JP7032855B2 - Compositions for altering the expression of clock genes - Google Patents

Description

The present invention relates to a composition for altering the expression of a clock gene, which comprises a water extract of Albizia bark as an active ingredient. The present invention also relates to a composition for changing the circadian rhythm, which comprises the extract as an active ingredient.

Most living organisms on the earth control physiological functions (sleep awakening, thermoregulation, feeding excretion, hormone secretion, etc.) in a cycle of about 24 hours according to the external environment (mainly the light-dark cycle). Such biological diurnal variation is called "circadian rhythm".

In recent years, it has become a social problem that various physical and mental disorders are caused by this abnormality in circadian rhythm. For example, living environment such as jet lag due to movement of aircraft, sleep disorders caused by irregular life patterns due to shift work (shift work), and modulation of sleep awakening rhythm due to night life (night shift, night shift, etc.). There are many problems caused by the disturbance of circadian rhythm due to changes in the working environment. In addition, with aging, the amount of hormones such as melatonin that regulates the circadian rhythm decreases, so that various rhythms such as sleep awakening and thermoregulation are attenuated in the elderly. The decline in QOL due to sleep disorders in old age is also a concern as a social problem, coupled with the rapid aging of the population in recent years. In view of this situation, there is a need for a method that can eliminate the modulation and attenuation of the circadian rhythm and, by extension, treat and improve the sleep disorder, but such a method has not yet been established. be.

Circadian rhythm is a small nerve nucleus with a diameter of only 1 mm located on the suprachiasmatic nucleus (optic chiasm) where the optic nerve extending from the retinas of the left and right eyes intersects in the hypothalamus in the brain. A suprachiasmatic nucleus (SCN) is central to control. The circadian rhythm produced by SCN is formed by a group of genes called clock genes (Bmal1, Block, Period, Cryptochrome, etc.). More specifically, the transcriptional activators BMAL1 and CLOCK proteins activate the expression of the Period and Cryptochrome genes, thereby transcribed and translated Period (PER1, PER2) and Cryptochrome (CRY1, CRY2) proteins into BMAL1 and By interacting with the CLOCK protein, it forms negative feedback that suppresses its own transcription. After that, the PER and CRY proteins undergo phosphorylation and ubiquitination modification, and then are degraded by the proteasome, so that the transcriptional repression is released. Then, the next cycle by BMAL1 and CLOCK protein is started, and it takes about 24 hours for one cycle of this cycle, which is the molecular mechanism of circadian rhythm formation.

Such a clock gene feedback mechanism also exists in peripheral tissues such as the liver, muscles, lungs, and heart, and tissues other than SCN in the brain, and this mechanism also forms a circadian rhythm in peripheral tissues and the like. It has become clear. In addition, the rhythm is controlled by SCN, which is the center of control, by issuing signals by nerves, hormones, etc. via various tissues.

Thus, circadian rhythms are regulated by the expression of clock genes. Therefore, a substance that can change the circadian rhythm by changing the expression and can treat sleep disorders and the like due to abnormal circadian rhythm has been searched for (Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2008-266319 Japanese Unexamined Patent Publication No. 2015-110564

The present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a substance for changing the expression of a clock gene and changing the circadian rhythm by the change in the expression.

As a result of diligent research to achieve the above object, the present inventors added a water extract of nemunoki bark to mouse embryonic fibroblasts, and as a result, the expression rhythm of the clock gene (Per2 gene) of the cells. It was found that the transitional attenuation of the amplitude can be suppressed. Furthermore, the present inventors have shown that the extract can advance or decrease the peak (phase) of the expression rhythm of the Per2 gene in a concentration-dependent manner depending on the timing of administration to cells. Revealed.

Furthermore, a water extract of Albizia bark was orally administered to mice, and the expression of clock genes (Per1, Per2, Bmal1) in the peripheral tissues (liver) thereof was analyzed. As a result, the present inventors have found that the expression level and phase of the clock gene are changed by the extract even in peripheral tissues.

In addition, as a result of oral administration of a water extract of Albizia bark to humans, the morning-type tendency of the subjects became stronger (faster sleep onset, as if reflecting the above-mentioned effect of changing the expression of the clock gene of the extract. We found that it became easier to get up early in the morning), drowsiness in the afternoon decreased, and activity (awakening, motivation, mood and concentration) improved, and the present invention was completed.

