JP7748115B2 - Mushroom-derived circadian rhythm regulators - Google Patents

Description

The present invention relates to a mushroom-derived physiologically active substance and its uses. In particular, the present invention relates to a mushroom-derived circadian rhythm regulating substance and food compositions, pharmaceutical compositions, cosmetic compositions, etc. containing the same. This application claims priority to Japanese Patent Application No. 2021-072719, the entire contents of which are incorporated herein by reference.

Circadian rhythms are rhythms that living organisms naturally maintain, with a cycle of approximately 24 hours. Disruptions in circadian rhythms not only cause circadian rhythm sleep disorders (including social circadian rhythm sleep disorders caused by long-distance air travel and shift work), but are also increasingly known to be associated with lifestyle-related diseases and cancer. Numerous studies have been published showing that circadian rhythms affect a wide range of physiological functions, including the intestinal environment, immunity, cognition, learning function, and drug metabolism. These factors have led to an increasing societal need for improving circadian rhythms. To meet this need, researchers are exploring circadian rhythm-regulating substances (e.g., Patent Documents 1 to 4). However, these substances are not necessarily satisfactory in terms of efficacy or safety.

Japanese Patent Application Publication No. 06-72874 Japanese Patent Application Publication No. 11-515014 Japanese Patent Application Laid-Open No. 2003-81829 JP 2010-215561 A

There was a need to discover new substances that have circadian rhythm regulating activity. There was also a need to provide compositions for circadian rhythm regulation containing such substances, and methods for circadian rhythm regulation using such substances.

To solve the above problems, the inventors created a library of extracts from nearly 2,000 species of mushrooms and screened them for circadian rhythm regulating substances. They found that extracts from the edible mushroom Tsuchinako (Cyclocybe erebia) have extremely strong circadian rhythm regulating properties. The inventors then isolated circadian rhythm regulating substances from this extract, determined their structures, and discovered that these substances are the same as the previously undescribed diterpenoid, (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-si The inventors identified these substances as cyclopropa[e]azulene-5,6-diol and (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2'oxirane]-5,6-diol. Furthermore, the inventors confirmed the circadian rhythm-regulating activity of these substances in animal experiments. Thus, the inventors have completed the present invention.

Thus, the present invention provides:
(1) Formula I:
A compound represented by the formula: [wherein R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a ; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen; A is CH ₂ , O, S, or NH; R ^a is hydrogen or C _1-3 alkyl; and R ^b and R ^c are each independently hydrogen or C _1-3 alkyl].
(2) The compound according to (1), wherein R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl or acetyl, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl, and A is CH ₂ .
(3) The compound according to (2), wherein R ¹ , R ² and R ³ are hydrogen, R ⁴ , R ⁵ , R ⁶ and R ⁷ are methyl, and A is CH ₂ .
(4) The compound according to any one of (1) to (3), having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS).
(5) Formula I':
The compound according to (4), which is represented by the formula:
(6) Formula II:
[wherein R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a ; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen; R ^a is hydrogen or C _1-3 alkyl; R ^b and R ^c are each independently hydrogen or C _1-3 alkyl; and n is an integer of 1 to 3.
(7) The compound according to (6), wherein R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl or acetyl, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl, and n is 1 or 2.
(8) The compound according to (7), wherein R ¹ , R ² and R ³ are hydrogen, R ⁴ , R ⁵ , R ⁶ and R ⁷ are methyl, and n is 1.
(9) The compound according to any one of (6) to (8), having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS).
(10) Formula II':
The compound according to (9), which is represented by the formula:
(11) A composition for regulating circadian rhythm, comprising the compound according to any one of (1) to (10).
(12) The composition according to (11), comprising the compound according to (5) and/or the compound according to (10).
(13) A composition for regulating circadian rhythms, comprising a culture of a mushroom of the genus Cyclocybe.
(14) The composition described in (13), wherein the mushroom of the genus Cyclocybe is Tsuchinako.
(15) The composition according to (14), wherein the Tsuchinako mushroom is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453.
(16) The composition according to any one of (11) to (15), which is a food composition, a pharmaceutical composition, or a cosmetic composition.
(17) The composition according to any one of (11) to (16) for improving, treating or preventing health disorders caused by disruption of circadian rhythm.
(18) The composition according to (17), wherein the health disorder is a circadian rhythm sleep disorder.
(19) A method for producing the compound according to any one of (1) to (10), comprising culturing a mushroom of the genus Cyclocybe and purifying the compound according to any one of (1) to (10) from the culture.
(20) The method according to (19), wherein the mushroom of the genus Cyclocybe is Tsuchinako.
(21) The method according to (20), wherein the Tsuchinako mushroom is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453.
(22) The method according to any one of (19) to (21), wherein the compound is the compound according to (5) and/or the compound according to (10).
(23) Tsuchinameko, deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation under the accession number NITE BP-03453.

