JP2019043912A - Circadian rhythm regulator - Google Patents

Abstract

To provide a circadian rhythm regulator that has high safety and can be taken continuously.SOLUTION: The circadian rhythm regulator contains a specific triterpene as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a circadian rhythm regulator comprising a specific triterpene as an active ingredient.

Circadian rhythm refers to the pattern of physiological phenomena of about 24 hours cycle that many organisms on earth including human being show. This rhythm is not only sleep and awakening-based activity cycle, but also body temperature, blood pressure, hormone secretion and autonomy. It also exists in nervous activity. The mechanism that generates circadian rhythm is called the internal clock. It is the suprachiasmatic nucleus (SCN) of the hypothalamus that receives the light and dark information from the eye and determines the activity cycle, which is called the central clock. On the other hand, a clock mechanism also exists in peripheral tissues such as liver, kidney, and lung, and this is called a peripheral clock. Basically, the central clock and the peripheral clock maintain a constant phase relationship and chop up the rhythm, but the peripheral clock is independent of the central clock due to stimulation with glucocorticoid, insulin, etc. and also by a specific food component. It is reported that it is affected (nonpatent literature 1, 2, 3). Circadian rhythms are produced by variations in expression of a group of genes called clock genes, which are expressed almost in whole-body cells. For each tissue, the clock gene is timed (this is called synchronization) and repeats the expression fluctuation with a 24-hour cycle, creating a rhythm of metabolism downstream of it. In mammals, transcription and translation regulation with the negative feedback of clock genes such as Per, Cry, Bmal, and Clock as the core loop produces a 24-hour rhythm.
In recent years, it has been pointed out that physical and mental disorders occur due to irregular activities and meals brought about by shift work, and effects such as long distance movement by aircraft (non-patent documents 4 and 5). One of the factors is that there is a discrepancy between the actual sleep and meal times and the rhythms of the central clock and peripheral clock up to that point, and there is a difference in the time taken for correction from tissue to tissue. Be

In order to adjust the circadian rhythm, in particular to cope with jet lag, the airline takes measures such as bringing the lighting time of lighting and the timing of meals closer to the arrival place. In Japan, melatonin is prescribed as a medicine. Melatonin is an endogenous hormone that induces sleep, and its secretion rises at night and shows diurnal fluctuation before waking up in the morning. Precursors, receptor agonists and melatonin secretion rhythm improvers are used for the purpose of adjusting melatonin rhythm as treatment of sleep disorder caused by jet lag. Because melatonin is an endogenous hormone, its use requires caution, and it can not be said that it has a specific circadian rhythm regulating action. With regard to serotonin, which is a precursor of melatonin, it has been reported that the digestive and circulatory system have an adverse effect on oral intake (Non-patent Document 6), and these have not been approved as food in the country.
In addition, as an evaluation system of circadian rhythm, an evaluation system using fluctuation in the phase of expression rhythms of clock genes of cultured cells and animals as an index has been established, and so far clock genes have been targeted for the purpose of regulating circadian rhythm. Many compounds have been reported (for example, Patent Documents 1, 2 and 3). However, the novel compounds that modulate these circadian rhythms are currently not suitable for daily intake because their safety has not been established or their use requires caution.
Therefore, as a means to adjust the circadian rhythm in order to improve the incongruity of the biological clock and keep it normal, it can be taken on a daily basis and the disturbance of the circadian rhythm can be effectively suppressed, safety Higher food forms are desired.

Triterpenes are a group of compounds widely distributed in the plant world, and are one of terpenes composed of six isoprenes and having a molecular formula of C30 H48. The triterpenes include those having no cyclic structure such as squalene, and a compound group having five cyclic structures such as oleanane type, ursan type and lupan type. The oleanane triterpenes include maslinic acid, oleanolic acid and the like, and the ursan triterpenes include ursolic acid, corosolic acid, tolmentic acid and the like.
Triterpenes, which are biological substances widely contained in plants, have recently been attracting attention as functional components having physiological activity, for example, skin lightening of maslinic acid for oleanane type triterpenes and ursan type triterpenes (Patent Document 4), Inhibiting differentiation of osteoclasts and preventing arthritis (Patent Document 5), composition containing corosolic acid, maslinic acid and the like as active ingredients and suppressing elevation of blood glucose level (Patent Document 6), and caries of oleanolic acid (Patent Document 7) Prevention, treatment or treatment of osteoarthritis (Patent Document 9), etc., anti-cancer effect (Patent Document 8), etc., oleanolic acid, corosolic acid, ursolic acid, maslinic acid etc., muscle enhancement, improvement of metabolic syndrome and QOL (patent document) 10) has been reported.
However, it has not been known that oleanane-type triterpenes and ursan-type triterpenes are effective in regulating circadian rhythm.