That is, the present invention provides the following.
<1> A composition for altering the expression of a clock gene, which comprises a water extract of Albizia bark as an active ingredient.
<2> The composition according to <1>, wherein the clock gene is at least one gene selected from the group consisting of the Per1 gene, the Per2 gene and the Bmal1 gene.
<3> The composition according to <1> or <2>, which is a composition for changing a circadian rhythm.

According to the present invention, it is possible to change the expression of the clock gene and change the circadian rhythm by the change in the expression. Then, since the abnormality in the circadian rhythm can be eliminated by such a change, according to the present invention, the disease (circadian rhythm disorder, etc.) caused by the abnormality is treated, improved (alleviated) or prevented. Is also possible.

It is a waveform graph which shows the outline of the expression rhythm of a clock gene, or the circadian rhythm controlled by it. FIG. 6 is a waveform graph showing the expression rhythm of the Per2 gene in mouse embryonic fibroblasts supplemented with a water extract of Albizia bark (50 μg / ml or 100 μg / ml) or distilled water (DW) as a control. The horizontal axis in the figure shows the number of days since dexamethasone was added to the medium in order to reset and synchronize the expression cycle of clock genes such as the Per2 gene in each cell. It is a schematic diagram which shows the schedule of the water extract administration test of the silk tree bark to the mouse. It is a graph which shows the expression level of the Per1 gene in the mouse which orally administered the water extract (1 mg or 10 mg) of the Albizia bark or distilled water as a control (cont.). It is a graph which shows the expression level of Per2 gene in the mouse to which the water extract (1 mg or 10 mg) of the Albizia bark or distilled water was orally administered as a control (cont.). It is a graph which shows the expression level of the Bmal1 gene in the mouse which orally administered the water extract (1 mg or 10 mg) of the Albizia bark or distilled water as a control (cont.). It is a graph which shows the time-dependent change of the expression level of Per1 gene in a mouse. The vertical axis shows the expression level of the Per1 gene as a relative value based on that of the Gapdh gene. The horizontal axis indicates the time under light and dark conditions (ZT; Zeitgeber Time, where the light period start time is ZT0). It is a graph which shows the result of having undergone the sleep test (Kwansei Gakuin University drowsiness scale, KSS) in the human who ingested the water extract of Albizia bark in the morning. It is a graph which shows the result (arousal degree) which a person who ingested the water extract of Albizia bark in the morning underwent a sleep test (visual analog evaluation scale, VAS). It is a graph which shows the result (motivation) which a person who ingested the water extract of Albizia bark in the morning underwent a sleep test (visual analog evaluation scale, VAS). It is a graph which shows the result (mood) which a person who ingested the water extract of Albizia bark in the morning underwent a sleep test (visual analog evaluation scale, VAS). It is a graph which shows the result (concentration ability) which a person who ingested the water extract of Albizia bark in the morning underwent a sleep test (visual analog evaluation scale, VAS). It is a graph which shows the result of having undergone a sleep test (morning type / night type questionnaire, MEQ) in the human who ingested the water extract of Albizia bark in the morning. It is a graph which shows the result of having undergone the sleep test (morning type / night type questionnaire, MEQ) of the human who ingested the water extract of Albizia bark at the time of dinner.

As shown in Examples described later, it was revealed that in the present invention, the water extract of Albizia japonica can change the expression level and phase of the clock gene. The extract can also change the circadian rhythm controlled by the clock gene through changes in the expression of the clock gene. Therefore, the present invention provides a composition for altering the expression of a clock gene, which comprises a water extract of Albizia bark as an active ingredient. The present invention also provides a composition for changing the circadian rhythm, which comprises the extract as an active ingredient.

Water extract of Albizia bark First, "Water extract of Albizia bark" contained as an active ingredient in the composition of the present invention will be described. "Albizia julibrissin" means a deciduous tree plant (Albizia julibrissin) belonging to the subfamily Mimosaceae of the family Leguminosae. When the bark of Albizia japonica is subjected to the following extraction treatment, the bark may be used as it is, but from the viewpoint of further improving the extraction efficiency, it is extracted after being crushed by a mixer, a blender, a homogenizer, a mortar, an ultrasonic crusher or the like. It may be used for processing.