The present invention further provides:
(24) A method for regulating circadian rhythm, comprising administering the compound according to any one of (1) to (10) to a subject in need of regulating circadian rhythm.
(25) The method according to (24), wherein the compound is the compound according to (5) and/or the compound according to (10).
(26) A method for regulating circadian rhythm, comprising administering a culture of a mushroom of the genus Cyclocybe to a subject in need of circadian rhythm regulation.
(27) The method described in (26), wherein the mushroom of the genus Cyclocybe is Tsuchinako.
(28) The method according to (27), wherein the Tsuchinameko is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453.
(29) The method according to any one of (24) to (28) for improving, treating, or preventing a health disorder caused by a disruption of circadian rhythm.
(30) The method according to (29), wherein the health disorder is a circadian rhythm sleep disorder.

The present invention further provides:
(31) The compound according to any one of (1) to (10) for use in regulating circadian rhythm.
(32) The compound according to (32), which is the compound according to (5) and/or the compound according to (10).
(33) A culture of a mushroom of the genus Cyclocybe for use in circadian rhythm regulation.
(34) The culture described in (33), wherein the mushroom of the genus Cyclocybe is Tsuchinako.
(35) The culture according to (34), wherein the Tsuchinako mushroom is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453.
(36) The compound according to (31) or (32), or the culture according to any one of (33) to (35), for use in improving, treating, or preventing a health disorder caused by disruption of circadian rhythm.
(37) The compound or culture according to (36), wherein the health disorder is a circadian rhythm sleep disorder.

The present invention further provides:
(38) Use of the compound according to any one of (1) to (10) for producing a composition for regulating circadian rhythm.
(39) The use according to (40), wherein the compound is a compound according to (5) and/or a compound according to (10).
(40) Use of a culture of a mushroom of the genus Cyclocybe for producing a composition for regulating circadian rhythm.
(41) The use according to (40), wherein the mushroom of the genus Cyclocybe is Tsuchinako.
(42) The use according to (43), wherein the Tsuchinako mushroom is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453.
(43) The use according to any one of (38) to (42), wherein the composition is used for improving, treating, or preventing a health disorder caused by disruption of circadian rhythm.
(44) The use according to (43), wherein the health disorder is a circadian rhythm sleep disorder.

The present invention provides novel circadian rhythm regulating substances, as well as circadian rhythm regulating compositions containing the same. The circadian rhythm regulating substances of the present invention have a strong circadian rhythm regulating effect. Moreover, because the substances are derived from the edible mushroom Tsuchinomeko, they are highly safe. Therefore, circadian rhythm regulating compositions containing the substances have a strong circadian rhythm regulating effect and are highly safe. Furthermore, the present invention provides a circadian rhythm regulating composition containing a culture of a Cyclocybe mushroom. Because many mushrooms of the Cyclocybe genus, including Tsuchinomeko, are edible, a circadian rhythm regulating composition containing a culture of a Cyclocybe mushroom also has a strong circadian rhythm regulating effect and is highly safe. The compounds of the present invention represented by Formula I and Formula II and their analogs, as well as compositions containing a culture of a Cyclocybe mushroom, are expected to have a significant impact in fields such as food, pharmaceuticals, cosmetics, and chrononutrition.

