-His Tag", product number: 11058-V07B-50, Life Technology Corporation). In addition, 4-methylumbelliferone sodium (product name “4-methylumbelliferone sodium salt”, product number M1508, Sigma) was used for optimization of the analysis system.

《Various triterpenoids derived from Ganoderma》
Thirty- three triterpenoids (purity> 98%) from Ganoderuma lingzhi were purchased from Chemfaces (Wuhan, Hubei, China) and Quality Phytochemicals (LLC, New Jersey, USA). These triterpenoids (1) to (33) were dissolved in DMSO so as to be 25 mM to 2.5 mM in dimethyl sulfoxide (DMSO).

<< Known NA inhibitor >>
“Oseltamivir phosphate” (product number: 1479304), a phosphate of Tamiflu (registered trademark), was purchased from Sigma.

[Test Example 1]
About 33 types of triterpenoids derived from Ganoderuma lingzhi , the inhibitory activity of neuraminidase (H1N1, H5N1) of influenza virus was investigated, and the inhibition rate at various concentrations of compounds (1) to (33) of each triterpenoid and IC ₅₀ was determined.

(material)
In addition, chloroform extract and hot water extract from Ganoderma lingzhi (preparation method for each extract is according to the extraction method described later) in dimethyl sulfoxide (DMSO), respectively, from 0 μg / mL to 1600 μg / mL. And dissolved. In addition, “Oseltamivir phosphate” (product number: 1479304, Sigma) (20 mM to 2.5 mM), which is a phosphate of Tamiflu (registered trademark), was used as a positive control.

(Method)
(1) Measurement of NA inhibitory activity The principle of measuring the inhibitory activity of neuraminidase (NA) is shown in FIG. Analysis of inhibitory activity against neuraminidase (H1N1 type) and neuraminidase (H5N1 type) of the above-mentioned triterpenoids (compounds (1) to (33)) derived from Ganoderma lucidum “Product Description of NA-Fluor Influenza Neurminidase Assay Kit” Based on the procedure described in, the following changes were made.

(Preparation of reagent and NA recombinant solution)
The volume ratio of “2 × NA-Fluor ^™ Assay Buffer” (66.6 mM 2- [N-morpholino] ethanesulfonic acid [MES] buffer, 8 mM CaCl ₂ , pH 6.5) contained in the kit and distilled water 1: 1 was mixed to prepare 20 mL of 1 × analytical buffer for virus dilution. In addition, a solution containing NA recombinant at 300 ng / mL was prepared using 1 × analysis buffer as a solvent.

"procedure"
The inhibition rate (%) and IC ₅₀ (μM) for each neuraminidase were specified as follows.

  I) 1 μL of each compound (1) to (33) (concentration 0 μM, 25 μM, 50 μM, 100 μM, 200 μM) dissolved in DMSO is mixed with 24 μL of 1 × analysis buffer for each of the above concentrations, and neuraminidase (H1N1) Type) solution (300 ng / mL) was mixed with 25 μL, and a total of 50 μL of the mixed solution was prepared for each concentration.

On the other hand, for the neuraminidase (H1N1 type) solution, a total of 50 μL of the mixture was similarly adjusted for each concentration.
Also, a negative control mixture containing no NA recombinant was prepared using 25 μL of 1 × analysis buffer instead of 25 μL of each neuraminidase solution (300 ng / mL).
Each of the prepared mixed solutions was stored in each well of a 96-well plate and shaken at 37 ° C. for 20 minutes.

  II) 50 μM of 4-MUNANA (4- [methylumbelliferyl] -N-acetylneuraminic acid) solution (6.1 mg of 4-MUNANA in 5 mL of 1 × analytical buffer for the above mixture after shaking. 50 μL of the dissolved one) was added to obtain a total of 100 μL of the mixed solution. Thereafter, the 100 μL of the mixed solution was further shaken at 37 ° C. for 60 minutes.

III) The reaction shown in FIG. 1 was stopped by adding 100 μL of a stop solution (0.2 M NaCO ₃ solution) to the above mixture after shaking for 60 minutes. Thereafter, the mixture solution was placed under excitation light having a wavelength of 355 nm, and fluorescence (460 nm) emitted from the mixture solution was measured.

IV) The average amount (RFU _background ) of the relative fluorescence units (RFU: Relative Fluorescent Unit) of the negative control was calculated by averaging the fluorescence amounts measured for a plurality of negative controls.

