JP7538495B2 - Circadian rhythm regulators - Google Patents

Description

The present invention relates to a circadian rhythm regulator that contains specific triterpenes as active ingredients.

Many living organisms, including humans, have a 24-hour behavioral pattern called the circadian rhythm, and all physiological phenomena, including not only the sleep-wake cycle but also body temperature, blood pressure, hormone secretion, and autonomic nerve activity, fluctuate according to this rhythm. The mechanism that generates the circadian rhythm is called the body clock, and a group of genes called clock genes synchronize with each tissue (this is called entrainment), and repeat expression fluctuations in a 24-hour cycle, creating a circadian metabolic rhythm downstream. For example, in plants, various activities such as growth, flower formation, stomata opening and closing, and nitrogen metabolism show circadian fluctuations under the control of these clock genes, and attempts have been made to regulate these by controlling the expression of clock genes (Patent Document 1). In mammals, a 24-hour rhythm is created by transcription-translation regulation with the negative feedback of clock genes PER, CRY, BMAL, and CLOCK as the core loop. Mammalian body clocks are broadly divided into a central clock (suprachiasmatic nucleus) that determines activity times from light and dark information, and peripheral clocks that exist in various peripheral tissues. Generally, the central clock and peripheral clock maintain a constant phase relationship and beat in rhythm; however, it has been reported that the peripheral clock can be influenced independently of the central clock by stimuli such as glucocorticoids and insulin, as well as by certain food components (Non-patent literature 1 to 3).
In recent years, it has been pointed out that physical and mental disorders are caused by irregular activities and meals due to shift work and the habit of staying up late (Non-Patent Document 4). In addition, it is well known that jet lag occurs due to long-distance travel by airplane, as a familiar example. One of the common factors in these is that when a discrepancy occurs between the actual sleep and meal times and the rhythms set by the central and peripheral clocks, the time it takes to correct it varies from tissue to tissue. If the circadian rhythm can be adjusted and kept normal, the disharmony of the body clock can be improved. In particular, in order to suppress the long-term adverse effects on health such as the former, it is desirable to use a circadian rhythm regulator that is in the form of a highly safe food that can be taken daily and can effectively suppress circadian rhythm disturbances.

To adjust circadian rhythms, especially to deal with jet lag, airlines take measures such as changing the lighting hours and timing meals closer to the destination. Melatonin is also prescribed as a medicine in Japan. Melatonin is an endogenous hormone that induces sleep, and shows a diurnal variation in which its secretion increases at night and decreases before waking in the morning. To treat sleep disorders caused by circadian rhythm disruptions, melatonin precursors, receptor agonists, and melatonin secretion rhythm improvers are used to regulate the melatonin rhythm.
However, since melatonin is an endogenous hormone, it must be used with caution, and it is difficult to say that it has a specific circadian rhythm regulating effect. In addition, it has been pointed out that serotonin, a precursor of melatonin, has adverse effects on the digestive and circulatory systems when orally ingested, and these substances have not been approved as foods in Japan.

To solve the above problems, the biological clock regulating function of food-derived or food-usable ingredients has been actively evaluated. For example, oleacein, a polyphenol contained in olives, regulates circadian rhythms by regulating the expression of clock genes BMAL1 and PER1 (Patent Document 2). Furthermore, alkylresorcinols derived from wheat bran regulate circadian rhythms by changing the expression rhythm of the clock gene BMAL1 and altering its period (Patent Document 3). It has also been reported that cryptoxanthin extracted from Unshu mandarins and/or its esters have the ability to regulate circadian rhythms (Patent Document 4).

Triterpenes are a group of compounds widely distributed in the plant kingdom, and are a type of terpene composed of six isoprenes and having the molecular formula C ₃₀ H _48. Triterpenes include many compounds, such as those without a ring structure, such as squalene, compounds with five ring structures, such as oleanane type, ursane type, lupane type, hopane type, selatan type, friedelane type, taraxerane type, taraxastan type, multiflorane type, and germanican type, and compounds with four ring structures, such as cucurbitane type, dammarane type, lanostane type, and protostane type. Hopane type triterpenes include celastrol, hopane, pristimerin, etc., and cucurbitane type triterpenes include cucurbitacins A to L, O to T, and cuguacins A to S, etc.