Figure 1 shows a graph showing the circadian rhythm-regulating activity of the hexane/acetone = 60/40 (v/v) fraction obtained by silica gel column chromatography of an ethyl acetate extract of the culture filtrate of Tsuchinako mushroom (TUFC 32157 strain). The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active fraction was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. Figure 2 is a graph showing the circadian rhythm-regulating activity of the methanol/water = 80/20 (v/v) fraction obtained by subjecting the hexane/acetone = 60/40 (v/v) fraction to ODS column chromatography. The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active fraction was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. The upper panel of Figure 3 is an HPLC chart of the above-mentioned methanol/water = 80/20 (v/v) fraction. The lower panel of Figure 3 is a graph showing the circadian rhythm-regulating activity of the active substance in Fraction 2 purified by preparative HPLC using an ODS column. The horizontal axis of the graph in the lower panel of Figure 3 represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active fraction was added to cells, and the dashed line represents the circadian rhythm when only solvent was added to cells. Figure 4 shows an HPLC chart of the fractions with retention times of 4.5 to 5.0 minutes shown in the upper panel of Figure 3, obtained by using an ODS column with 80% acetonitrile/0.1% acetic acid. Fractions 1 and 2 were collected. Figure 5 shows the ¹ H NMR spectrum (upper panel) and ¹³ C NMR spectrum (lower panel) of the active substance in isolated Fr. 2. 6 is a graph showing the circadian rhythm-regulating activity of the active substance in Fr. 1. The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active substance in Fr. 1 was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. The upper left panel of Figure 7 is an HPLC chart obtained by subjecting Fraction 1 to an ODS column using 50% acetonitrile/0.1% acetic acid. The lower left panel of Figure 7 is an HPLC chart obtained by subjecting Fraction 1-2 to an ODS column using 70% methanol/water. The upper right panel of Figure 7 is a graph showing the circadian rhythm-regulating activity of the active substance in a fraction (Fr. 1-2) obtained by fractionating the peak at a retention time of approximately 11 minutes in the HPLC chart at the upper left of Figure 7. The lower right panel of Figure 7 is a graph showing the circadian rhythm-regulating activity of the active substance in a fraction (Fr. 1-2-6) obtained by fractionating the peak at a retention time of approximately 20 minutes in the HPLC chart at the lower left of Figure 7. In the upper right and upper left panels, the horizontal axis of the graphs represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line indicates the circadian rhythm when an active substance is added to the cells, and the dashed line indicates the circadian rhythm when only the solvent is added to the cells. 8 shows a comparison of ^{the 1} H and ¹³ C NMR spectra of the active material in Fr. 2 and the active material in Fr. 1-2-6. 9 is a graph showing the concentration-dependent amplitude-increasing activity of the active substance in Fr. 2. The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The fine dashed line represents the results for the system in which 3 μg/ml of the active substance in Fr. 2 was added, the coarse dashed line represents the results for the system in which 10 μg/ml of the active substance in Fr. 2 was added, the solid line represents the results for the system in which 30 μg/ml of the active substance in Fr. 2 was added, and the dotted line represents the results for the system in which only the solvent was added. 10 is a graph showing changes in circadian rhythms when the active substance in Fr. 2 is added to cells before the peak of the circadian rhythm. The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active substance in Fr. 2 is added to cells, and the dashed line represents the circadian rhythm when only the solvent is added to cells. 11 is a graph showing the change in circadian rhythm when the active substance in Fr. 2 was added to cells after the peak of the circadian rhythm. The horizontal axis of the graph represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active substance in Fr. 2 was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. Figure 12 is a photograph showing the change in luminescence in the mouse liver at each time point when the active substance in Fr. 2 was administered to Bmal1 reporter mice (GPC Research Institute, Inc.) one hour before the change from light to dark (ZT11) (administration was performed at ZT11 for three days). In the figure, blue indicates low luminescence values, and red indicates high luminescence values. The three mice on the left are the group administered with the active substance in Fr. 2, and the two mice on the right are the control group. 13 is a graph showing the change in luminescence intensity of mouse liver at each time point when the active substance in Fr. 2 was administered to Bmal1 reporter mice at ZT11 (administration was performed at ZT11 for 3 days). The horizontal axis represents the measurement time point, and the vertical axis represents the relative luminescence intensity. The solid line represents the luminescence intensity when the active substance in Fr. 2 was administered to Bmal1 reporter mice, and the dashed line represents the luminescence intensity when only the solvent was administered to Bmal1 reporter mice. Figure 14 is a photograph showing the change in luminescence in the mouse liver at each time point when the active substance in Fr. 2 was administered to Bmal1 reporter mice one hour after the light-to-dark phase (ZT13) (administration was performed at ZT13 for three days). In the figure, blue indicates low luminescence values, and red indicates high luminescence values. The three mice on the left are the group administered with the active substance in Fr. 2, and the two mice on the right are the control group. 15 is a graph showing the change in luminescence intensity of mouse liver at each time point when the active substance in Fr. 2 was administered to Bmal1 reporter mice at ZT13 (administration was performed at ZT13 for 3 days). The horizontal axis represents the measurement time point, and the vertical axis represents the relative luminescence intensity. The solid line represents the luminescence intensity when the active substance in Fr. 2 was administered to Bmal1 reporter mice, and the dashed line represents the luminescence intensity when only the solvent was administered to Bmal1 reporter mice. Figure 16 is a graph showing the circadian rhythm-regulating activity of the active substances in Fr. 1-2-6. The horizontal axis represents measurement time (h), and the vertical axis represents detrended bioluminescence (counts/min). The solid line represents the circadian rhythm when the active substance in Fr. 1-2-6 was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells.

In one aspect, the present invention provides a compound represented by formula I:

The compound of Formula I includes 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol and its analogs. The compound of Formula I also includes its derivatives. The compound of Formula I also includes its structural isomers and stereoisomers. Furthermore, when the compound of Formula I can form salts and solvates, these salts and solvates are also included in the compound of Formula I.

In the compound of formula I, R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a . R ^a is hydrogen or C _1-3 alkyl. Preferably, R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl, or acetyl. More preferably, R ¹ , R ² and R ³ are hydrogen.

C _1-3 alkyl means an alkyl group having from 1 to 3 carbon atoms and includes methyl, ethyl, propyl and isopropyl groups.

In the compound of formula I, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen. ^{R b} and R ^c are each independently hydrogen or C _1-3 alkyl. C _1-3 alkyl is as defined above. Halogen includes fluorine, chlorine, bromine, and iodine. Preferably, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl. More preferably, R ⁴ , R ⁵ , R ⁶ and R ⁷ are methyl.

A is _CH2 , O, S, or NH. Preferably, A is _CH2 .

A preferred compound of formula I is one in which R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl or acetyl, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl, and A is CH _2. A further preferred compound of formula I is one in which R ¹ , R ² and R ³ are each independently hydrogen, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl, and A is CH ₂ , i.e., 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol. The preferred configuration of this compound is (1R,1aS,4aR,5R,6S,7S,7aR,7bS). Thus, one particularly preferred compound of formula I of the present invention is (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol (compound of formula I').