V) Next, the RFU value (RFU-minus-background _sample ) obtained by subtracting the blank value by subtracting the RFU average value of the negative control from the RFU value measured for each of the compounds (1) to (33) was calculated. Using these values, the inhibition rate IR (%) was calculated by the following formula.
The inhibition rate IR was calculated using the following formula.
IR (%) = (1-(RFU-minus-background _sample / RFU-minus-background _DMSO )) x 100 (%)

Next, the inhibition rate (IR) calculated for each of the compounds (1) to (33) according to the concentration (concentration: 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM) was plotted, and the compounds (compounds (1) to (33) were plotted. )) Separately, 33 graphs (inhibitor concentration-inhibition rate graph, horizontal axis: inhibitor concentration, vertical axis: inhibition rate (%)) were prepared. From the graph, the concentration (IC ₅₀ ) that inhibits the activity of NA by 50% was calculated for each compound (compounds (1) to (33)).

  Moreover, based on the inhibition rate IR of Tamiflu (registered trademark) of neuraminidase (H1N1) and the inhibition rate IR of the compounds (1) to (33), each compound (1) to (33) (50 μM) is equivalent. Tamiflu concentration (nM OE) was calculated and shown in Table 8 below.

  Δ24 and 25 indicate the position of the double bond, (E) indicates that the conformation of the double bond is different, and (Z) and (R) indicate that the conformation of the double bond is the same. (In the simple example, (E) means trans type, (Z) and (R) mean cis type).

(Results and discussion)
In order to examine the influence of each compound (1) to (33) on each activity of neuraminidase (H1N1 type), in this Test Example 1, each compound (1) to (33) was added in stepwise concentrations (0 μM, 25 μM). , 50 μM, 100 μM, 200 μM) to determine the inhibition rate (%) (see Table 8 and FIG. 3 (not shown in FIG. 3)). In addition, the vertical axis | shaft of FIG. 3 shows RFU value (= grade of NA inhibitory ability) of the product (4-MU) of FIG. Similarly, IC ₅₀ for neuraminidase (H5N1 type) in each of the compounds (1) to (33) was also identified (see Table 8).

  2 (A) and 2 (B) are graphs showing changes in the inhibition rate of Tamiflu against neuraminidase (H1N1 type) shown when the concentration of Tamiflu used as a positive control is changed.

(Results and discussion)
All the inhibition rates (%) and IC ₅₀ of the compounds (1) to (33) are shown in Table 8 above. This result indicates that a specific triterpenoid derived from Ganoderma lingzhi has an inhibitory ability against a specific influenza virus neuraminidase (H1N1 type, H5N1 type). Of these triterpenoids, Ganodermic Acid TQ (14) and Ganoderic Acid TR (28) are particularly low in the RFU value of the produced 4-MU (FIG. 1) and particularly high in NA inhibition ability. is there.

(1) Inhibitory ability against neuraminidase (H1N1 (N295S) type) As shown in Table 8, against neuraminidase (H1N1) of influenza virus, compounds (3), (9), (10), (13), (13) 14), (19), (22) to (29), and (30) to (33) were found to have inhibitory ability. Therefore, any one or two of compounds (3), (9), (10), (13), (14), (19), (22) to (29), and (30) to (33) It turns out that NA inhibitor containing the above is effective with respect to influenza virus (H1N1) as an anti-influenza agent.

Furthermore, among the above compounds, the compounds having an IC ₅₀ of 100 μM or less are compounds (14) and (27), and one or two selected from the group consisting of these compounds (14) and (27) It can be seen that NA inhibitors containing as an active ingredient are particularly effective against influenza viruses (H1N1 type) as anti-influenza agents.

[Test Example 2]
Ganodermic acid TQ and ganoderic acid TR, which showed particularly strong NA inhibitory activity against specific influenza neuraminidase (H1N1 type), were further used for other influenza virus neuraminidases (NA (H1N1, N295S), NA (H3N2, E119V) and NA (H7N9)) were examined for IC ₅₀ in the same manner as in Test Example 1. The results are shown in Table 9 below.

(Results and discussion)
As a result of this test, it was revealed that ganoderic acid TQ and ganoderic acid TR inhibit all of these neuraminidase subtypes (types in parentheses shown in Table 9). It can be seen that the inhibition ability varies depending on the type of neuraminidase targeted. Ganodermic acid TQ exhibited NA inhibitory ability equal to or higher than Tamiflu against neuraminidase (N5N1 type). In addition, as shown in Table 9, since the IC ₅₀ value of Tamiflu against neuraminidase (NA (H1N1, N295S), NA (H3N2, E119V)) is high, the analysis system of Test Example 2 is reliable. I understand.