In the present invention, the solvent used for the extraction treatment may be water alone or a mixed solvent of water and another solvent. The water used for extraction in the present invention is not particularly limited, and examples thereof include distilled water, deionized water, RO water, and ultrapure water. The other solvent to be mixed with water is not particularly limited, and examples thereof include alcohols such as ethanol and methanol, polyhydric alcohols such as propylene glycol, and esters such as methyl acetate. The mixing ratio is not particularly limited, but water: other solvent = 90 to 50:10 to 50 (volume ratio) is preferable.

The extraction conditions are not particularly limited and vary depending on the solvent used. For example, when only water is used, 1 to 100 parts by weight of water is used with respect to 1 part by weight of the bark of Albizia japonica, and 4 to 100 ° C. It is preferable to extract at the temperature of 10 minutes to 7 days, using 5 to 20 parts by weight of water for 1 part by weight of the bark of Albizia japonica, and taking 30 minutes to 2 hours at a temperature of 70 to 100 ° C. It is more preferable to extract the water. Further, from the viewpoint of further improving the extraction efficiency, pressurization, stirring, ultrasonic treatment and the like may be performed at the same time as the extraction treatment. Further, after such an extraction treatment, it may be separated from the residue by filtration or centrifugation.

The "water extract of Nemunoki bark" according to the present invention can be used in the composition of the present invention in the form of an extract, but if necessary, an ion exchange method, a gel filtration method, a membrane fractionation method, or electricity. It may be concentrated by a dialysis method, a solvent extraction method, a vacuum concentration method, a heat treatment method, or the like.

Further, the "water extract of Albizia bark" may be dried by subjecting it to a treatment such as spray drying or freeze drying, if necessary, to form a powder.

Further, from the viewpoint of safety, the "water extract of Albizia bark" may be sterilized. Examples of such sterilization treatment include heat sterilization, pressure sterilization, radiation sterilization, filter sterilization and the like.

Further, the "water extract of Albizia bark" prepared in this manner may be used as it is, for example, as the composition of the present invention, or as described later, by combining with other components, the present invention. It may be used as a composition of.

Composition for Changing Clock Gene Expression or Circadian Rhythm Next, embodiments of the composition of the present invention will be described. The above-mentioned water extract of Albizia bark has an activity of changing the circadian rhythm by changing the expression of the clock gene, as shown in Examples described later. Therefore, the composition of the present invention containing this as an active ingredient can be used as a pharmaceutical composition, a food or drink for treating, improving (alleviating) or preventing a circadian rhythm disorder described later. It can also be used as a reagent for changing the expression of a clock gene and a reagent for changing the circadian rhythm.

In the present invention, the "clock gene" may be any gene having a function of controlling the circadian rhythm (biological clock), for example, a Period gene (Per1, Per2, Per3, etc.), a Bmal gene (Arntl, Mop3, Tic). , Jap3, Pasd3, bHLHe5 genes, Bmal1, Bmal2, etc.), Cryptchrome genes (Cry1, Cry2, etc.), Lock gene, Ror gene, Rev-erb gene, E4BP gene, GSK3β gene, CK1 gene, Dec gene. However, in the present invention, the Per1 gene, the Per2 gene, and the Bmal1 gene are preferable.

Typically, the human Per1 gene is a gene encoding a protein consisting of the amino acid sequence specified by RefSeq ID: NP_002607 (a gene containing the nucleotide sequence specified by RefSeq ID: NM_002616), and the human Per2 gene is , RefSeq ID: a gene encoding a protein consisting of an amino acid sequence specified by NP_073728 (a gene containing a nucleotide sequence specified by RefSeq ID: NM_003894 or NM_022817), and the human Bmal1 gene is RefSeq ID: NP_0010254443, NP_001025444. , NP_001169, NP_001284648 or a gene encoding a protein consisting of the amino acid sequence specified by NP_001284651 (RefSeq ID: NM_001030272, NM_001030273, NM_001178, NM_001297719 or a gene containing the nucleotide sequence specified by NM_0012977722). , RefSeq ID: NP_001152839 or a gene encoding a protein consisting of an amino acid sequence specified by NP_305195 (a gene containing a nucleotide sequence specified by RefSeq ID: NM_001159367 or NM_011065), and the mouse Per2 gene is RefSeq ID: NP_035196. It is a gene encoding a protein consisting of the amino acid sequence specified in RefSeq ID: a gene containing the nucleotide sequence specified by NM_011066, and the mouse Bmal1 gene is derived from the amino acid sequence specified by RefSeq ID: NP_0012229977 or NP_031515. A gene encoding a protein (RefSeq ID: NM_001243048 or a gene containing a nucleotide sequence specified by NM_007489). It should be noted that the nucleotide sequence changes in nature (that is, non-artificially). Therefore, the clock gene according to the present invention also includes such a natural variant without being specified in the above sequence given as a typical example.