In another aspect, the present invention provides a compound represented by formula II:

The compound of formula II includes 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2'oxirane]-5,6-diol and its analogs. The compound of formula I includes its derivatives. The compound of formula II includes its structural isomers and stereoisomers. Furthermore, if the compound of formula II can form salts and solvates, these salts and solvates are also included in the compound of formula II.

In the compound of formula II, R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a . R ^a is hydrogen or C _1-3 alkyl. Preferably, R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl, or acetyl. More preferably, R ¹ , R ² and R ³ are hydrogen.

C _1-3 alkyl means an alkyl group having from 1 to 3 carbon atoms and includes methyl, ethyl, propyl and isopropyl groups.

In the compound of formula II, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen. ^{R b} and R ^c are each independently hydrogen or C _1-3 alkyl. C _1-3 alkyl is as defined above. Halogen includes fluorine, chlorine, bromine, and iodine. Preferably, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl. More preferably, R ⁴ , R ⁵ , R ⁶ and R ⁷ are methyl.

In compounds of formula II, n is 0, 1, 2, or 3. Preferably, n is 1 or 2. More preferably, n is 1.

A preferred compound of formula II is one in which R ¹ , R ² and R ³ are each independently hydrogen, methyl, ethyl or acetyl, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl or ethyl, and n is 1 or 2. A further preferred compound of formula II is one in which R ¹ , R ² and R ³ are each independently hydrogen, R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently methyl, and n is 1, i.e., 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2′oxirane]-5,6-diol. The preferred configuration of this compound is (1R,1aS,4aR,5R,6S,7S,7aR,7bS). Thus, one particularly preferred compound of formula II of the present invention is (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2′oxirane]-5,6-diol (compound of formula II′).

The compounds of Formula I and Formula II have circadian rhythm-regulating activity. Circadian rhythms are rhythms that living organisms naturally maintain, with a period of approximately 24 hours. As used herein, regulating circadian rhythms includes shortening or lengthening the period of the circadian rhythm, advancing or delaying the phase, or increasing the amplitude.

Disruptions to circadian rhythms not only cause jet lag caused by long-distance air travel, social jet lag caused by shift work, and circadian rhythm sleep disorders, but are also increasingly being linked to lifestyle-related diseases and cancer. Numerous studies have been published showing that circadian rhythms affect a wide range of physiological functions, including intestinal bacteria, immunity, cognitive and learning functions, skin function, energy metabolism, and the absorption, metabolism, and excretion of drugs and nutrients. For these reasons, consumer interest in substances that improve circadian rhythms is extremely high, and societal demand is also rapidly increasing.

By regulating circadian rhythm using a compound of Formula I and/or a compound of Formula II, various diseases and disorders caused by disruption of circadian rhythm can be improved, treated, or prevented. Disorders caused by disruption of circadian rhythm include, but are not limited to, circadian rhythm sleep disorders (e.g., delayed sleep phase syndrome, advanced sleep phase syndrome, non-24-hour sleep-wake syndrome, irregular sleep-wake patterns, shift work sleep disorders, jet lag, etc.), intestinal flora disorders, impaired immune function, impaired cognitive and learning function, drug metabolism disorders, lifestyle-related diseases, and cancer. For example, a compound of Formula I and/or a compound of Formula II may be used to improve, treat, or prevent circadian rhythm sleep disorders. To regulate circadian rhythm, a compound of Formula I or a compound of Formula II may be used, or a compound of Formula I and a compound of Formula II may be used in combination, or a compound of Formula I and a compound of Formula II may be used in combination with other circadian rhythm regulators or sleep-improving agents.

For example, the compound of formula I' can delay the phase of the circadian rhythm in animals (see Example (7)(iii), Figures 12, 13, 14, and 15). Thus, for example, the compound of formula I' may be used to delay the phase of the circadian rhythm. For example, the compound of formula I' may be used to inhibit the body clock from advancing too fast. By inhibiting the body clock from advancing too fast, it is possible to improve lifestyle patterns such as excessively early bedtimes and early rises. Such uses may be particularly useful in elderly people.

In this invention, circadian rhythm regulating substances were screened using cells (GP-M02-P2) derived from a circadian rhythm reporter mouse developed by GPC Laboratories, Inc. This mouse has a luciferase gene inserted downstream of the promoter of the bmal1 gene (bmal1), one of the clock genes that regulates circadian rhythms. By adding a sample to cells derived from this mouse and examining changes in luminescence, it is possible to identify substances that directly affect circadian rhythms. The circadian rhythm regulating activity of the compounds of formula I and formula II of the present invention was confirmed using cells derived from the above mouse, and therefore the compounds of formula I and formula II of the present invention are substances that directly affect circadian rhythms. In contrast, existing sleep-improving products, such as functional foods and supplements, contain amino acids, neurotransmitters, aroma oils, etc., and are thought to have mechanisms of action such as a calming effect on the central nervous system, changes in core body temperature, and stress relief. Therefore, when the compound of formula I and/or the compound of formula II obtained in the present invention is applied to a sleep-improving product, a product having a new mechanism of action that induces natural sleep by improving the balance between day and night biological rhythms and circadian rhythms, which is different from existing sleep-improving products, can be obtained.