Japanese Patent Application Publication No. 2016-40231 JP, 2016-183189, A JP, 2009-183197, A International Publication 2002/043736 JP 2014-141444 Japanese Patent Application Publication No. 2006-34967 Japanese Patent Application Laid-Open No. 1-290619 JP-A-8-119866 Japanese Patent Application Laid-Open No. 2016-172695 Japanese Patent Application Laid-Open No. 2016-199536

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 Togo F et al., Chronobiol Int. 2017; 34 (3): 349-359 van Zwieten PA, Blauw GJ, van Brummelen P., Cardiovasc Drugs Ther 1990 Dec; 4 (6): 1443-7 Balsalobre A, Cell. 1998 Jun 12; 93 (6): 929-37

  An object of the present invention is to provide a circadian rhythm regulator that is highly safe and can be continuously taken.

The present inventors have found that the oleanane type triterpene and the ursane type triterpene are effective for regulating the circadian rhythm by regulating the phase of the expression rhythm of the clock gene of mammals.
That is, the present invention is as follows.
[1] A circadian rhythm regulator comprising an oleanane type triterpene, an ursan type triterpene or a pharmacologically acceptable salt thereof as an active ingredient.
[2] The circadian rhythm regulator according to the above [1], which regulates the phase of expression rhythm of PER2 gene.
[3] The circadian date described in the above [1] or [2], wherein the oleanane type triterpene is selected from maslinic acid and oleanolic acid or a mixture thereof, and the ursan type triterpene is selected from corosolic acid, ursolic acid or a mixture thereof Rhythm regulator.
[4] A food or beverage for regulating circadian rhythm comprising the circadian rhythm regulator according to any one of claims 1 to 3.
[5] A pharmaceutical composition for preventing, alleviating or treating sleep disorder or jet lag which is caused by disturbance of circadian rhythm, comprising the circadian rhythm regulator according to any one of claims 1 to 3.

  According to the present invention, it is possible to provide a circadian rhythm regulator capable of regulating the phase of expression rhythm of a clock gene of a mammal and regulating circadian rhythm and taking it in a highly safe food form. It can.

The change of the circadian rhythm of human PER2 gene expression when triterpenes (corosolic acid, maslinic acid, oleanolic acid, ursolic acid) is added at the timing of the arrow in the figure is shown. When corosolic acid is added to cells, an example in which the phase of the expression rhythm of PER2 gene is retracted (left) and an example in which the phase is advanced (right) are shown. The time phase from serum stimulation is taken on the horizontal axis, and the vertical axis is taken on the phase mutation to show a phase response curve plotted. The expression rhythm of human PER2 gene when betulinic acid, squalane and squalene are added at the timing of the arrow in the figure is shown.

The circadian rhythm regulator of the present invention contains, as an active ingredient, an oleanane type triterpene, an ursan type triterpene or a pharmacologically acceptable salt thereof.
In the present invention, a triterpene is a compound having a carbon number of 30 as a basic skeleton, and a pentacyclic triterpene is mentioned as a typical example thereof. Here, a pentacyclic triterpene is one kind of triterpenes, and is a pentacyclic compound consisting of six isoprene units, which has a carbon number of 30 as a base, but is rearranged, oxidized, Also included are those which are eliminated or alkylated and the carbon number fluctuates.
These can be obtained from natural plants or artificially. Moreover, a commercial item can also be utilized suitably. Pentacyclic triterpenes are generally classified according to their skeletons, and include, for example, oleanane type triterpenes, ursan type triterpenes, lupan type triterpenes, hopane type triterpenes, serratan type triterpenes, friedellan type triterpenes, taraxelan type triterpenes, taraxastane type triterpenes, and mulch Examples include florane-type triterpenes and germanican-type triterpenes.
Among the pentacyclic triterpenes, maslinic acid, oleanolic acid and the like can be mentioned as representative examples of the oleanane type triterpenes, and corosolic acid, ursolic acid and the like can be mentioned as examples of the ursan type triterpenes.