The hopane triterpene celastrol (2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydropicene-2-carboxylic acid; CAS 34157-83-0) is contained in Laikoto (Formosan Crane), a traditional Chinese medicine used to treat inflammation, swelling, and psoriasis. It has high antioxidant properties and is expected to have therapeutic effects on obesity and neurodegenerative diseases, as well as anti-inflammatory properties (Non-Patent Document 5). Pristimerin (methyl(2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydropicene-2-carboxylate; CAS 1258-84-0) is a triterpenoid quinone methide that can be isolated from plants of the Celastraceae family (Gymnosporia heterophylla) and the like, and exhibits activity as an anticancer agent and an antiviral agent (Non-Patent Document 6), and is also known to have an effect as a contraceptive (Non-Patent Document 7). Cucurbitacin B ([(E,6R)-6-[(2S,8S,9R,10R,13R,14S,16R,17R)-2,16-dihydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-2,7,8,10,12,15,16,17-octahydro-1H-cyclopenta[a]phenentren-17-yl]-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl]acetic acid salt; CAS 6199-67-3), cucurbitacin D ((2S,8S,9R,10R,13R,14S,16R,17R)-17-[(E,2R)-2,6-dihydroxy-6-methyl-3-oxohept-4-en-2-yl]-2,16-dihydroxy-4,4,9,13,14-pentamethyl-2,7,8,10,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthrene e-3,11-dione; CAS 3877-86-9), cucurbitacin E ([(E,6R)-6-[(8S,9R,10R,13R,14S,16R,17R)-2,16-dihydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-8,10,12,15,16,17-hexahydro-7H-cyclopenta[a]phenanthrene -17-yl]-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl]acetate; CAS 18444-66-1), cucurbitacin I ((8S,9R,10R,13R,14S,16R,17R)-17-[(E,2R)-2,6-dihydroxy-6-methyl-3-oxohept-4-en-2-yl]-2,16-dihydroxy-4,4,9,13,14-pentamethyl-8,10,12,15,16,17-hexahydro-7H-cyclopenta[a]phenanthrene e-3,11-dione; CAS 2222-07-3), cucurbitacin S ((1S,2S,4R,6S,9S,10R,11R,14R,15R)-17-hydroxy-6-(2-hydroxypropan-2-yl)-2,9,11,14,19,19-hexamethyl-5-oxapentacyclo[12.8.0.02,11.04,10.015,20]docosa-16,20-diene-8,13,18-trione; CAS 60137-06-6), isocucurbitacin B ([(E,6R)-6-[(3R,8S,9R ,10R,13R,14S,16R,17R)-3,16-dihydroxy-4,4,9,13,14-pentamethyl-2,11-dioxo-3,7,8,10,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthren-17-yl]-6-hydroxy-2-methyl-5-oxohept-3-en-2-yl]acetate; CAS 17278-28-3) is a cucurbitane-type triterpene that is expected to have anticancer effects, and is one of the bitter components contained in plants of the Cucurbitaceae family in general (Non-Patent Document 8). Among triterpenes, it is known that oleanane-type triterpenes such as corosolic acid, maslinic acid, and oleanolic acid, and ursane-type triterpenes such as ursolic acid are effective in regulating circadian rhythms (Patent Document 5), but it was not previously known that hopane-type triterpenes and cucurbitane-type triterpenes are effective in regulating circadian rhythms.