Therefore, in another aspect, the present invention provides a composition for regulating circadian rhythm, comprising a compound of formula I and/or a compound of formula II. The composition of this aspect may be used to improve, treat, or prevent health disorders caused by disruption of circadian rhythm. For example, the composition of this aspect may be used to improve, treat, or prevent circadian rhythm sleep disorders.

The composition may be a food composition, a pharmaceutical composition, or a cosmetic composition.

When the composition of the present invention is a food composition, its form is not particularly limited and may be any form, such as solid, semi-solid, or liquid. Foods also include beverages. Food compositions also include health foods and supplements, such as so-called FOSHU and functional food products. Methods for producing food compositions are known to those skilled in the art. Health foods and supplements may be in the form of pharmaceutical compositions, such as tablets, capsules, powders, granules, drinks, drops, etc. Health foods and supplements may be produced by methods similar to those for producing pharmaceutical compositions.

When the composition of the present invention is a pharmaceutical composition, the dosage form is not particularly limited, and examples include oral preparations such as tablets, granules, powders, capsules, drinks, syrups, and drops; topical preparations such as creams, gels, ointments, and pastes; and injections and infusions. Pharmaceutical compositions are formulated with appropriate carriers or excipients. Methods for producing pharmaceutical compositions are known to those skilled in the art.

When the composition of the present invention is a cosmetic composition, its form is not particularly limited and may be, for example, a lotion, cream, gel, emulsion, foundation, shampoo, rinse, soap, body wash, bath additive, etc. Methods for producing cosmetic compositions are known to those skilled in the art.

The intake, administration or use amount of the composition of the present invention can be determined and changed as appropriate while observing the circadian rhythm regulating effect.

The compounds of Formula I and Formula II of the present invention were obtained from a culture of Cyclocybe erebia, a mushroom of the Cyclocybe genus. Accordingly, in another aspect, the present invention provides a composition for circadian rhythm regulation comprising a culture of a mushroom of the Cyclocybe genus. Examples of mushrooms of the Cyclocybe genus include, but are not limited to, Cyclocybe erebia, Cyclocybe aegerita, and Cyclocybe cylindracea. Preferably, the composition of this aspect comprises a culture of Cyclocybe erebia. More preferably, the composition of this embodiment contains a culture of Tsuchinako mushroom deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under Accession Number NITE BP-03453. The composition of this embodiment may be used for improving, treating, or preventing circadian rhythm sleep disorders. The composition of this embodiment may be a pharmaceutical composition, a food composition, or a cosmetic composition.

In the present invention, the culture of a mushroom of the genus Cyclocybe may be any of the whole culture (mycelia and medium), mycelia, or culture filtrate obtained by culturing a mushroom of the genus Cyclocybe in a medium. The mushroom culture may also be an extract obtained by disrupting the mycelia using known means such as a homogenizer. The mushroom culture may be formed into a concentrate (extract), a dried product (e.g., a freeze-dried product), a powder, a pellet, or granules using known means or methods. Preferably, the mushroom culture is a culture filtrate. The culture filtrate is obtained by removing the mycelia from a mushroom culture using a filter or centrifuge. The culture filtrate may be purified using known means or methods such as solvent extraction or chromatography, or may be concentrated using known means or methods such as freeze-drying. In the present invention, the culture of a mushroom of the genus Cyclocybe preferably contains a compound of Formula I and/or a compound of Formula II.

In a further aspect, the present invention provides a method for regulating circadian rhythm in a subject, comprising administering to the subject a compound of formula I and/or a compound of formula II. The regulation of circadian rhythm may be for the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

In a further aspect, the present invention provides a compound of formula I and/or a compound of formula II for use in regulating circadian rhythm in a subject. The regulation of circadian rhythm may be for the amelioration, treatment, or prevention of a circadian rhythm sleep disorder.

In a further aspect, the present invention provides use of a compound of formula I and/or a compound of formula II in the manufacture of a composition for regulating circadian rhythm in a subject. The regulation of circadian rhythm may be for the amelioration, treatment, or prevention of a circadian rhythm sleep disorder.

In a further aspect, the present invention provides a method for regulating circadian rhythm in a subject, comprising administering to the subject a culture of a mushroom of the genus Cyclocybe. The regulation of circadian rhythm may be for the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

In a further aspect, the present invention provides a culture of a mushroom of the genus Cyclocybe for use in regulating circadian rhythm in a subject. The regulation of circadian rhythm may be for the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

In a further aspect, the present invention provides use of a culture of a mushroom of the genus Cyclocybe in the manufacture of a composition for regulating circadian rhythm in a subject. The regulation of circadian rhythm may be the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

In yet another aspect, the present invention provides a method for producing a compound of formula I and/or a compound of formula II, comprising culturing a mushroom of the genus Cyclocybe and purifying the compound of formula I and/or the compound of formula II from the culture.

Methods for culturing mushrooms of the genus Cyclocybe are known. Culture conditions can be appropriately selected taking into consideration the type of mushroom used, the amount of compound of formula I and/or compound of formula II required, and other factors. For example, Tsuchinameko may be cultured in a malt liquid medium at 20-30°C for two weeks to two months. The culture is as described above. Tsuchinameko is preferably used to produce the compound of formula I and/or compound of formula II, and it is even more preferable to use Tsuchinameko deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE BP-03453. When the deposited strain is used, (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol and (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2'oxirane]-5,6-diol are obtained as major products having circadian rhythm regulating activity.