[Test Example 3]
Based on the results of Test Example 2 in which some of the compounds derived from Ganoderma (Ganoderic Acid TQ, Ganodermic Acid TR) were confirmed to be inhibitory to neuraminidase (H1N1), the remaining compounds (1) to (13) , (15) to (27) and (29) to (33) also show the NA inhibitory ability of the neuraminidase (NA) subtypes (H3N2 (E119V), H5N1, H7N9), etc. It investigated similarly using the evaluation method. In addition, each said compound was used by the final concentration of 200 micromol as an inhibitor shown in FIG. The results of Test Example 3 are shown in FIGS.

(Results and discussion)
(2) Inhibitory ability against neuraminidase (H1N1 (N295S) type) As shown in Table 10 and FIG. 6, against neuraminidase (H1N1 (N295S)) of influenza virus, compounds (1), (3) to (33) It became clear that all of these have inhibitory ability. “N295S” indicates that the 295th amino acid of neuraminidase is substituted from asparagine (N) to serine (S).

  Therefore, it can be seen that NA inhibitors containing any one or more of compounds (1) and (3) to (33) are effective against influenza virus (H1N1 (N295S)) as anti-influenza agents. .

  Among the above compounds, compounds having an inhibition rate of 30% or higher against neuraminidase of influenza virus A (H1N1 (N295S) type) include Ganodermic Acid S (3), Ganodermic Acid TQ (14), and Ganodermic Acid TR. (28) (partially not shown), an NA inhibitor containing one or more selected from the group consisting of these compounds (3), (14) and (28) as an active ingredient is an anti-influenza It can be seen that it is particularly effective against influenza virus (H1N1 (N295S) type) as an agent.

  Further, the compounds having an inhibition rate exceeding 40% and 50% are ganoderic acid TQ (14) and ganoderic acid TR (28), which are selected from the group consisting of the compounds (14) and (28). It turns out that the anti-influenza agent which contains a seed | species or 2 types as an active ingredient is very effective with respect to influenza virus (H1N1 type | mold (N295S)).

(3) Inhibitory ability against neuraminidase (H3N2 (E119V) type) As shown in Table 10 and FIG. 8, all of compounds (1), (3) to (33) were against neuraminidase (H3N2 (E119V)). It became clear that it has inhibitory ability. Therefore, an NA inhibitor containing any one or more of compounds (1) and (3) to (33) is effective against influenza virus (H3N2 (E119V)) as an anti-influenza agent. I understand.

  Among the above compounds, useful compounds having an inhibition rate of more than 30% against neuraminidase of influenza virus (H3N2 (E119V) type) include Ganodermic Acid S (3), Ganodermic Acid A (8), Ganoderma Derrick Acid AM1 (9), Ganodermic Acid B (10), Ganoderic Acid TN (13), Ganoderic Acid TQ (14), Ganoderin Manone Diol (17), Lucylaldehyde B (22), Ganodermic Acid C1 (23), ganoderic acid C2 (24), ganoderic acid K (27), ganoderic acid TR (28), ganoderic acid C (30), ganoderic acid D (31), ganoderin sun H (33), NA inhibitor containing one or more of these compounds It is found to be particularly effective against the influenza virus (H3N2 (E119V)) as an anti-influenza agent.

  Further, as very useful compounds having an inhibition rate exceeding 40%, ganodermic acid S (3), ganodermic acid TN (13), ganodermic acid TQ (14), lucylaldehyde B (22), ganodermic acid C2 (24) and ganoderic acid TR (28), and NA inhibitors containing one or more of these compounds are highly effective against influenza virus (H3N2 (E119V)) as anti-influenza agents I understand.

  Furthermore, extremely useful compounds having an inhibition rate exceeding 50% are ganoderic acid TQ (14) and ganoderic acid TR (28), and NA inhibitors containing one or two of these compounds are anti-influenza agents. It turns out that it is very effective with respect to influenza virus (H3N2 (E119V)).

(4) Inhibitory ability against neuraminidase (H5N1 type) As shown in Table 10 and FIG. 7, compound (1), which is a triterpenoid derived from Ganoderma lingzhi , against neuraminidase (H5N1 type), (3) It became clear that compound (3), (4), (6)-(10), (12)-(33) has inhibitory ability among -33. Therefore, NA inhibitor containing any one or more of compounds (3), (4), (6) to (10), (12) to (33) is an influenza virus (H5N1) as an anti-influenza agent It can be seen that it is effective against the type).