Patent Publication No. 2016-40231 Patent Publication 2016-183189 Patent Publication 2009-183197 International Publication No. 2002/043736 Patent Publication No. 2019-43912

Balsalobre A et al., Science, 2000 Sep;289(5488):2344-7 Oishi K et al., Biochem Biophys Res Commun. 2017 Jan 29;483(1):165-170 Oike H, Biosci Biotechnol Biochem. 2017 May;81(5):863-870 Brum MC et al., Diabetol Metab Syndr. 2015 May 17;7:45 R Cascao et al., Front Med, 2017 Jun 15;4:69. Murayama T et al., Antivir Chem Chemother 2007 18 (3): 133-139 Mannowetza N et al., PNAS May 30, 2017 114 (22) 5743-5748 S Garg et al., Int J Oncol. 2018 Jan;52(1):19-37. Balsalobre A, Cell. 1998 Jun 12;93(6):929-37

The objective of the present invention is to provide a composition that regulates the circadian rhythm of humans and animals by regulating the expression level of mammalian clock genes and the phase or period length of the expression rhythm of clock genes, thereby preventing various symptoms caused by disturbance of the circadian rhythm.

The present inventors have discovered that hopane-type triterpenes and cucurbitane-type triterpenes are effective in regulating circadian rhythms by regulating the expression levels of mammalian clock genes and the phase or period length of the expression rhythm of clock genes.
That is, the present invention is as follows.
[1] A circadian rhythm regulating agent comprising, as an active ingredient, one or more selected from the group consisting of hopane-type triterpenes, cucurbitane-type triterpenes, and pharmacologically acceptable salts thereof.
[2] The circadian rhythm regulating agent of claim 1, which regulates the expression level, phase of the expression rhythm, or period length of the expression rhythm of PER2.
[3] The circadian rhythm regulator described in [1] or [2] above, wherein the hopane-type triterpene is one or more selected from the group consisting of celastrol and pristimerin, and the cucurbitane-type triterpene is one or more selected from the group consisting of cucurbitacin B, cucurbitacin D, cucurbitacin E, cucurbitacin I, cucurbitacin S and isocucurbitacin B.
[4] A food or drink for circadian rhythm regulation, comprising the circadian rhythm regulating agent according to any one of [1] to [3] above.
[5] A circadian rhythm regulating animal feed comprising the circadian rhythm regulating agent according to any one of [1] to [3] above.
[6] A pharmaceutical composition for preventing, alleviating, or treating a disease mediated by the regulation of circadian rhythm, comprising the circadian rhythm regulator according to any one of [1] to [3] above.
[7] A pharmaceutical composition for animals for preventing, alleviating, or treating a disease mediated by the regulation of circadian rhythm, comprising the circadian rhythm regulating agent according to any one of [1] to [3] above.

According to the present invention, it is possible to provide a composition that can regulate the expression level, phase or period length of the expression rhythm of mammalian clock genes, particularly PER2, and can regulate the circadian rhythm, thereby preventing various symptoms caused by circadian rhythm disturbance. The composition of the present invention can move the circadian rhythm forward or backward depending on the timing of administration. The body clock can be disrupted by external stimuli (food, strong light, time difference, etc.), and it is known that there are time periods when the rhythm tends to advance or retard depending on the timing of the stimulus. The triterpene of the present invention moves the rhythm not only in one direction but also in both directions, so that it can effectively regulate a disturbed circadian rhythm.

The figure shows the change in the circadian rhythm of human PER2 expression when triterpenes ((A) celastrol, (B) cucurbitacin B, (C) glycyrrhetinic acid) were added at the times indicated by the arrows. Changes in the circadian rhythm of PER2 expression due to the addition of 16 randomly selected terpenes (including two diterpenes) are shown in Fig. 1A, showing the phase of the second peak that appears after sample addition, and in Fig. 1B, showing the phase of the third peak that appears after sample addition. 4 shows the concentration dependence of the phase-delaying effect of (A) celastrol and (B) cucurbitacin B. The effect of addition of (A) celastrol or (B) cucurbitacin B on the phase of the circadian rhythm at various times (15, 21, 27, and 33 hours) after serum stimulation is shown. The figure shows changes in the circadian rhythm of mouse PER2 expression when triterpenes ((A) celastrol, (B) cucurbitacin B) were added at the times indicated by the arrows. The figure shows changes in the circadian rhythm of human PER2 expression when celastrol and pristimerin were added at the times indicated by the arrows. The figure shows changes in the circadian rhythm of mouse PER2 expression when cucurbitacin D, cucurbitacin E, or cucurbitacin I was added at the times indicated by the arrows. The figure shows the change in the circadian rhythm of mouse PER2 expression when cucurbitacin S was added at the time indicated by the arrow. The figure shows changes in the circadian rhythm of mouse PER2 expression when isocucurbitacin B was added at the times indicated by the arrows. The effect of adding different concentrations of celastrol at the times indicated by the arrows in the figure is to enhance the expression level of mouse PER2 (A) and shorten the period length (B). The effect of enhancing the expression level of mouse PER2 when different concentrations of pristimerin were added at the timing indicated by the arrows in the figure.

