Mushroom-derived circadian rhythm regulators

Abstract

The present invention provides: a compound represented by formula I, formula I', formula II, or formula II'; and a composition that is for regulating circadian rhythm and that contains said compound or an Agrocybe erebia culture.

Description

[Technical Field]

[0001] The present invention relates to a mushroom-derived physiologically active substance and its use. Specifically, the present invention relates to a mushroom-derived circadian rhythm regulating substance and a food composition, pharmaceutical composition, cosmetic composition, 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. [Background technology]

[0002] Circadian rhythms are rhythms that living organisms naturally maintain, with a cycle of approximately 24 hours. It has become clear that disruption of circadian rhythms not only causes circadian rhythm sleep disorders (including social circadian rhythm sleep disorders caused by long-distance air travel or shift work), but is also 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 social need for improving circadian rhythms. To address this need, circadian rhythm-regulating substances have been explored (e.g., Patent Documents 1 to 4). However, these substances are not necessarily satisfactory in terms of efficacy or safety. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 06-72874 [Patent Document 2] Japanese Patent Application Publication No. 11-515014 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-81829 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-215561 Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need to discover new substances that have circadian rhythm regulating activity, and there is also a need to provide compositions for circadian rhythm regulation that contain such substances, methods for circadian rhythm regulation that use such substances, etc. [Means for solving the problem]

[0005] To solve the above problems, the present inventors created a library of extracts from nearly 2,000 mushrooms and screened them for circadian rhythm regulating substances. They found that extracts from the edible mushroom, Cyclocybe erebia, have very strong circadian rhythm regulating properties. They then isolated circadian rhythm regulating substances from this extract, determined their structures, and found that these substances were 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-diterpenoid. The inventors identified these compounds 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 compounds in animal experiments. Thus, the inventors have completed the present invention.

[0006] Thus, the present invention provides: (1) Formula I: [ka] [In the formula, R 1 , R 2 and R 3 are independently hydrogen, C 1-3 Alkyl, or COR a and R 4 , R5 , R 6 and R 7 are independently hydrogen, C 1-3 Alkyl, OH, COOH, NR b R c , NO2, SO3H, or halogen, A is CH2, O, S, or NH, and R a is hydrogen or C 1-3 alkyl, and R b and R c are independently hydrogen or C 1-3 A compound represented by the formula: wherein R is an alkyl group. (2)R 1 , R 2 and R 3 are independently hydrogen, methyl, ethyl or acetyl, and R 4 , R 5 , R 6 and R 7 are each independently methyl or ethyl, and A is CH2. (3)R 1 , R 2 and R 3 is hydrogen and R 4 , R 5 , R 6 and R 7 The compound according to (2), wherein is methyl and A is CH2. (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': [ka] The compound according to (4), (6) Formula II: [ka] [In the formula, R 1 , R 2 and R 3 are independently hydrogen, C 1-3 Alkyl, or COR a and R 4 , R5 , R 6 and R 7 are independently hydrogen, C 1-3 Alkyl, OH, COOH, NR b R c , NO2, SO3H, or halogen, and R a is hydrogen or C 1-3 alkyl, and R b and R c are independently hydrogen or C 1-3 and n is an integer of 1 to 3. (7)R 1 , R 2 and R 3 are independently hydrogen, methyl, ethyl or acetyl, and R 4 , R 5 , R 6 and R 7 are each independently methyl or ethyl, and n is 1 or 2. (8)R 1 , R 2 and R 3 is hydrogen and R 4 , R 5 , R 6 and R 7 is 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': [ka] The compound according to (9), (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 according to (13), wherein the mushroom of the genus Cyclocybe is Tsuchinako. (15) The composition according to (14), wherein the Tsuchinako mushroom is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation 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 described in (19), wherein the mushroom of the genus Cyclocybe is Tsuchinako. (21) The method according to (20), wherein the Tsuchinameko mushroom is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation 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 Microorganisms Deposit Center of the National Institute of Technology and Evaluation under accession number NITE BP-03453.