  Further, among the compounds that have been confirmed to be inhibitory, compounds having an inhibition rate of 30% or higher against neuraminidase of influenza virus (H5N1 type) include compounds (5), (10), (11) and (25). All of the compounds are excluded, and contain one or more compounds (3), (4), (6) to (9), (12) to (24), and (26) to (33). It can be seen that NA inhibitors are particularly effective against influenza viruses (H5N1 type) as anti-influenza agents.

  Further, as very useful compounds having an inhibition rate exceeding 40%, ganoderic acid S (3), ganoderic acid F (4), ganoderic acid LM2 (6), ganoderic acid DM (7), ganoderic Acid AM1 (9), Ganodermic Acid Y (12), Ganodermic Acid TN (13), Ganodermic Acid TQ (14), Ganolic Acid A (15), Ganodermanone Triol (16), Ganodermanone Diol (17), Ganoderol A (18), Ganoderol B (19), Rucylaldehyde A (21), Rucylaldehyde B (22), Ganodermic Acid C1 (23), Ganoderic Acid C2 (24), Ganoderic Acid H (26), Ganoderic Acid K (27), Ganoderic Acid NA inhibitor containing R (28), ganoderenic acid D (31), ganoderenic acid F (32) and ganoderenic acid H (33), and containing one or more of these compounds as active ingredients It turns out that it is very effective with respect to influenza virus (H5N1 type) as an agent.

  Further, as extremely useful compounds having an inhibition rate exceeding 50%, the compounds (3), (6), (7), (9), (12) to (14), (16) to (19), (21 ), (22), (24), (26) to (28), (31) to (33), and an anti-influenza agent containing one or more of these compounds as an active ingredient is influenza virus It turns out that it is very useful with respect to (H5N1 type). Similarly, an NA inhibitor containing, as an active ingredient, one or more compounds included in each group having an inhibition rate of 60% or more, 70% or more, and 80% or more is an influenza virus (H5N1 type) as an anti-influenza agent It is found that the compound is extremely effective against NA, and in particular, a compound having a very strong NA inhibitory activity with an inhibition rate of 90% or more (Ganodermic Acid TN (13), Ganodermic Acid TQ (14), Ganoderma). NA inhibitor containing one or more of derrick acid TR (28) as an active ingredient is effective against influenza virus (H5N1 type) as an anti-influenza agent, comparable to a known anti-influenza agent (Tamiflu) It turns out that it is.

(5) Inhibitory ability against neuraminidase (H7N9 type) As shown in Table 10 and FIG. 9, all of compounds (1), (3) to (33) have inhibitory ability against neuraminidase (H7N9). It became clear. Therefore, it can be seen that NA inhibitors containing any one or more of compounds (1) and (3) to (33) are effective against influenza virus (H7N9 type) as anti-influenza agents.

  Further, among the compounds that have been confirmed to have an inhibitory ability, compounds having an inhibition rate of 30% or more against neuraminidase of influenza virus (H7N9 type) include ganoderic acid S (3), lucylaldehyde B (22), and ganodelenic acid. NA inhibitor containing one or more of these compounds, particularly D (31), ganoderenic acid F (32) and ganoderenic acid (33) as an anti-influenza agent against influenza virus (H7N9 type) It turns out that it is effective.

(Multi target)
It can be said that exhibiting inhibitory ability against more types of neuraminidase (NA) is highly valuable as an active ingredient of NA inhibitors and anti-influenza agents. As shown in Table 10, for example, ganodermic acid S (3), ganodermic acid DM (7), ganodermic acid TN (13), ganodermic acid TQ (14), ganodermanone diol (17) , And ganoderic acid TR (28), etc. are high in inhibitory ability against all four types of neuraminidase (H1N1 (N295S) type, H3N2 (E119V) type, H5N1 type, H7N9 type), and are compounds. (3), (7), (13), (14), (17) and (28) NA inhibitors and anti-influenza agents containing any one or more of the group consisting of two or more active ingredients are widely used. It can be said that it is a very useful NA inhibitor and anti-influenza agent acting on influenza.

  Similarly, for example, any one or two of the group consisting of lucylaldehyde B (22), ganoderic acid C2 (24), ganoderic acid D (31), ganoderic acid F (32) and ganodelenic acid H (33) NA inhibitors and anti-influenza agents containing more than one species should be excellent NA inhibitors and anti-influenza agents that effectively act on three types of neuraminidases (H3N2 (E119V) type, H5N1 type, H7N9 type) I understand.