The circadian rhythm regulating agent of the present invention contains, as an active ingredient, one or more selected from the group consisting of hopane-type triterpenes, cucurbitane-type triterpenes, and pharmacologically acceptable salts thereof.
In the present invention, triterpene is a compound having a basic skeleton of 30 carbon atoms, and representative examples thereof include pentacyclic triterpenes and tetracyclic triterpenes. Here, the pentacyclic triterpene and tetracyclic triterpene are a type of triterpene, which are pentacyclic and tetracyclic compounds, respectively, consisting of six isoprene units, and have a basic carbon number of 30, but also include those with a larger or smaller carbon number due to rearrangement, oxidation, elimination, or alkylation during the biosynthesis process.
Pentacyclic and tetracyclic triterpenes are generally classified according to their skeletons. Examples of pentacyclic triterpenes include oleanane-type triterpenes, ursane-type triterpenes, lupane-type triterpenes, hopane-type triterpenes, seratan-type triterpenes, friedelane-type triterpenes, taraxerane-type triterpenes, taraxastane-type triterpenes, multiflorane-type triterpenes, and germanicane-type triterpenes. Examples of tetracyclic triterpenes include cucurbitane-type, dammarane-type, lanostane-type, and protostane-type.
Among the pentacyclic triterpenes, representative examples of hopane-type triterpenes include celastrol, hopane, pristimerin, etc., and examples of cucurbitane-type triterpenes include cucurbitacins A to L, O to T, cuguacins A to S, etc.

Celastrol is a hopane-type triterpene and is contained in plants such as Formosan crane and Celastrus regelii (a species closely related to Celastrus regelii).
Pristimerin is a triterpenoid quinone methide that can be isolated from plants of the Celastraceae family (Gymnosporia heterophylla) and the like. Pristimerin is a methyl ester of celasterol and is classified as a hopane-type triterpene.

Examples of cucurbitane-type triterpenes include cucurbitacin B, cucurbitacin D, cucurbitacin E, cucurbitacin I, cucurbitacin S, and isocucurbitacin B, which are contained in plants of the Cucurbitaceae family (melon, bottle gourd, snake gourd, loofah, bitter melon, Cucurbita pepo, colocynth, ruffa, etc.).

In the present invention, the hopane-type triterpene is preferably one or more selected from the group consisting of celastrol and pristimerin. The cucurbitane-type triterpene is preferably one or more selected from the group consisting of cucurbitacin B, cucurbitacin D, cucurbitacin E, cucurbitacin I, cucurbitacin S, and isocucurbitacin B.

Hopane-type triterpenes and cucurbitane-type triterpenes can be obtained by extraction and purification from natural plants known to contain them by known methods, or can be artificially obtained by chemical synthesis. They can also be used in the form of pharmacologically acceptable salts and/or derivatives. Commercially available products can also be used suitably.
The hopane-type triterpenes and cucurbitane-type triterpenes may be used in the form of plant extracts.

When used in the form of a plant extract, hopane-type triterpenes and cucurbitane-type triterpenes can be obtained by a method suitable for the plant used as the raw material. For example, celastrol can be obtained by using Formosan Crane as the extraction raw material and the method disclosed in Kutney et al., Can. J. Chem. 59:2677, 1981. Cucurbitacin B can be obtained by using a Cucurbitaceae plant as the extraction raw material, crushing the plant body, and extracting it with an appropriate organic solvent such as a lower alcohol (ethanol, methanol, etc.) or its aqueous alcohol, a ketone (acetone), or an ester (diethyl ether, ethyl acetate, acetonitrile).