In the present invention, "expression of a clock gene" includes not only expression at the transcription level (expression as mRNA) but also expression at the translation level (expression as a protein). In addition, the "change in clock gene expression" includes an increase or decrease in the expression level of the clock gene and a change in the expression rhythm of the clock gene (at least one of amplitude, phase and cycle length). Is done.

As shown in FIG. 1, the "amplitude" is the length from the peak to the bottom (magnitude of vibration) in the rhythm, and in the expression rhythm of the clock gene, from the peak to the peak or from the bottom to the bottom. The range of the expression level of the clock gene in the period (one cycle) of is shown. It is known that the amplitudes of clock gene expression rhythms and circadian rhythms controlled by them diminish with aging. On the other hand, as shown in Examples described later, the bark water extract of Albizia japonica can suppress such attenuation.

"Phase" is a specific position in one cycle (eg, peak, bottom). Further, as shown in FIG. 1, the "cycle length" is the length (period) of one cycle in the rhythm, and is originally (normal) about 24.3 hours in humans. As shown in Examples below, the Nemunoki bark water extract advances the phase of the clock gene expression rhythm and the circadian rhythm controlled by it depending on the timing of administration or ingestion (the waveform is on the left side in FIG. 1). Since it is possible to shift) or reverse (shift the waveform to the right in FIG. 1), according to the present invention, the phase shifted due to the circadian rhythm disorder described later is adjusted to the original (normal) phase. Can be done.

As described above, the water extract of Nemunoki bark can eliminate abnormalities (attenuation of amplitude, phase shift, etc.) in the expression rhythm of the clock gene and the circadian rhythm controlled by the expression rhythm. According to the composition of the present invention containing the active ingredient, it is possible to treat, improve (alleviate) or prevent a disease (circadian rhythm disorder, etc.) caused by the abnormality.

Examples of circadian rhythm disorders include delayed sleep phase syndrome (ASPS), delayed sleep phase syndrome (DSPS), non-24-hour sleep-wake disorder, irregular sleep-wake disorder, and jet lag (time band change syndrome, jet lag syndrome). ), Rhythm modulation due to shift work (shift work sleep disorder), life rhythm modulation due to night life (night shift, night shift, etc.), and rhythm disappearance in the elderly. Furthermore, the composition of the present invention is also effective in treating, improving (alleviating) or preventing various symptoms such as insomnia, illness, attention deficit, decreased motivation, and rough skin associated with these circadian rhythm disorders.

In addition, as shown in the examples described later, even in a healthy person, by ingesting a water extract of Albizia bark, the subject's morning-type tendency becomes stronger, afternoon drowsiness decreases, and activity (awakening). Degree, motivation, mood and concentration) increase. Therefore, the composition of the present invention is also useful as a composition for shifting the lifestyle rhythm (lifestyle) to the morning type. In the present invention, the "morning type" means a lifestyle rhythm (lifestyle) in which a person wakes up early in the morning, is active in the morning, and falls asleep early at night. For example, a morning type / night type questionnaire. It can be evaluated by (MEQ; Morningness-Eveningness Questionnaire) (Kaneyoshi Ishihara et al., "Results of Survey by Japanese Morning-Eveningness Questionnaire", Psychology Research, 1986, Vol. 57. , Pages 87-91).