[0007] 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 mushroom is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation 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.

[0008] 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) Cultures of mushrooms of the genus Cyclocybe for use in circadian rhythm regulation. (34) The culture according to (33), wherein the mushroom of the genus Cyclocybe is Tsuchinako. (35) The culture according to (34), wherein the Tsuchinako mushroom is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation 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.

[0009] 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 rhythms. (41) The use according to (40), wherein the mushroom of the genus Cyclocybe is Tsuchinako. (42) The use according to (43), wherein the Tsuchinameko mushroom is deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE BP-03453. (43) The use according to any one of (38) to (42), wherein the composition is used to improve, treat, or prevent 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. [Effects of the Invention]

[0010] The present invention provides novel circadian rhythm regulating substances and compositions for circadian rhythm regulating containing them. 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 composition for circadian rhythm regulating containing a culture of a mushroom of the genus Cyclocybe. Because mushrooms of the genus Cyclocybe, including Tsuchinomeko, are often edible, a composition for circadian rhythm regulating containing a culture of a mushroom of the genus Cyclocybe 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 mushroom of the genus Cyclocybe, are expected to have a significant impact in fields such as food, medicine, cosmetics, and chrononutrition. [Brief explanation of the drawings]

[0011] [Figure 1]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] Figure 2 shows the circadian rhythm-regulating activity of the methanol / water = 80 / 20 (v / v) fraction obtained by ODS column chromatography of the hexane / acetone = 60 / 40 (v / v) fraction. 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 3] 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 Fr. 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] Figure 4 shows the HPLC chart of the fractions with retention times of 4.5 to 5.0 minutes shown in the upper panel of Figure 3, using an ODS column with 80% acetonitrile / 0.1% acetic acid. Fractions 1 and 2 were collected. [Figure 5] FIG. 5 shows the 1H NMR spectrum (upper panel) and 13C NMR spectrum (lower panel) of the active substance in isolated Fr. 2. [Figure 6]Figure 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. [Figure 7] The upper left panel of Figure 7 is an HPLC chart obtained by subjecting Fr. 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 Fr. 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 upper left HPLC chart 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 lower left HPLC chart of Figure 7. In both the upper right and upper left panels, 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 was added to the cells, and the dashed line represents the circadian rhythm when only solvent was added to the cells. [Figure 8] FIG. 8 shows a comparison of the 1H and 13C NMR spectra of the active substance in Fr. 2 and the active substance in Fr. 1-2-6. [Figure 9] Figure 9 is a graph showing the concentration-dependent amplitude-increasing activity of the active substance in F.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 active substance was added to F.2, the coarse dashed line represents the results for the system in which 10 μg / ml of active substance was added to F.2, the solid line represents the system in which 30 μg / ml of active substance was added to F.2, and the dotted line represents the results for the system in which only solvent was added. [Figure 10]Figure 10 is a graph showing the change in circadian rhythm when the active substance in F.2 was 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 F.2 was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. [Figure 11] Figure 11 is a graph showing the change in circadian rhythm when the active substance in F.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 F.2 was added to cells, and the dashed line represents the circadian rhythm when only the solvent was added to cells. [Figure 12] Figure 12 is a set of photographs 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 carried out 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. [Figure 13] Figure 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]Figure 14 is a photograph showing the change in luminescence in the mouse liver at each time point when the active substance in F.2 was administered to Bmal1 reporter mice one hour after the light-to-dark phase (ZT13) (administration was carried out 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 F.2, and the two mice on the right are the control group. [Figure 15] Figure 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] 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. DETAILED DESCRIPTION OF THE INVENTION

[0012] In one aspect, the present invention provides compounds of formula I:

[0013] 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, the salts and solvates are also included in the compound of formula I.