The amount of hopane-type triterpenes and cucurbitane-type triterpenes that a human should take varies depending on the age, sex, severity of symptoms, and form of intake of the patient, but for adults (body weight approximately 60 kg), the upper limit for hopane-type triterpenes per day is preferably 5,000 mg, more preferably 1 mg to 500 mg, and the upper limit for cucurbitane-type triterpenes is preferably 10 mg, more preferably 0.01 mg to 1 mg.

The amount of hopane-type triterpenes and cucurbitane-type triterpenes that an animal should take will vary depending on the type, age, sex, severity of symptoms, and form of intake. For example, in a mouse (body weight approximately 30 g), the upper limit for hopane-type triterpenes per day is preferably 30 mg, more preferably 6 μg to 3 mg, and the upper limit for cucurbitane-type triterpenes is preferably 60 μg, more preferably 0.06 to 6 μg.

The circadian rhythm regulator of the present invention may be in the form of a pharmaceutical composition, and may be formulated together with an auxiliary agent into any form to produce a pharmaceutical product that can be administered orally or parenterally. The subject of administration of the pharmaceutical composition is not limited to humans, and includes veterinary pharmaceutical compositions intended for administration to animals. For example, oral dosage forms may include solid dosage forms such as tablets, orally rapidly disintegrating tablets, capsules, granules, and fine granules, and liquid dosage forms such as syrups and suspensions. Parenteral dosage forms may be administered in the form of injections, eye drops, nasal drops, patches, suppositories, and topical skin preparations. Note that pharmaceutical products also include quasi-drugs.

When the solid dosage form is used, it may contain appropriate amounts of various additives used in the manufacture of general pharmaceutical preparations. Such additives include, for example, excipients, binders, acidulants, foaming agents, artificial sweeteners, flavors, lubricants, colorants, stabilizers, pH adjusters, surfactants, etc.

When formulated in a liquid dosage form, the circadian rhythm regulating agent of the present invention can be formulated by conventional methods in the presence of pH adjusters, buffers, solubilizers, suspending agents, isotonicity agents, stabilizers, preservatives, etc., as necessary.

The form of the skin topical preparation is not particularly limited, and can be, for example, cosmetics such as emulsions, creams, lotions, packs, dispersions, cleansers, makeup cosmetics, scalp and hair products, and pharmaceuticals such as ointments, creams, and topical liquids. In addition to the above ingredients, ingredients normally used in skin topical preparations such as cosmetics and pharmaceuticals, such as whitening agents, moisturizers, various skin nutrient ingredients, UV absorbers, antioxidants, oily ingredients, surfactants, thickeners, alcohols, coloring agents, water, preservatives, fragrances, etc., can be appropriately blended as needed.

The circadian rhythm regulator of the present invention may be prepared as a functional food, health food, food for specified health uses, nutritional functional food, or other health functional food, special purpose food (e.g., food for the sick), dietary supplement, supplement, etc. As a supplement, it can be in the form of, for example, a tablet, pill, capsule (including hard capsule, soft capsule, and microcapsule), powder, granule, fine granule, lozenge, liquid (including syrup, milk, and suspension) together with various additives used in the manufacture of general supplements.

The circadian rhythm regulator of the present invention can be incorporated into food and beverages. There is no particular limitation on the foods that can be incorporated, but examples include confectionery such as candy, gummies, and chewing gum; confectionery such as cookies, crackers, biscuits, chocolate, pudding, jelly, snacks, rice crackers, buns, and yokan; cold desserts such as ice cream, popsicles, sorbets, and gelato; bakery foods such as donuts, cakes, bread, French bread, and croissants; noodles such as udon, soba, Chinese noodles, and Kishimen; rice dishes such as white rice, red rice, and pilaf; sauces such as curry, stew, and dressing; paste products such as ham, sausage, kamaboko, chikuwa, and fish sausage; various side dishes such as tempura, croquettes, and hamburger steaks; and beverages such as juice and tea.