The composition of the present invention can be formulated by a known pharmaceutical method. For example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chewing agents, buccal agents, pastes, syrups, suspensions, etc. Orally or parenterally as an elixir agent, emulsion, coating agent, ointment agent, plaster agent, pap agent, transdermal absorption type preparation, lotion agent, inhalant agent, aerosol agent, injection agent, suppository, etc. Can be done. However, the composition of the present invention is preferably used as an oral composition from the viewpoint of reducing the burden on the administration subject.

In these formulations, carriers that are pharmacologically or acceptable as food and drink, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, etc. Flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, tonics, soothing agents, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweetness It can be appropriately combined with an agent, a viscous agent, a flavoring agent, a solubilizing agent, or other additives.

When the composition of the present invention is used as a food and drink, the food and drink is, for example, a health food, a functional food, a food for specified health use, a food with a functional claim, a nutritional supplement, a food for the sick, and a food additive. Or it can be animal feed. The food and drink of the present invention can be ingested as the composition as described above, and can also be ingested as various foods and drinks. Specific examples of foods and drinks include edible oils, dressings, mayonnaise, margarine and other oil-containing products; soups, dairy drinks, soft drinks, tea drinks, alcoholic drinks, drinks, jelly-like drinks, functional drinks, etc. Liquid foods; carbohydrate-containing foods such as rice, noodles and breads; processed livestock foods such as ham and sausage; processed marine foods such as kamaboko, dried foods and salted foods; processed vegetable foods such as pickles; half of jelly and yogurt Solid foods; Fermented foods such as miso and fermented beverages; Western confectionery, Japanese confectionery, candy, gums, gummy, chilled confectionery, ice confectionery and other confectionery; curry, ankake, Chinese soup and other retort products; instant soup, Examples include instant foods such as instant miso soup and foods compatible with microwave ovens. Further, healthy foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly can be mentioned.

The composition of the present invention can be used for any organism, but is particularly preferably used for animals including humans. The animals other than humans are not particularly limited, and various livestock, poultry, pets, experimental animals and the like can be targeted. Specific examples include, but are not limited to, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats and monkeys.

The food and drink in the present invention can be manufactured by a manufacturing technique known in the art. In the food or drink, one or more kinds of ingredients or other functional foods effective for improving or preventing circadian rhythm disorder may be added. Further, it may be a multifunctional food or drink by combining it with other ingredients or other functional foods that exhibit functions other than the improvement.

When the composition of the present invention is administered or ingested, the dose or ingestion thereof is appropriately selected according to the age, body weight, symptoms, health condition, type of composition (pharmaceutical products, foods and drinks, etc.) of the subject. However, in the case of humans (adults), it is preferable to administer 10 to 1000 mg of the water extract of Nemunoki bark once or several times a day, preferably 50 to 200 mg. More preferred. The timing of administration of the composition of the present invention depends on how the circadian rhythm is deviated (forward or backward), but is preferably in the morning, more preferably between 7 and 10 am, and even more preferably. It is between 8 and 9 am. Furthermore, it is desirable that the composition of the present invention be administered continuously (for at least 3 days, more preferably for 10 days or more).

The products (pharmaceuticals, foods and drinks, reagents) of the compositions of the present invention or their instructions are labeled as being used to change the expression of clock genes and / or to change the circadian rhythm. possible. Here, "marked on the product or instruction manual" means that the label is attached to the main body, container, packaging, etc. of the product, or the instruction manual, package insert, advertisement, or other printed matter that discloses the information of the product. It means that a display is attached to such as.

Further, the present invention is characterized in that the pharmaceutical composition of the present invention is administered to a subject (for example, a human suffering from a circadian rhythm disorder), accompanied by a change in the circadian rhythm of the subject. It also provides a method of treating or preventing the disease.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

(Example 1)
<Preparation of water extract of Albizia bark>
To the bark of Albizia japonica (manufactured by Young Forest Co., Ltd.), 10 times the weight of distilled water was added, and the mixture was treated in an autoclave (100 ° C., 60 minutes). The obtained extract was passed through a stainless mesh (opening: 150 μm, manufactured by Tokyo Screen Co., Ltd., mesh sieve, product number: 635-54-18-41). The obtained filtrate was referred to as No. After further passing through 193 filter paper (manufactured by ADVANTEC) and performing a total of two filtration treatments, it is concentrated to a solid content of 5% using a rotary evaporator (manufactured by Tokyo Rika Kikai Co., Ltd., N-1300 type). did. Next, the obtained concentrate was mixed with dextrin (Sandeck, manufactured by Sanwa Cornstarch Co., Ltd.), subjected to heat sterilization treatment (90 ° C., 60 minutes), and then freeze-dried (AGC Technoglass Co., Ltd.). Manufactured by FRD-82M) to make a dry powder.