[Test Example 4] (Rough extract)
In order to confirm the inhibitory effect of Ganoderma lingzhi crude extract on neuraminidase, four types of extracts (chloroform, chloroform, water, 99.5% ethanol and 30% ethanol) were used from Ganoderma. Extract, hot water extract, 99.5% ethanol extract and 30% ethanol extract).

(Preparation of each extract)
・ Chloroform extract (Chloroform-Ex . )
The chloroform extract was prepared by immersing 320 g of Ganoderma lingzhi powder in 9.6 L of chloroform (purity> 99.9%, Wako Pure Chemical), shaking for 3 days, and removing the solvent from the shaken chloroform solution. The chloroform (boiling point 61 ° C.) was removed by degassing and boiling (solution temperature> 61 ° C.) to prepare 3.585 g of chloroform extract (Chloroform-Ex.) (Yield 1.12%). This was dissolved in dimethyl sulfoxide (DMSO) at various concentrations (160 mg / mL to 40 mg / mL).

・ Hot water extract (water extract; Water-Ex . )
The hot water extract was prepared by immersing 30 g of Ganoderma lingzhi in 200 mL of distilled water and autoclaving (treating with water vapor at 2 atm for 20 minutes at 121 ° C.). After autoclaving, the solvent was extracted from the aqueous solution. Water (boiling point: 100 ° C.) was removed by boiling (solution temperature: 100 ° C.) to prepare 1.351 g of hot water extract (Water-Ex.) (Yield: 4.5%). This was dissolved in DMSO at various concentrations (160 mg / mL to 40 mg / mL).

・ 99.5% ethanol extract (Ethanol-Ex.)
The 99.5% ethanol extract (Ethanol-Ex.) Was prepared by immersing 5.047 g of Ganoderma lingzhi in 100 mL of 99.5% ethanol (purity: 99.5%) and extracting by shaking for 48 hours. The soaked liquid was subjected to suction filtration to obtain a filtrate. Ethanol extract (Ethanol-Ex.) Was prepared from this filtrate by removing ethanol (boiling point 78.37 ° C.) as a solvent by degassing and boiling (> 79 ° C.). This was dissolved in DMSO at various concentrations (160 mg / mL to 40 mg / mL).

・ 30% ethanol extract (30% Ethanol-Ex.)
30% ethanol extract (30% Ethanol-Ex.) Was prepared by preparing 30% ethanol from the above 99.5% ethanol using distilled water, and adding 5.035 g of Ganoderma lingzhi to the above 30.0%. By soaking in 100 mL of% ethanol and shaking for 48 hours, the solvent water and ethanol (boiling points: 100 ° C., 78.37 ° C.) are removed from the shaken ethanol solution by degassing and boiling (> 100 ° C.). An ethanol extract (Ethanol-Ex.) Was prepared. This was dissolved in DMSO at various concentrations (160 mg / mL to 40 mg / mL).

(NA inhibition assay)
Each extract was used as an inhibitor in FIG. 1 at various final concentrations (400 μg / mL, 800 μg / mL, 1600 μg / mL), and each neuraminidase (H1N1 (N295S) type, An inhibition assay (see FIG. 1) for H3N2 (E119V), H5N1, H7N9, and H1N1) was performed, and the inhibition rate (%) and IC ₅₀ for each neuraminidase of each extract were calculated and determined. The results are shown in FIGS. 10 to 13, FIG. 15, and Table 11.

(Results and discussion)
(1) Inhibitory ability against neuraminidase (H1N1 (N295S) type) As shown in FIG. 10, four types of extracts (chloroform extract (Chloroform-Ex.), Hot water extract ( Water-Ex.), 99.5% ethanol extract (Ethanol-Ex.)) And 30% ethanol extract (30% Ethanol-Ex.)) All have lower relative fluorescence intensity (RFU) than DMSO. Value, and neuraminidase (H1N1, N295S) was inhibited. Therefore, NA inhibitors and anti-influenza agents containing any one or more of the above four types of extracts are useful as anti-influenza agents against influenza virus (H1N1 (N295S). Furthermore, since the IC ₅₀ value for 99.5% ethanol extract is the lowest for neuraminidase (H1N1 (N295S)), NA inhibitor containing 99.5% ethanol extract as an active ingredient, or anti-influenza It can be seen that the agent is particularly effective against influenza virus (H1N1 (N295S) type).