The circadian rhythm regulator of the present invention can be added to animal feed. It may be added to any animal feed, or may be added to the raw materials during the feed manufacturing process. When used as animal feed, the amount of addition is not particularly limited and may be determined appropriately depending on the type of animal feed.

The circadian rhythm regulator of the present invention can regulate the expression level, phase of the expression rhythm, or period length of the expression rhythm of a clock gene. In this specification, the "expression rhythm" of a gene refers to a rhythm in which the gene repeats activation and inhibition of expression at a certain period, and "regulating the phase of the expression rhythm of a clock gene" refers to delaying (retreating) or advancing (advancing) the phase of the expression rhythm (or the period of the expression rhythm) in a chronological order when the clock gene shows activation and inhibition of expression along a certain expression rhythm (or the period of the expression rhythm). Therefore, a preferred embodiment of "regulating the phase of the expression rhythm" of a clock gene is to regulate by delaying or advancing the phase along the time axis by the amount of the phase difference compared to the original expression rhythm (or the period of the expression rhythm). "Regulating the period length of the expression rhythm" of a clock gene refers to lengthening or shortening the period length when the clock gene shows activation and inhibition of expression along a certain expression rhythm (or the period of the expression rhythm). The circadian rhythm improving agent of the present invention can adjust, for example, the phase of the expression rhythm of PER2, one of the clock genes, by delaying or advancing it along the time axis, or by lengthening or shortening the period length of the expression rhythm.

Clock genes are genes that govern circadian rhythms. Examples of clock genes include PER (PER1, PER2, etc.), CRY (CRY1, CRY2, etc.), BMAL (BMAL1, etc.), and CLOCK (CLOCK). These genes are found in various animals, including humans and mice, and are known to be expressed in almost all cells of the body.
Circadian rhythms are generated by the expression fluctuations between the above clock genes. Clock genes, which are expressed in almost all cells of the body, repeat expression fluctuations in a 24-hour cycle, synchronizing with each tissue (this is called entrainment), and create a metabolic rhythm downstream. The 24-hour rhythm is generated by transcription-translation regulation with the negative feedback of clock genes as the core loop. By controlling the expression rhythm of PER2, which plays a central role, the expression of a series of clock genes can be controlled, and as a result, it is possible to control the circadian rhythm.

For example, shift work can severely disrupt circadian rhythms, which can be a risk factor for obesity and cancer (van Drongelen et al., Scand J Work Environ Health 2011;37(4):263-275, Wegrzyn LR et al., Am J Epidemiol. 2017 Sep 1;186(5):532-540). In addition, people with disrupted circadian rhythms are more likely to develop symptoms such as major depression, bipolar disorder, significant loneliness, lack of happiness, health anxiety, poor reaction time, and mood instability (Jones, Lane et al., Nat Commun. 2019 Jan 29;10(1):343). Furthermore, it has been reported that people who continue to live a nocturnal circadian rhythm are at higher risk of diabetes, neurological disorders, mental disorders, and digestive and respiratory diseases than morning people (Knutson and von Schantz et al., Chronobiol Int. 2018 August; 35(8): 1045-1053). Since the circadian rhythm regulator of the present invention can regulate circadian rhythm, it can be used to prevent, alleviate, and treat diseases mediated by circadian rhythm regulation. Examples of such diseases include, but are not limited to, sleep disorders, insomnia, jet lag, cancer, obesity, hypertension, cerebral infarction, cerebral hemorrhage, coronary spasm angina, autonomic imbalance, manic depression, depression, major depression, bipolar disorder, severe loneliness, lack of happiness, health anxiety, deterioration of reaction time, mood instability, digestive and respiratory diseases, neurological disorders, neurological disorders, and diabetes.

The following examples are provided to specifically explain the present invention, but the present invention is not limited to the following examples.