The compound of Formula I and/or the compound of Formula II can be purified from the culture using known means and methods, such as solvent extraction and chromatography. For example, the culture may be extracted with an organic solvent such as ethyl acetate to obtain an active fraction. Examples of chromatography include reverse-phase column chromatography using an ODS column or the like, normal-phase column chromatography using a silica gel column or the like, adsorption chromatography, ion exchange chromatography, and gel filtration chromatography. By performing purification, the compound of Formula I and/or the compound of Formula II may be isolated from the culture, or a fraction enriched in the compound of Formula I and/or the compound of Formula II may be obtained.

In yet another aspect, the present invention provides Tsuchinameko, which has been deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE BP-03453. This fungus can be cultivated to obtain a culture, from which the compound of formula I and the compound of formula II can be obtained.

Unless otherwise specified, terms used in this specification shall be interpreted in the sense commonly understood in the fields of biology, biochemistry, chemistry, pharmacology, medicine, chrononutrition, etc.

The present invention will be explained in more detail and specifically using the following examples, but the examples are not intended to limit the scope of the present invention.

(1) Screening method for circadian rhythm regulators Mushroom strains owned by the Fungus and Mushroom Genetic Resource Research Center, Tottori University, were cultured in liquid, and extracts were prepared from the mycelium and culture filtrate. For strains that are difficult to cultivate artificially, extracts were prepared from fruiting bodies collected in the field.

The experiments used cells (GP-M02-P2) derived from a circadian rhythm reporter mouse developed by GPC Research Institute, Inc. These cells were derived from a mouse in which a luciferase gene was inserted downstream of the promoter of the bmal1 gene (bmal1), a clock gene that forms circadian rhythms. A mushroom extract sample was added to these cells, and changes in luminescence were examined to determine the effect on circadian rhythms.

(2) Screening Results for Circadian Rhythm Regulators 1,760 samples were screened, and 24 samples were found to affect circadian rhythms.

(3) Isolation and Identification of Circadian Rhythm Regulating Substances. The edible mushroom, Tsuchinako (TUFC 32157 strain), was cultured in large quantities, and active substances were isolated and identified. Tsuchinako mushrooms were transferred to malt medium and cultured at 25°C for one month. The culture medium was separated into filtrate and mycelium by suction filtration. The culture filtrate was extracted with ethyl acetate, yielding 450 mg of extract. The ethyl acetate extract exhibited circadian rhythm regulating activity, which was then fractionated by silica gel column chromatography using hexane and acetone, followed by ODS column chromatography using water and methanol. Silica gel column chromatography revealed activity in the hexane/acetone = 60/40 (v/v) fraction (yield: 145.7 mg), and ODS column chromatography revealed activity in the methanol/water = 80/20 (v/v) fraction (yield: 57.6 mg). The circadian rhythm regulating activities of the above fractions are shown in Figures 1 and 2. The fraction with a retention time of 4.5 to 5.0 minutes contained in the methanol/water = 80/20 (v/v) fraction obtained by ODS column chromatography (see the upper panel of Figure 3) was purified by preparative HPLC on an ODS column using 80% acetonitrile/0.1% acetic acid (the HPLC chart is shown in Figure 4). 2.2 mg of the active substance was obtained from the fraction with a retention time of 7 minutes (Fr. 1). 16.9 mg of the active substance was obtained from the fraction with a retention time of 10 minutes (Fr. 2). The circadian rhythm-regulating activity of the active substance in Fr. 2 is shown in the lower panel of Figure 3.

(4) Identification of the Active Substance in Fr. 2 Subsequently, the active substance in Fr. 2 was analyzed by electrospray ionization mass spectrometry (ESI-MS) and high-resolution mass spectrometry (HR ESI-MS), revealing that the molecular weight was _{318 and} the molecular formula was _C20H30O3 .
ESI-MS
Positive: m/z 341[M+Na]+, 382[M+Na+ACN]+
Negative: m/z 318 [M+Cl]-, 363 [M+HCOO]-
HR ESI MS
Positive: m/z 341.2078 C20H30O3Na (Δ=-0.876)
Negative: m/z 353.1890 C20H30O3Cl (Δ=1.181)

Furthermore, one-dimensional and two-dimensional NMR analyses revealed that this compound has the following formula:
This compound was identified as an undescribed diterpenoid, 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol, having the structure shown in Figure 5. The ^1H and ^13C NMR spectra of this compound are shown in the upper and lower panels of Figure 5, respectively.