(2) Inhibitory ability to neuraminidase (H3N2 (E119V) type) As shown in FIG. 11, four types of extracts (chloroform extract (Chloroform-Ex.), Hot water extract ( Water-Ex.) And 99.5% ethanol extract (Ethanol-Ex.)) Had a relative fluorescence intensity (RFU) lower than that of DMSO and inhibited neuraminidase (H3N2 (E119V)). Therefore, an NA inhibitor containing any one or more of the above four types of extracts is useful as an anti-influenza agent against influenza virus (H3N2 (E119V) type). Moreover, as shown in Table 11, for neuraminidase (H3N2 (E119V) type), the IC ₅₀ values for water extract (hot water extract) and 99.5% ethanol extract are lower than others. From the above, an NA inhibitor or an anti-influenza agent containing one or two of water extract (hot water extract) and 99.5% ethanol extract as an active ingredient is influenza virus (H3N2 (E119V) type) It can be seen that this is particularly effective.

(3) Inhibitory ability against neuraminidase (H5N1 type) As shown in FIG. 12, four types of extracts (chloroform extract (Chloroform-Ex.), Hot water extract (Water-Ex) used as inhibitors in FIG. .) And 99.5% ethanol extract (Ethanol-Ex.)) Had a relatively low relative fluorescence intensity (RFU) than DMSO, and strongly inhibited neuraminidase (H5N1). Therefore, an NA inhibitor containing any one or more of the above four types of extracts is useful as an anti-influenza agent against influenza virus (H5N1 type). Further, as shown in Table 11, for neuraminidase (H5N1), the IC ₅₀ values for the water extract (hot water extract) and the 99.5% ethanol extract are lower than others (IC 50 < 50 μg / mL), NA inhibitor containing one or two of water extract (hot water extract) and 99.5% ethanol extract as an active ingredient, or anti-influenza agent is influenza virus (H5N1 type) It can be seen that this is particularly effective. Here, since the IC50 value of the 30% ethanol extract is also 50 μg / mL and the IC50 value is sufficiently low, the water extract (hot water extract), 99.5% ethanol extract and 30% ethanol extract are used. It can also be seen that NA inhibitors containing any one or more of these are useful as anti-influenza agents.

(4) Inhibitory ability against neuraminidase (H7N9 type) As shown in FIG. 13, four types of extracts (chloroform extract, hot water extract, 99.5% ethanol extract and 30) used as the inhibitors in FIG. % Ethanol extract) had a lower relative fluorescence intensity (RFU) than DMSO and inhibited NA (H7N9). Therefore, an NA inhibitor containing any one or more of the above four types of extracts is useful as an anti-influenza agent against influenza (H7N9). Further, as shown in Table 11, for NA (H7N9), the IC ₅₀ values for 99.5% ethanol extract and 30% ethanol extract were lower than others, and 99.5% ethanol extraction was performed. It can be seen that NA inhibitors containing any one or two of the product and 30% ethanol extract as an active ingredient are particularly effective against influenza virus (H7N9 type) as an anti-influenza agent.

(Multi target)
It can be said that exhibiting inhibitory ability against more types of neuraminidase (NA) is highly valuable as an active ingredient of NA inhibitors and anti-influenza agents. As shown in Table 12, hot water extract (water extract) and 99.5% ethanol extract were mixed with neuraminidase (NA (H1N1, N295S), NA (H3N2, E119V), NA (H5N1), NA ( H7N9)) is highly effective in inhibiting all of NA inhibitors and anti-influenza drugs containing any one or more of hot water extract and 99.5% ethanol extract as active ingredients It can be said that it is a very useful NA inhibitor and anti-influenza agent acting on a wide range of influenza.

[Test Example 6] (Docking simulation)
As shown below, the active sites of different types of neuraminidase (H1N1, H3N2, H5N1, H7N9 and H1N1 (N295S)) neuraminidases present in influenza viruses are calculated on the computer ( In Silico). ) To dock each compound (1) to (33).

  By this docking simulation, each of the compounds (1) to (33) can be easily bound to the active site of each neuraminidase, and the inhibition mode (competitive inhibition, non-competitive inhibition, or non-antagonistic inhibition is any inhibition mode). Can be determined.

Protein structure data bank (Protein Data Bank: http: 3D structure of each type of influenza virus neuraminidase (H1N1, H3N2, H5N1, H7N9 and H1N1 (N295S)), which is the docking target for inhibitors //Pdbj.org/)
For the docking simulation, PC software “CLC Drug Discovery Workbench” (CLC bio) was used.