Test Example 1: Circadian rhythm regulating effect of celastrol, pristimerin, cucurbitacin B, cucurbitacin D, cucurbitacin E, cucurbitacin I, cucurbitacin S, or isocucurbitacin B. Focusing on PER, one of the representative clock genes, the expression rhythm of human PER2 was used as an indicator of circadian rhythm to examine the circadian rhythm regulating function of triterpenes. Using the expression rhythm of human PER2 as an indicator of circadian rhythm has been established as a system for evaluating the regulating effect of circadian rhythm, and is disclosed in, for example, Isojima et al., PNAS 2009 Sep;106(37):15744-15749, Hirota et al., Science 2012 Aug;337:1094-1097, etc.

(Test substance)
Celastrol (Tokyo Chemical Industry Co., Ltd., C2737), pristimerin (MedChemexpress, HY-N1937), cucurbitacin B (Tokyo Chemical Industry Co., Ltd., C3442), cucurbitacin D (ChemFaces, CFN90209), cucurbitacin E (Cayman Chemical, 14821), cucurbitacin I (Cayman Chemical, 14747), cucurbitacin S (ChemFaces, CFN90535), and isocucurbitacin B (ChemFaces, CFN91003) were commercially available products.
Each reagent was dissolved in dimethyl sulfoxide (DMSO; Wako, 048-21985) so that the final DMSO concentration was 0.1% when each reagent was added to the cells.

(Test Method)
As the test system, human osteosarcoma-derived U2OS cells (U2OS-PER2-Luc) stably expressing a circadian rhythm reporter in which a luciferase gene was incorporated under the human PER2 promoter, mouse-derived 3T3 cells (3T3-PER2-Luc) stably expressing a circadian rhythm reporter in which a luciferase gene was incorporated under the mouse PER2 promoter, or cultured tissue pieces of PER2::LUC knock-in mice in which a luciferase gene was knocked in downstream of mouse PER2 were used. The change in luminescence value due to the luciferin-luciferase reaction represents the change in transcriptional activity of PER2. Luminescence was measured for several days, and a continuous luminescence waveform was obtained as an index of circadian rhythm. PER2::LUC is a fusion protein in which luciferase is linked to the C-terminus of PER2.
(1) U2OS-PER2-Luc cells or 3T3-PER2-Luc cells were seeded on a 35 mm dish and cultured until confluent in 2 mL of DMEM medium (high glucose) supplemented with 10% fetal bovine serum and penicillin-streptomycin.
(2) The cells were cultured for 2 hours in DMEM medium supplemented with 50% horse serum to synchronize the rhythm of the cells (Non-Patent Document 9). This 2-hour culture in medium containing 50% horse serum is hereinafter referred to as serum stimulation. After serum stimulation, the medium was replaced with 2 mL of DMEM medium supplemented with 2 μL of 0.1 M luciferin and penicillin-streptomycin (final concentration: 0.1 mM luciferin), and luminescence was measured continuously for more than 3 days under conditions of 37°C and 5% _CO2 . For the measurement, a luminescence measuring device Kronos dio from ATTO was used. The amount of luminescence was measured at 10-minute intervals as an integrated value for 1 minute.
(3) After dissection, the PER2::LUC knock-in mice were carefully transferred to 1 mL of DMEM medium (High glucose) (final concentration: 0.1 mM luciferin) containing 1 μL of penicillin-streptomycin and 0.1 M luciferin, and luminescence was measured continuously for more than 3 days under conditions of 37°C and 5% _CO2 . For measurement, the luminescence measuring device Kronos dio from ATTO was used, as in the case of cells. The amount of luminescence was measured at 10-minute intervals as an integrated value for 1 minute.
(4) During the measurement, the device was stopped and a reagent was added to the medium. After the addition, the device was restarted and the luminescence was continuously recorded.
(5) Using the analysis system installed in the Kronos dio, noise was removed from the waveform of the time-dependent change in the obtained luminescence value, and the luminescence rhythm was obtained by detrending. The phase of the obtained luminescence rhythm was determined and compared with a control group (one to which an equal amount of dimethyl sulfoxide, which was used as the solvent, was added).