  Maslinic acid is contained in olive, jujube, almond, banaba leaf, sage, almond, apple, cranberry, cullin and the like. Oleanolic acid is olive, grape, beet, jujube, almond, banaba leaf, olive leaf, sage, hawthorn, raspberry, karin, rosemary leaf, guava, shiso leaf, blueberry, prune, loquat, pomegranate, lemon balm, basil, rose It is included in hips, oysters, slivers, etc. Corosolic acid is contained in Banaba leaves, loquat, karin, almonds, cranberries and the like. Ursolic acid is contained in olives, almonds, apples, cranberries and the like.

  In the present invention, the oleanane-type triterpenes are preferably selected from maslinic acid, oleanolic acid and mixtures thereof. Also, ursan-type triterpenes are preferably selected from corosolic acid, ursolic acid and mixtures thereof.

These oleanane-type triterpenes or ursan-type triterpenes can be obtained by extraction and purification from the above-mentioned plants by a known method, and can also be obtained by chemical synthesis. The oleanane triterpene or ursan triterpene may be a single component or a mixture of several components. They can also be used in the form of pharmacologically acceptable salts and / or derivatives.
Oleanane type triterpene or ursan type triterpene may be used in the form of the extract of the said plant.

  When used in the form of a plant extract, the oleanane type triterpene or ursan type triterpene can be obtained by a method suitable for the plant used as a raw material. For example, corosolic acid can be obtained by a method disclosed in JP-A-2005-263650 using banaba leaves as an extraction raw material. Maslinic acid and oleanolic acid can be obtained by methods disclosed in WO 02/012159 using olive plants as extraction raw materials. Corosolic acid and ursolic acid can be obtained by the method disclosed in JP-A-2005-263650 using loquat leaves as an extraction raw material. In addition, the type and composition ratio of the components of the oleanane type triterpene or ursan type triterpene to be obtained depend on the plant used as the raw material.

  The intake amount of the oleanane triterpene or ursan triterpene varies depending on the age, sex, degree of symptoms and intake form of patients to be ingested, and is preferably 0.1 mg / kg or more per day for adults. There is no upper limit of intake, but it is not preferable from the viewpoint of product cost if it exceeds 50 mg / kg.

  The circadian rhythm regulator of the present invention may be in the form of a composition, and may be formulated into an optional form together with an auxiliary agent to provide a pharmaceutical which can be orally or parenterally administered. For example, oral dosage forms can be tablets, oral fast disintegrating tablets, capsules, solid dosage forms such as granules, granules, liquid dosage forms such as syrups and suspensions. As a parenteral dosage form, it is administered in the form of injection, eye drop, nasal drop, patch, suppository, external preparation for skin. Pharmaceuticals also include quasi drugs.

  In the case of solid dosage forms, appropriate amounts of various additives used in the preparation of general formulations may be contained. Examples of such additives include excipients, binders, acidulants, foaming agents, artificial sweeteners, flavors, lubricants, coloring agents, stabilizers, pH adjusters, surfactants and the like.

  When the liquid dosage form is used, the circadian rhythm regulator of the present invention is optionally in the presence of a pH regulator, a buffer, a solubilizer, a suspending agent, etc., an isotonicity agent, a stabilizer, a preservative, etc. And can be formulated by conventional methods.

  The form of the external preparation for skin is not particularly limited. For example, emulsions, creams, lotions, packs, dispersions, detergents, makeup cosmetics, cosmetics such as scalp and hair products, ointments, creams, external use It can be used as a drug such as a liquid drug. In addition to the above components, components usually used for external skin preparations such as cosmetics and pharmaceuticals, for example, skin lightening agents, moisturizers, various skin nutritional components, ultraviolet light absorbers, antioxidants, oil components, surfactants, thickeners Alcohols, coloring agents, water, preservatives, perfumes and the like can be appropriately blended as required.

  The circadian rhythm regulator of the present invention is used as a functional food, a health food, a food for specified health use, a health function food such as a nutrition function food, a food for special use (for example, a food for a sick person), a health food supplement, a supplement etc. It may be prepared. As a supplement, for example, tablets, rounds, capsules (including hard capsules, soft capsules, and microcapsules), powders, granules, fine particles, troches, and liquids, together with various additives used for the production of common supplements It can be in the form of syrup (including syrup, milk, suspension) and the like.