[0014] In compounds of formula I, R 1 , R 2 and R 3 are independently hydrogen, C 1-3 Alkyl, or COR a R a is hydrogen or C 1-3 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 is hydrogen.

[0015] C 1-3 Alkyl means an alkyl group having 1 to 3 carbon atoms, and includes methyl, ethyl, propyl and isopropyl groups.

[0016] In compounds of formula I, R 4 , R 5 , R 6 and R 7 are independently hydrogen, C 1-3 Alkyl, OH, COOH, NR b R c , NO2, SO3H, or halogen. b and R c are independently hydrogen or C 1-3 It is an alkyl. C 1-3 Alkyl is as described 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 is methyl.

[0017] A is CH2, O, S, or NH. Preferably, A is CH2.

[0018] Preferred compounds of formula I are those having R 1 , R 2 and R 3 are independently hydrogen, methyl, ethyl or acetyl, and R 4 , R 5 , R 6 and R 7 are each independently methyl or ethyl and A is CH. Further preferred compounds of formula I are compounds in which R 1 , R 2 and R 3 is hydrogen and R 4 , R 5 , R 6 and R 7 The compound where A is methyl and A is CH is 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').

[0019] In another aspect, the present invention provides compounds of formula II:

[0020] 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, when the compound of formula II can form salts and solvates, the salts and solvates are also included in the compound of formula II.

[0021] In the compound of formula II, R 1 , R 2 and R 3 are independently hydrogen, C 1-3 Alkyl, or COR a R a is hydrogen or C 1-3 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 is hydrogen.

[0022] C 1-3 Alkyl means an alkyl group having 1 to 3 carbon atoms, and includes methyl, ethyl, propyl and isopropyl groups.

[0023] In the compound of formula II, R 4 , R 5 , R 6 and R 7 are independently hydrogen, C 1-3 Alkyl, OH, COOH, NR b R c , NO2, SO3H, or halogen. b and R c are independently hydrogen or C 1-3 It is an alkyl. C 1-3Alkyl is as described 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 is methyl.

[0024] In compounds of formula II, n is 0, 1, 2, or 3. Preferably, n is 1 or 2. More preferably, n is 1.

[0025] Preferred compounds of formula II are those of R 1 , R 2 and R 3 are independently hydrogen, methyl, ethyl or acetyl, and R 4 , R 5 , R 6 and R 7 are each independently methyl or ethyl and n is 1 or 2. Further preferred compounds of formula II are those in which R 1 , R 2 , and R 3 is hydrogen and R 4 , R 5 , R 6 and R 7is 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′).

[0026] The compound of formula I and the compound of formula II have circadian rhythm regulating activity.Circadian rhythm is the rhythm that organisms naturally have, with a period of about 24 hours.In this specification, regulating circadian rhythm includes shortening or lengthening the period of circadian rhythm, advancing or delaying the phase, or increasing the amplitude.

[0027] It is becoming clear that circadian rhythm disruption not only causes jet lag caused by long-distance air travel, social jet lag caused by shift work, and circadian rhythm sleep disorders, but is also related 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. Due to these factors, consumer interest in substances that improve circadian rhythms is extremely high, and societal demand is also rapidly increasing.

[0028] By regulating circadian rhythm using a compound of Formula I and / or a compound of Formula II, various diseases and disorders caused by circadian rhythm disruption can be improved, treated, or prevented. Disorders caused by circadian rhythm disruption 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 syndrome, etc.), intestinal microbiota disorders, impaired immune function, impaired cognitive and learning function, drug metabolic 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.

[0029] 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, for example, a lifestyle pattern of going to bed too early and getting up too early can be improved. Such use may be particularly useful in elderly people.

[0030] In the present invention, circadian rhythm-regulating substances were screened using cells (GP-M02-P2) derived from a circadian rhythm reporter mouse developed by GPC Research Institute, Inc. A luciferase gene was 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 became 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 mouse, indicating that 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 central nervous system sedation, 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 are 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.

[0031] 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.