(result)
The phase of the circadian rhythm is characterized by the time of the peak of the waveform shown by the luminescence (horizontal axis: time elapsed since the start of luminescence measurement with Kronos). Immediately after the addition of the reagent, the luminescence is somewhat disturbed due to the stimulation of the addition, so the phase was analyzed based on the peak phase after the luminescence value recovered. When celastrol (final concentration 0.5 μM) or cucurbitacin B (final concentration 0.1 μM) were added 24 hours after serum stimulation and the peak phases obtained after several days of measurement were compared, the phase of the circadian rhythm was delayed by up to about 10 hours with celastrol and up to about 5 hours with cucurbitacin B (Figure 1). Figure 1 shows representative waveform data at this time and waveform data of glycyrrhetinic acid (final concentration 5 μM) as an example of the same triterpenes that do not affect the phase. The reagent was added at the timing indicated by the arrow in the figure.
When a similar test was performed with 16 randomly selected terpenes (including two diterpenes), the phase of the second and third peaks appearing after the addition of serum was significantly delayed by two triterpenes, celastrol and corosolic acid (n=6-10, analyzed by one-way analysis of variance followed by Tukey HSD test). In addition, cucurbitacin B showed a tendency to delay the third peak (p=0.079) (Figure 2). As for corosolic acid, its phase mutation effect in cells is publicly known as mentioned above (Patent Document 5).
The phase-delaying effect of celastrol and cucurbitacin B was concentration-dependent (Fig. 3).Addition of celastrol or cucurbitacin B at 15, 21, 27, or 33 hours after serum stimulation advanced or delayed the phase of the circadian rhythm depending on the timing (Fig. 4).
Furthermore, when celastrol or cucurbitacin B was added to cultured lung tissue explants from PER2::LUC knock-in mice, the phase of the circadian expression rhythm of PER2 was changed, as in human cultured cells (Figure 5).
The addition of pristimerin (final concentration 0.5 μM) significantly altered the phase of U2OS-PER2-Luc cells, as did the addition of celastrol (final concentration 0.5 μM) (FIG. 6).
Addition of cucurbitacin D (final concentration 0.5 μM), cucurbitacin E (final concentration 0.1 μM), cucurbitacin I (final concentration 0.1 μM), cucurbitacin S (final concentration 5 μM), or isocucurbitacin B (final concentration 5 μM) altered the phase of 3T3-PER2-Luc cells (Figures 7, 8, and 9).
Furthermore, celastrol and pristimerin increased the amount of luminescence in 3T3-PER2-Luc cells in a concentration-dependent manner (FIG. 10A, FIG. 11), which means that they increased the expression level of PER2.
Furthermore, celastrol concentration-dependently shortened the period length of the circadian clock in 3T3-PER2-Luc cells (FIG. 10B).

Claims (6)

A circadian rhythm regulator comprising as an active ingredient one or more selected from the group consisting of hopane-type triterpenes, cucurbitane-type triterpenes, and pharmacologically acceptable salts thereof , wherein the hopane-type triterpene is pristimerin, and the cucurbitane-type triterpene is one or more selected from the group consisting of cucurbitacin B, cucurbitacin D, cucurbitacin E, cucurbitacin I, cucurbitacin S, and isocucurbitacin B. The circadian rhythm regulator according to claim 1, which regulates the expression level, phase of the expression rhythm, or period length of the expression rhythm of PER2. A food or drink for regulating circadian rhythm, comprising the circadian rhythm regulating agent according to claim 1 or 2 . 3. An animal feed for regulating circadian rhythm, comprising the circadian rhythm regulating agent according to claim 1 or 2 . A pharmaceutical composition for preventing, alleviating, or treating a disease mediated by the regulation of circadian rhythm, comprising the circadian rhythm regulating agent according to claim 1 or 2 . 3. A pharmaceutical composition for animals for preventing, alleviating, or treating a disease mediated by the regulation of circadian rhythm, comprising the circadian rhythm regulating agent according to claim 1 or 2 .

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