  The circadian rhythm regulator of the present invention can be formulated into food. There are no particular limitations on the foods that can be incorporated, but, for example, sweets such as sweet potato, gummi, chewing gum, etc .; cookies, crackers, biscuits, chocolate, puddings, jellies, snacks, rice confectionery, sweets such as bald meat, sheep porridge; Ice cream candy, sherbet, gelato frozen desserts; bakery foods such as donuts, cakes, bread, french bread, croissants etc .; noodles such as udon, buckwheat noodles, Chinese noodles, Chinese noodles; cooked rice such as white rice, red rice, pilaf; curry, stew , Sauces such as dressings; Braised products such as ham, sausages, kamaboko, chikuwa and fish sausages; Various vegetables such as tempuras, croquettes and hamburgs; Beverages such as juices and teas.

  According to the circadian rhythm regulator of the present invention, the phase of the expression rhythm of the clock gene can be regulated. In the present specification, "the expression rhythm of a gene" refers to a rhythm in which a gene repeats activation and suppression of expression in a constant cycle, and "regulates the phase of the expression rhythm of a clock gene" means a clock gene. When showing the activation of expression and the suppression of expression along a certain expression rhythm (or cycle of expression rhythm), the phase of the expression rhythm (or cycle of expression rhythm) is temporally delayed (recessed) Or, it means to advance (advance). Therefore, as a preferred embodiment of “regulate the phase of the expression rhythm of the clock gene”, the phase is set along the time axis by the amount of the phase where the difference occurs in comparison with the original expression rhythm (or the cycle of the expression rhythm). To adjust by delaying or advancing. The agent for improving circadian rhythm of the present invention can be regulated, for example, by delaying or advancing the phase of the expression rhythm of PER2 gene of clock genes along the time axis.

The clock gene is a gene that controls the circadian rhythm (circuladian rhythm). Examples of clock genes include Per gene (PER1, PER2, etc.), Cry gene (CRY1, CRY2, etc.), Bmal gene (BMAL1, etc.), Clock gene (CLOCK). These genes are known to be contained in various animals such as humans and mice, and to be expressed almost in whole cells.
Circadian rhythms are caused by expression fluctuations between the clock genes. The clock gene, which is expressed in almost whole-body cells, repeats the expression fluctuation in a 24-hour cycle at the same time for each tissue (this is called synchronization) and creates the rhythm of metabolism downstream of it. Transcriptional translation regulation with the negative feedback of the clock gene as the core loop produces a 24-hour rhythm. By controlling the expression rhythm of the PER2 gene, which plays a central role, the expression of a series of clock genes is controlled, and as a result, circadian rhythm control is possible.

  Further, since the circadian rhythm regulator of the present invention can regulate the circadian rhythm, it is possible to prevent physical disorder or disease caused by disturbance of the circadian rhythm such as sleep disorder, jet lag, etc. It is possible to improve diseases such as alleviation and treatment.

  EXAMPLES The present invention will be illustrated specifically by the following examples, but the present invention is not limited to the following examples.