[0032] The composition may be a food composition, a pharmaceutical composition, or a cosmetic composition.

[0033] 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. The form of health foods and supplements may be similar to that 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.

[0034] 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, as well as injections and infusions. Pharmaceutical compositions are formulated with suitable carriers or excipients. Methods for preparing pharmaceutical compositions are known to those skilled in the art.

[0035] 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.

[0036] The intake amount, administration amount or use amount of the composition of the present invention can be appropriately determined and changed while observing the circadian rhythm regulating effect.

[0037] 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 Cyclocybe mushrooms. Examples of Cyclocybe mushrooms 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 aspect comprises a culture of Cyclocybe deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under Accession Number NITE BP-03453. The composition of this aspect may be used for improving, treating, or preventing circadian rhythm sleep disorders. The composition of this aspect may be a pharmaceutical composition, a food composition, or a cosmetic composition.

[0038] In the present invention, the culture of a mushroom of the genus Cyclocybe may be any of the whole culture (mycelia and medium), mycelia, and 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 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), powder, pellets, granules, or the like using known means or methods. Preferably, the mushroom culture is a culture filtrate. The culture filtrate is obtained by removing 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.

[0039] 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.

[0040] 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.

[0041] 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 improvement, treatment, or prevention of a circadian rhythm sleep disorder.

[0042] In a further aspect, the present invention provides a method for regulating circadian rhythm in a subject, comprising administering a culture of a Cyclocybe mushroom to the subject. The regulation of circadian rhythm may be for the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

[0043] 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.

[0044] 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 for the improvement, treatment, or prevention of a circadian rhythm sleep disorder.

[0045] 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.

[0046] 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 mushrooms may be cultured in a malt liquid medium at 20-30°C for 2 weeks to 2 months. The culture is as described above. Tsuchinameko mushrooms are preferably used to produce the compound of formula I and / or compound of formula II, and Tsuchinameko mushrooms deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under accession number NITE BP-03453 are even more preferably used. When the deposited strain is used, the main products having circadian rhythm regulating activity are (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.

[0047] The compound of formula I and / or the compound of formula II can be purified from the culture by 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, gel filtration chromatography, and the like. By performing purification, the compound of formula I and / or the compound of formula II may be isolated from the culture, or a fraction rich in the compound of formula I and / or the compound of formula II may be obtained.

[0048] In yet another aspect, the present invention provides Tsuchinako, which has been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation under Accession No. 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.

[0049] Unless otherwise specified, terms used in this specification shall be interpreted as having the meanings commonly understood in the fields of biology, biochemistry, chemistry, pharmacology, medicine, chrononutrition, and the like.

[0050] The present invention will be described in more detail and specifically below with reference to examples, but the examples are not intended to limit the scope of the present invention. [Example]

[0051] (1) Screening method for circadian rhythm regulators Mushroom strains held by the Fungus and Mushroom Genetic Resource Research Center, Tottori University, were cultured in liquid, and extracts were made from the mycelium and culture filtrate. For strains that are difficult to cultivate artificially, extracts were made from fruiting bodies collected in the wild.

[0052] The experiment 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. The mushroom extract sample was added to these cells, and changes in luminescence were examined to determine the effect on circadian rhythms.

[0053] (2) Screening results for circadian rhythm regulators They screened 1,760 samples and found that 24 samples affected circadian rhythms.

[0054] (3) Isolation and identification of circadian rhythm regulators The edible mushroom Tsuchinako (TUFC 32157 strain) was cultured in large quantities, and active substances were isolated and identified. The 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), while ODS column chromatography revealed activity in the methanol / water = 80 / 20 (v / v) fraction (yield 57.6 mg). The circadian rhythm-regulating activities of these fractions are shown in Figures 1 and 2. The fraction with a retention time of 4.5 to 5.0 minutes (see the upper panel of Figure 3) contained in the methanol / water = 80 / 20 (v / v) fraction obtained by ODS column chromatography was purified by preparative HPLC using an ODS column with 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.