(5) Determination of the stereostructure of the active substance in Fr. 2 Using the same method as above, Tsuchinako (TUFC 32157 strain) was cultured, and Fr. 2 was obtained from the culture solution. The active substance in Fr. 2 was dissolved in a small amount of toluene by heating, allowed to cool to room temperature, and then further cooled to 4°C to obtain a single crystal. When the single crystal was subjected to X-ray crystal structure analysis, it was found that two molecules of the compound formed a donut-shaped dodecamer with a hollow structure in the crystal due to intermolecular hydrogen bonds, and a toluene molecule was enclosed within it. Furthermore, data processing and structural analysis were performed at 2θ _max = 152.334°, and it was found that 12 molecules of the compound and 7 molecules of toluene were present in the asymmetric unit. The structures of the obtained 12 molecules all matched the predicted structure and had the same stereoconfiguration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS). From these results, it was determined that Fr. The active substance in 2 was identified as (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-7-methylene-1a,2,4a,5,6,7,7a,7b-octahydro-1H-cyclopropa[e]azulene-5,6-diol. The structural formula of this compound, including its stereochemistry, is shown below.

(6) Identification of the Active Substance in Fr. 1 Using the same method as above, Tsuchinako (TUFC 32157 strain) was cultivated, and Fr. 1 was obtained from the culture medium. The circadian rhythm-regulating activity of the active substance in Fr. 1 was examined, and a phase-delaying effect was observed ( FIG. 6 ). When Fr. 1 was applied to an ODS column using 50% acetonitrile/0.1% acetic acid, five peaks were observed on the HPLC chart ( FIG. 7 , upper left panel). A fraction with a retention time of approximately 11 minutes (Fr. 1-2) was collected. The circadian rhythm-regulating activity of the active substance contained in Fr. 1-2 is shown in the upper right panel of FIG. 7 . When Fr. 1-2 was applied to an ODS column using 70% methanol/water, six peaks were observed on the HPLC chart ( FIG. 7 , lower left panel). A fraction with a retention time of approximately 20 minutes (Fr. 1-2-6) was collected. Fr. The circadian rhythm-regulating activity of the active substance contained in Fr. 1-2-6 is shown in the lower right panel of Figure 7. The active substance contained in Fr. 1-2-6 was found to have amplitude-increasing and phase-delaying effects. Structural analysis of the active substance contained in Fr. 1-2-6 was performed.

^{The 1} H NMR and ¹³ C NMR spectra of the active substance in Fr. 2 and the active substance in Fr. 1-2-6 were compared ( FIG. 8 ), and the structure and configuration of the active substance in Fr. 1-2-6 were determined by taking into consideration ¹ H ¹ H COSY, HMQC, HMBC, etc. This compound has the following formula:
It was revealed that the compound was an undescribed diterpenoid, 1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2'oxirane]-5,6-diol, having the structure shown in Figure 1. This compound had the configuration (1R,1aS,4aR,5R,6S,7S,7aR,7bS). From these results, it was confirmed that Fr. The active substance in 1-2-6 was identified as (1R,1aS,4aR,5R,6S,7S,7aR,7bS)-1-(1-hydroxy-4-methylpent-3-en-1-yl)-1,4-dimethyl-1,1a,2,4a,5,6,7a,7b-octahydrospiro[cyclopropa[e]azulene-7,2'oxirane]-5,6-diol. The structural formula of this compound, including its stereochemistry, is shown below.

(7) Circadian rhythm-regulating activity of the active substance (formula I') in Fr. 2 In the following experiments (i), (ii-1), and (ii-2), cells derived from a circadian rhythm reporter mouse (GP-M02-P2) developed by GPC Research Institute, Inc. were used. The basic experimental procedures are described below.
Day 1: Cells (GP-M02-P2) were seeded in a dish for measurement.
Day 2: 24 hours after cell seeding, dexamethasone was used for synchronization for 2 hours. After 2 hours, the medium was replaced with a substrate-containing medium, and rhythm measurement was initiated.
Days 3 to 7: Cell rhythm data was obtained by measuring luminescence for 5 days.

(i) Concentration dependence of circadian rhythm-regulating activity On day 2, rhythm measurement was initiated using medium supplemented with the active substance in Fr. 2. The concentrations of the active substance in Fr. 2 in the medium were 3 μg/ml, 10 μg/ml, and 30 μg/ml. The results are shown in Figure 9. It was confirmed that the active substance in Fr. 2 changed the amplitude in a concentration-dependent manner.

(ii) Differences in effects depending on the timing of addition to cells Experiments were conducted in advance to predict the time when the first peak of the circadian rhythm, which had been continuously measured from the second day onwards, would be reached (it was found that the first peak would occur in the morning of the third day).
(ii-1) Pre-peak addition The active substance in Fr. 2 was added to the medium of the cells being measured 2 hours before the predicted time of peak arrival on day 3, and measurement was resumed. The results are shown in Figure 10. In this experiment, the active substance in Fr. 2 exhibited a phase-delaying effect.
(ii-2) Post-peak addition Four hours after the predicted peak time on day 3, the active substance in Fr. 2 was added to the culture medium of the cells being measured, and measurement was resumed. The results are shown in Figure 11. In this experiment, the active substance in Fr. 2 exhibited an amplitude increase and phase advance effect.
These experiments revealed that the active substance in Fr. 2 could advance or delay the phase, or not cause any change in the phase, depending on the timing of its addition.