Test Example 1 Circadian Rhythm-Regulating Effect of Triterpenes Focusing on Per gene, a representative clock gene, the expression rhythm of human PER2 gene was used as an index of circadian rhythm, and the circadian rhythm-regulating function of triterpenes was examined . Using the expression rhythm of human PER2 gene as an indicator of circadian rhythm has been established as a system for evaluating the regulatory action of circadian rhythm, for example, Isojima et al., PNAS 2009 Sep; 106 (37): 15744- 15749, Hirota et al., Science 2012 Aug; 337: 1094-1097 and the like.
(Test substance)
Corosolic acid (Tokiwa Phytochemical Laboratories, P 2302), Maslinic acid (Cayman Chemical Co., 10009645), oleanolic acid (SIGMA-ALDRICH, O5504), ursolic acid (SIGMA-ALDRICH, U6753), betulinic acid (Tokyo) As Chemical Industry Co., Ltd., squalene (Tokyo Chemical Industry Co., Ltd.) and squalane (Tokyo Chemical Industry Co., Ltd.), commercially available products were used.
Each triterpene 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 a test system, human osteosarcoma-derived U2OS cells (U2OS-hPER2-Luc) stably expressing a circadian rhythm reporter incorporating a luciferase gene under the human PER2 gene (hPER2) promoter were used. The change in luminescence value due to the luciferin-luciferase reaction represents the change in promoter activity of the PER2 gene. The luminescence was measured for several days, and a waveform of continuous luminescence was obtained and used as an index of circadian rhythm.
(1) U2OS-hPER2-Luc cells were seeded in a 35 mm diameter dish and cultured to confluence in 2 mL of DMEM medium (High glucose) supplemented with 10% fetal calf serum and penicillin-streptomycin.
(2) The cells were cultured for 2 hours in DMEM medium supplemented with 50% horse serum, and the rhythms of the cells were matched (Non-patent Document 7). The culture for 2 hours in a medium containing 50% horse serum is hereinafter referred to as serum stimulation. After serum stimulation, replace with 2 μL of 0.1 M luciferin, 2 mL of DMEM medium supplemented with penicillin-streptomycin (final concentration: 0.1 mM luciferin), continuously for 3 days or more under 37 ° C., 5% CO ₂ conditions The luminescence was measured. For measurement, a luminescence measurement apparatus Kronos dio manufactured by ATTO was used. The luminescence amount was measured at 10-minute intervals for 1 minute of integrated value.
(3) During the measurement, the device was stopped and a reagent (triterpenes) was added to the culture medium. The device was restarted after the addition and the luminescence was subsequently recorded.
(4) Noise was removed from the waveform of the time-dependent change of the light emission value obtained by the analysis system mounted on Kronos dio, and detrended to obtain a light emission rhythm. The phase of the obtained luminescence rhythm was determined, and comparison with the control group was performed.
(result)
The influence of corosolic acid, maslinic acid, oleanolic acid and ursolic acid on the time course of luminescence is shown in FIG. The phase of the circadian rhythm is characterized by the time (peak time, horizontal axis: culture time relative to the serum stimulation start time) of the waveform indicated by light emission. The reagent (triterpenes) is added at the timing of the arrow shown in the figure, but the light emission is slightly disturbed by the addition stimulus immediately after the addition of the reagent, so the peak phase after the light emission value recovers (the second and subsequent peaks) As a reference, it is determined whether the phase is advanced or retracted by advancing or retracting the peak time relative to the control. The addition of corosolic acid, maslinic acid, oleanolic acid and ursolic acid reversed the phase of the expression rhythm (FIG. 1). Although corosolic acid is shown as a representative example in FIG. 2, the direction and size of the phase variation of the expression rhythm differ depending on the addition timing, and they tend to recede when added at the light emission rise and advance when the light emission decreased. The In FIG. 3, the above experiment using corosolic acid as a test substance is performed in multiple times, the time from the start of serum stimulation to the addition of corosolic acid is plotted on the horizontal axis, and the phase variation is plotted on the vertical axis A phase response curve is shown plotting forward +, backward-). The time on the horizontal axis is the start of serum stimulation as 0, and how many hours it has passed from serum stimulation is described by a 24-hour interval (when more than 24 hours, the time obtained by subtracting a multiple of 24 is the time). When corosolic acid stimulation is given 12 to 18 hours later, it shows a tendency to move forward, otherwise to move backward. This result indicates that the timing of corosolic acid stimulation can shift the phase of the expression rhythm not only in one direction but in both directions.
Figure 4 shows the results of the lupine-type triterpenes betulinic acid, linear triterpenes squalene, and squalane, but the phase after addition almost matches the control group, and corosolic acid, maslinic acid, oleanolic acid, ursolic acid There was no expression rhythm phase mutation action as seen with oleanane type triterpenes and ursan type triterpenes.

  Corosolic acid, maslinic acid, oleanolic acid and ursolic acid move the circadian rhythm forward or backward depending on the timing of administration. Although the biological clock may be disturbed by external stimuli (meal, strong light, time difference, etc.), it is known that there is a time zone in which the rhythm tends to advance or a time zone in which it tends to recede according to the timing of stimulation. . Since the composition of the present invention moves the rhythm not only in one direction but in both directions, the disturbed circadian rhythm can be effectively adjusted.

Claims (5)

  A circadian rhythm regulator comprising, as an active ingredient, an oleanane triterpene, ursan triterpene or a pharmacologically acceptable salt thereof.   The circadian rhythm regulator according to claim 1, which regulates the phase of expression rhythm of Per2 gene.   The circadian rhythm regulator according to claim 1 or 2, wherein the oleanane triterpene is selected from maslinic acid and oleanolic acid or a mixture thereof, and the ursan triterpene is selected from corosolic acid, ursolic acid or a mixture thereof.   Food and drink for regulating circadian rhythm, comprising the circadian rhythm regulator according to any one of claims 1 to 3.   A pharmaceutical composition for preventing, alleviating or treating sleep disorder or jet lag caused by disturbance of circadian rhythm, which comprises the circadian rhythm regulator according to any one of claims 1 to 3.