[0055] (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), and the molecular weight was found to be 318 and the molecular formula was C 20 H 30 It turned out to be O3. 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)

[0056] Furthermore, one-dimensional and two-dimensional NMR analyses revealed that this compound has the following formula: [ka] It was revealed that the compound is 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, with the structure shown below. 1 H NMR spectrum and 13 The C NMR spectra are shown in the upper and lower panels of Figure 5, respectively.

[0057] (5) Determination of the three-dimensional structure of the active substance in Fr.2 Using the same method as above, Tsuchinako mushroom (TUFC 32157 strain) was cultivated, and Fructose 2 was obtained from the culture solution. The active substance in Fructose 2 was dissolved by heating in a small amount of toluene, then 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 the two molecules of the compound formed a donut-shaped dodecamer with a hollow structure due to intermolecular hydrogen bonding in the crystal, and that a toluene molecule was enclosed inside. Furthermore, 2θ max Data processing and structural analysis at Λ = 152.334° revealed the presence of 12 molecules of the compound and 7 molecules of toluene in the asymmetric unit. The structures of all 12 molecules obtained were consistent with the predicted structure and had the same stereochemistry (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS). Based on these results, the active substance in Fr. 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, including the stereochemistry, of this compound is shown below. [ka]

[0058] (6) Identification of the active substance in Fr.1 Using the same method as above, Tsuchinako mushroom (TUFC 32157 strain) was cultivated, and Fraction 1 was obtained from the culture medium. The circadian rhythm-regulating activity of the active substance in Fraction 1 was examined, and a phase-delaying effect was observed (Figure 6). Fraction 1 was applied to an ODS column using 50% acetonitrile / 0.1% acetic acid, and five peaks were observed on the HPLC chart (Figure 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 Fraction 1-2 is shown in the upper right panel of Figure 7. Fraction 1-2 was applied to an ODS column using 70% methanol / water, and six peaks were observed on the HPLC chart (Figure 7, lower left panel). A fraction with a retention time of approximately 20 minutes (Fr. 1-2-6) was collected. The circadian rhythm-regulating activity of the active substance contained in Fraction 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.The structure of the active substance contained in Fr.1-2-6 was analyzed.

[0059] Active substances in Fr.2 and active substances in Fr.1-2-6 1 H NMR and 13 Comparing the C NMR spectra (Figure 8), 1 H 1 Taking into consideration H COSY, HMQC, HMBC, etc., the structure and configuration of the active substance in Fr. 1-2-6 were determined. This compound has the following formula: [ka] The compound was identified as 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, with the configuration (1R,1aS,4aR,5R,6S,7S,7aR,7bS). Based on these results, the active substance in Fr. 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 three-dimensional structure, is shown below. [ka]

[0060] (7) Circadian rhythm regulating activity of the active substance (formula I') in Fr. 2 In the following experiments (i), (ii-1), and (ii-2), we used cells derived from a circadian rhythm reporter mouse (GP-M02-P2) developed by GPC Research Institute, Inc. The basic experimental procedure is as follows. Day 1: Cells (GP-M02-P2) were seeded onto 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 were obtained by measuring luminescence for 5 days.

[0061] (i) Concentration dependence of circadian rhythm-regulating activity On the second day, 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.

[0062] (ii) Differences in effects depending on the timing of addition to cells A preliminary experiment was conducted to predict the time when the circadian rhythm, which had been measured from the second day onwards, would reach its first peak (it was found that the first peak would occur in the morning of the third day). (ii-1) Pre-peak addition On the third day, the active substance in F.2 was added to the medium of the cells being measured 2 hours before the predicted peak time, and the measurement was resumed. The results are shown in Figure 10. In this experiment, the active substance in F.2 exhibited a phase-delaying effect. (ii-2) Post-peak addition On the third day, 4 hours after the predicted peak time, 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 increased the amplitude and advanced the phase. 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.