<Streucute-activity relationship analysis>
Thirty-three types of triterpenoids (compounds (1) to (33)) (derived from Ganoderma lucidum) were divided into three groups (skeleton structures A to C) based on the difference in their skeleton structures. Skeletal structure A consists of two double bonds (double bond between 7th and 8th carbons (Δ ^7,8 ), between 9th and 11th carbons) It has a double bond (Δ ^9,11 )) in the tetracyclic ring and has a branched chain having a carboxyl group. Skeletal structure B has one double bond (double bond between 8th and 9th carbons (Δ ^8,9 )) in the tetracyclic ring, and carboxyl It has a branched chain having a group. The skeleton structure C has two double bonds (Δ ^7,8 , Δ ^9,11 ) in the tetracyclic ring and does not have a carboxyl group in the branched chain. The NA inhibitory ability of these skeletal structures A to C and their compounds was compared and analyzed.

  The neuraminidase (H1N1 type, which has 5 subtypes of influenza virus, each of the three-dimensional structures of compounds (1) to (33) and clinically used Tamiflu (registered trademark), Relenza (registered trademark) and laminamivir. A simulation was carried out to dock the three-dimensional structure (three-dimensional structure in the absence of water) of the neuraminidase of H3N2, H5N1, H7N9 and H1N1 (N295S). In the simulation, the pocket of the substrate binding site on neuraminidase (NA) is specified from the co-crystal of Tamiflu and neuraminidase when the neuraminidase and the substrate are bound, and the radius of 10 Å including the pocket serving as the binding site is specified. Spherical sites were selected as binding targets for inhibitors (compounds (1) to (33), Relenza, etc.).

(Results and discussion)
The results of structure-activity relationship analysis (SAR) performed by docking simulation are shown in FIGS. 5-1 to 5-2 and Table 12. FIG. Although the results of the structure-activity relationship analysis are not clear, some findings about the chemical structures important for NA inhibition were obtained.

  Four of six triterpenoids (compounds (14), (28), (13), (3), (22) and (17)) (compounds (14), (28), (13), (3)) ) Belonged to the group of skeletal structure A. This suggests that skeletal structure A is necessary for the neuraminidase inhibitory activity of these triterpenoids. For the groups with skeletal structures B and C, when R1 is oxygen, in most cases they showed low NA inhibitory activity or no activity.

  The results of docking simulation are shown in Table 12 as scores (Kcal / mol). Here, when the docking score is negative and the absolute value is large, it means that the affinity between the ligand (inhibitor) and the target protein (neuraminidase) is high (energy repelled between two molecules of neuraminidase and inhibitor) Means that the molecules are small and easy to bond strongly).

The results in Table 6 above show that Ganodermic Acid TQ and Ganodermic Acid TR have a low docking score (strong binding force) close to known NA inhibitors such as Tamiflu. The fact is that docking simulation itself docks Ganodermic Acid TQ, Ganodermic Acid TR, etc. in place of Tamiflu at the same binding site on NA as Tamiflu and neuraminidase (NA) binding site. Since the score (Kcal / mol) was obtained, it was suggested that ganoderic acid TQ (14) and ganoderic acid TR (28) were bound to neuraminidase through the same binding site as Tamiflu. Yes. That is, it is suggested that Ganodermic Acid TQ and Ganodermic Acid TR also compete with the substrate for neuraminidase and inhibit neuraminidase (NA) activity similarly to Tamiflu and the like. The binding site of the inhibitor (the site of the enzyme where the substrate only binds to the enzyme neuraminidase) is present in the active site of neuraminidase (the site on the enzyme neuraminidase where the substrate binds and the chemical reaction proceeds).

  Ganodermic acid TR (28) has a low docking score for each neuraminidase from influenza viruses (H3N2, H5N1, H7N9 and Tamiflu-resistant H1N1). This result not only suggests that Ganodermic Acid TR (28) is effective against human and avian influenza, and thus other influenza including influenza resistant to known drugs such as Tamiflu. This suggests that these subtypes may also be effective.

[Test Example 7]
(Preparation of each extract)
-Ganoderma hot water extract The same Ganoderma hot water extract as used in Test Example 4 was used. This hot water extract of Ganoderma lingzhi was dissolved in a solvent DMSO to prepare 1600 μg / mL hot water extract solutions.
-Ganoderma Chloroform Extract The same Ganoderma chloroform extract as in Test Example 4 was used. Further, the chloroform extract was dissolved in a solvent DMSO to prepare a chloroform extract solution having a concentration of 1600 μg / mL.

(Method)
Instead of “each compound (1) to (33) dissolved in DMSO (concentration 0 μM, 25 μM, 50 μM, 100 μM, 200 μM) 1 μL” used in Test Example 1, the above hot water extract solution (concentration: 1600 μg / (mL) 1 μL, or chloroform extract solution (concentration: 1600 μg / mL) 1 μL, the NA inhibition rate and IC ₅₀ value of the hot water extract or chloroform extract were measured in the same manner as in Test Example 1. .