(iii) Effects on Mouse Circadian Rhythms Experiments were conducted using mice (Bmal1 reporter mice) (GPC Research Institute, Inc.) in which a luciferase gene was inserted downstream of the promoter of the bmal1 gene (bmal1), one of the clock genes that regulates circadian rhythms. Bmal1 reporter mice were administered the active substance in Fr. 2 (5 mg/kg) one hour before the light-to-dark phase (ZT11) or one hour after the light-to-dark phase (ZT13). The active substance in Fr. 2 was administered at each timing over three days. In both experimental systems, luminescence intensity was measured every four hours for 24 hours, starting from ZT15 (three hours after the light-to-dark phase). A luminescent substrate (D-luciferin) was administered subcutaneously to mice, and luminescence intensity was measured 10 minutes after administration. In control systems, only the solvent (DMSO) was administered.
The results of administering an active substance in Fr. 2 at ZT11 are shown in Figures 12 and 13. The results of administering an active substance in Fr. 2 at ZT13 are shown in Figures 14 and 15. In both experimental systems, phase delay was confirmed.
From the above, it was found that the active substance in Fr. 2 alters the circadian rhythm in animals.

(8) Circadian rhythm regulating activity of the active substance (formula II') in Fr. 1-2-6 A test similar to that in (7)(i) above was carried out. The results are shown in Figure 16. It was found that the active substance in Fr. 1-2-6 exhibited amplitude-increasing and phase-delaying effects.

The present invention is extremely useful in the fields of food, pharmaceuticals and cosmetics, as well as in chrononutrition research.

The Tsuchinako mushroom (TUFC 32157 strain, Fungus and Mushroom Genetic Resource Center, Faculty of Agriculture, Tottori University) described in this specification was deposited at the Patent Microorganism Depositary, Biotechnology Center, National Institute of Technology and Evaluation, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, and was assigned accession number NITE AP-03453 on March 31, 2021. This fungus was assigned accession number NITE P-03453 on April 26, 2021 (deposit date: March 31, 2021) (domestic deposit). Subsequently, this fungus was assigned accession number NITE BP-03453 on March 15, 2022 (deposit date: March 31, 2021) (international deposit).

Claims (21)

Formula I:
A compound represented by the formula: [wherein R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a ; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen; A is CH ₂ , O, S, or NH; R ^a is hydrogen or C _1-3 alkyl; and R ^b and R ^c are each independently hydrogen or C _1-3 alkyl]. 2. The compound according to claim 1, wherein ^R1 , ^R2 and ^R3 are independently of one another hydrogen, methyl, ethyl or acetyl, ^R4 , ^R5 , ^R6 and ^R7 are independently of one another methyl or ethyl, and A is _CH2 . 3. The compound of claim 2, wherein ^R1 , ^R2 and ^R3 are hydrogen, ^R4 , ^R5 , ^R6 and ^R7 are methyl, and A is _CH2 . The compound according to any one of claims 1 to 3, having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS). Formula I':
The compound according to claim 4, which is represented by the formula: Formula II:
[wherein R ¹ , R ² and R ³ are each independently hydrogen, C _1-3 alkyl, or COR ^a ; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently hydrogen, C _1-3 alkyl, OH, COOH, NR ^b R ^c , NO ₂ , SO ₃ H, or halogen; R ^a is hydrogen or C _1-3 alkyl; R ^b and R ^c are each independently hydrogen or C _1-3 alkyl; and n is an integer of 0-3. 7. The compound according to claim 6, wherein ^R1 , ^R2 and ^R3 are independently hydrogen, methyl, ethyl or acetyl; ^R4 , ^R5 , ^R6 and ^R7 are independently methyl or ethyl; and n is 1 or 2. 8. The compound of claim 7, wherein ^R1 , ^R2 and ^R3 are hydrogen, ^R4 , ^R5 , ^R6 and ^R7 are methyl, and n is 1. The compound according to any one of claims 6 to 8, having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS). Formula II':
The compound according to claim 9, which is represented by the formula: A composition for regulating circadian rhythm, comprising a compound according to any one of claims 1 to 10. The composition according to claim 11, comprising the compound according to claim 5 and/or the compound according to claim 10. A composition for regulating circadian rhythms, comprising a culture of Tsuchinako, a mushroom of the genus Cyclocybe. The composition according to claim 13 , wherein the Tsuchinako mushroom is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453. The composition according to any one of claims 11 to 14 , which is a food composition, a pharmaceutical composition, or a cosmetic composition. The composition according to any one of claims 11 to 15 , for improving, treating or preventing health disorders caused by disruption of circadian rhythm. 17. The composition of claim 16 , wherein the health disorder is a circadian rhythm sleep disorder. A method for producing the compound according to any one of claims 1 to 10, comprising culturing a mushroom of the genus Cyclocybe, Tsuchinako , and purifying the compound according to any one of claims 1 to 10 from the culture. The method according to claim 18 , wherein the Tsuchinameko is deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center under the accession number NITE BP-03453. 20. The method of any one of claims 18 or 19 , wherein the compound is a compound according to claim 5 and/or a compound according to claim 10. Tsuchinaeko mushrooms are deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation under accession number NITE BP-03453.