[0063] (iii) Effects on mouse circadian rhythms Experiments were performed using mice (Bmal1 reporter mice) (GPC Laboratories, 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). The luminescence substrate (D-luciferin) was administered subcutaneously to mice, and luminescence intensity was measured 10 minutes after administration. Vehicle (DMSO) alone was administered as a control. The results of administering the active substance in Fr. 2 at ZT11 are shown in Figures 12 and 13. The results of administering the 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.

[0064] (8) Circadian rhythm regulating activity of the active substance (formula II') in Fr. 1-2-6 A test similar to that described 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 increase and phase delay effects. [Industrial Applicability]

[0065] The present invention is very useful in the fields of food, medicine and cosmetics, as well as in the field of chrononutrition research. [Accession number]

[0066] The Tsuchinako fungus described herein (TUFC 32157 strain, Fungus and Mushroom Genetic Resource Center, Faculty of Agriculture, Tottori University) 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 the accession number NITE AP-03453 on March 31, 2021. The fungus was assigned the accession number NITE P-03453 on April 26, 2021 (deposit date: March 31, 2021) (domestic deposit). Subsequently, the fungus was assigned the accession number NITE BP-03453 on March 15, 2022 (deposit date: March 31, 2021) (international deposit).

Claims

1. Formula I: 【Chemical 1】 [In the formula, R １ , R ２ and R ３ are each independently hydrogen, C １－３ Alkyl, or COR ａ and 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 , S.O. ３ H, or halogen, and A is CH ２ , O, S, or NH, and R ａ is hydrogen or C １－３ alkyl, and R ｂ and R ｃ are each independently hydrogen or C １－３ The compound represented by the formula: wherein R is an alkyl group.

2. R １ , R ２ and R ３ are 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 2. The compound of claim 1, wherein:

3. R １ , R ２ and R ３ is hydrogen, and R ４ , R ５ , R ６ and R ７ is methyl and A is CH ２ The compound of claim 2, wherein

4. The compound according to any one of claims 1 to 3, having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS).

5. Formula I': 【Chemistry 2】 The compound according to claim 4, which is represented by the formula:

6. Formula II: 【Chemistry 3】 [In the formula, R １ , R ２ and R ３ are each independently hydrogen, C １－３ Alkyl, or COR ａ and 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 , S.O. ３ H, or halogen, and R ａ is hydrogen or C １－３ alkyl, and R ｂ and R ｃ are each independently hydrogen or C １－３ alkyl, and n is an integer of 0-3.

7. R １ , R ２ and R ３ are independently hydrogen, methyl, ethyl or acetyl, R ４ , R ５ , R ６ and R ７ 7. The compound according to claim 6, wherein are each independently methyl or ethyl and n is 1 or 2.

8. R １ , R ２ and R ３ is hydrogen, and R ４ , R ５ , R ６ and R ７ 8. The compound of claim 7, wherein is methyl and n is 1.

9. The compound according to any one of claims 6 to 8, having the configuration (1R, 1aS, 4aR, 5R, 6S, 7S, 7aR, 7bS).

10. Formula II': 【Chemistry 4】 The compound according to claim 9, which is represented by the formula:

11. A composition for regulating circadian rhythm, comprising the compound according to any one of claims 1 to 10.

12. 12. The composition according to claim 11, comprising a compound according to claim 5 and / or a compound according to claim 10.

13. A composition for regulating circadian rhythms, comprising a culture of Tsuchinako, a mushroom of the genus Cyclocybe.

14. 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.

15. The composition according to any one of claims 11 to 14, which is a food composition, a pharmaceutical composition, or a cosmetic composition.

16. 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. 17. The composition of claim 16, wherein the health disorder is a circadian rhythm sleep disorder.

18. 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.

19. 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. 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.

21. Tsuchinameko is deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation under accession number NITE BP-03453.

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