(Results and discussion)
The crude extract of Ganoderma lingzhi contains many components. The chloroform extract and hot water extract of Ganoderma lingzhi both showed a slight inhibitory effect on neuraminidase activity at high concentrations (FIG. 15).

(Conclusion)
The inhibitory effect of ganoderic acid TQ and ganoderic acid TR on neuraminidase could be confirmed. Although the above extract showed a weak inhibitory effect on neuraminidase activity, it may be used to elucidate the mechanism that prevents the proliferation of influenza through other mechanisms.

  As described above, the neuraminidase inhibitor according to the present invention has been described in detail based on each test example and the like. However, the present invention is not limited to the above-described embodiments and the like, and the gist of the present invention described in the claims is as follows. Design changes are allowed as long as they do not deviate.

Claims (10)

A neuraminidase inhibitor comprising an extraction component obtained by extraction from Ganoderma lingzhi with an organic solvent or an aqueous solvent. Including tripenoids contained in the organic solvent extract as an active ingredient of a neuraminidase inhibitor,
The tripenoids are
Ganoderic acid Z, Ganoderic acid S, Ganoderic acid F, Ganoderic acid SZ, Ganoderic acid LM2, Ganoderic acid LM2, Ganoderic acid DM, Ganoderic acid A, Ganoderic acid AM1, Ganoderic acid B, Ganoderic acid D, Ganodermic acid Y, Ganoderic acid TN, Ganodermic acid TQ, Ganolucidic acid A, Ganodermanontriol, Gano Dermanondiol, ganoderol A, Nodelol B, Ganoderiol F, Lucialdehyde A, Lucialdehyde B, Ganoderic acid C1, Ganoderic acid C2, Ganoderic acid C6, Ganoderic acid H, Ganoderic acid K, Ganoderic acid TR, Ganoderenic acid A, Ganoderin One or more compounds selected from the group consisting of acid C (Ganoderenic acid C), ganoderic acid D (Ganoderenic acid D), ganoderic acid F (Ganoderenic acid F), and ganoderic acid H (Ganoderenic acid H) The neuraminidase inhibitor according to claim 1, wherein   Ganoderic acid Z, Ganoderic acid S, Ganoderic acid F, Ganoderic acid SZ, Ganoderic acid LM2, Ganoderic acid LM2, Ganoderic acid DM, Ganoderic acid A, Ganoderic acid AM1, Ganoderic acid B, Ganoderic acid D, Ganodermic acid Y, Ganoderic acid TN, Ganodermic acid TQ, Ganolucidic acid A, Ganodermanontriol, Gano Dermanondiol, ganoderol A, Nodelol B, Ganoderiol F, Lucialdehyde A, Lucialdehyde B, Ganoderic acid C1, Ganoderic acid C2, Ganoderic acid C6, Ganoderic acid H, Ganoderic acid K, Ganoderic acid TR, Ganoderenic acid A, Ganoderin One or more selected from the group consisting of Acid C (Ganoderenic acid C), Ganoderic acid D, Ganoderenic acid F, and Ganoderenic acid H A neuraminidase inhibitor comprising a tripenoid.   The neuraminidase inhibitor according to claim 1, wherein the aqueous solvent is hot water and the extraction component is a hot water extract. Extract (extract) extracted from Ganoderma lingzhi with an organic solvent or aqueous solvent in an amount of 400 μg / mL or more in terms of organic solvent extract (extract), or an aqueous solvent extract The neuraminidase inhibitor according to claim 1, which is contained in an amount of (extract) 1600 μg / mL or more in a converted amount.   A food (health supplement) or anti-influenza agent for preventing influenza infection or suppressing or reducing the progression of symptoms, comprising the neuraminidase inhibitor according to any one of claims 1 to 5.   The anti-influenza agent according to claim 6, wherein the symptom is pneumonia, acute encephalopathy or Reye syndrome, cardiac complication, acute myositis, otitis media, conjunctivitis, sinusitis, myocarditis prevention).   Bittering agent containing the neuraminidase inhibitor as described in any one of Claims 1-5. A method for producing an anti-influenza agent having an anti-influenza action, comprising a step of extracting a component having neuraminidase inhibitory ability from Ganoderma lingzhi using an organic solvent or an aqueous solvent.   The method for producing an anti-influenza agent according to claim 9, wherein the aqueous solvent is hot water.