The ratio of the water extract of Albizia bark to dextrin in the dry powder thus obtained is 1: 3. Since the water extract of Albizia bark has high hygroscopicity and its physical properties are liable to change (solidify), dextrin was added as an excipient as described above. The water extract of Albizia japonica bark in the dry powder was 6 to 8% by mass of the Albizia japonica bark subjected to the extraction treatment.

(Example 2)
<Cell sensitization test>
PER2 :: LUC MEF (mouse embryonic fibroblasts) was maintained and cultured in DMEM (Dulvecco-modified Eagle's medium) containing 10% fetal bovine serum (FBS) and 0.1 mM D-luciferin sodium salt. Then, dexamethasone was added to the medium to a concentration of 200 nM and cultured for 2 hours, and then the medium was replaced with a medium containing no dexamethasone and cultured. Then, within ± 2 hours 2.4 days after the addition of dexamethasone, the dry powder containing the water extract of Albizia bark prepared as described above was added to the medium to 50 μg / ml and 100 μg / ml. After adding and culturing for 30 minutes, the medium was replaced with the medium containing no water extract of Albizia bark and continued culturing. In addition, for 5.5 days after dexamethasone treatment, cells were set at 37 ° C. and 5% CO ₂ and cultured in an incubator equipped with a Lumicycle (LumiCycle32, Analytics), and the expression rhythm of the PER2 gene was observed. Was analyzed. The obtained results are shown in FIG.

PER2 :: LUC MEF is an embryonic fibroblast isolated from Per2 :: luciferase knock-in mouse. In addition, since the firefly luciferase gene has been introduced downstream of the Per2 gene in the mouse, the expression rhythm of the Per2 gene can be followed by the luminescence rhythm of the luciferin-luciferase reaction (Narishige S. et al., Br J Pharmacol. , December 2014, Vol. 171 and No. 24, pp. 5858-5869).

In addition, dexamethasone, which is a synthetic corticosteroid (synthetic glucocorticoid), also functions as a circadian rhythm synchronization factor for peripheral organs (cells), so that the expression cycle of clock genes in each cell is reset and synchronized. , As described above, added to PER2 :: LUC MEF.

Furthermore, in the analysis of the expression rhythm (luminescence rhythm) of the Per2 gene, detrending processing is performed in which the moving average value for 24 hours is subtracted from the measurement data (RAW data) in the lumino cycle, and then the moving average value for 2 hours is used. A smoothing process for smoothing was performed, and a peak value of the emission value of this waveform data was calculated (Hayasaka N. et al., Endocrinology, 2007, Vol. 148, No. 7, pp. 3316-3326, Ohta H. et al.,
PLoS ONE, 2008, 3: e2601). In addition, changes in the values of peak2 and peak3 shown in FIG. 2 were analyzed. The results are shown in Table 1.

(Example 3)
<Administration test to mice>
As shown in FIG. 3, 7-week-old female ICR mice were subjected to water extract of Albizia japonica bark (1 mg or 10 mg of dry powder prepared as described above dissolved in distilled water) or distilled water in ZT4 (12: At the timing of 00), oral administration was forcibly performed using a sonde for 3 days. Then, at ZT5.5 on the final day of administration, these mice were dissected and the liver was removed. Next, total RNA was extracted from the excised liver, reverse transcribed into cDNA, and then the expression levels of the Per1, Per2 and Bmal1 genes were analyzed by real-time PCR. The obtained results are shown in FIGS. 4 to 6.

(Example 4)
<Human administration test>
Under the following conditions, we asked humans to ingest the water extract of Albizia bark every day for 10 days and used it for sleep tests (edited by Masato Matsuura, "Basics and Clinical Practice of Sleep Tests", Co., Ltd. Emerging Medical Publisher, published August 10, 2009, pp. 149-155).
Subjects: 29-49 years old, healthy subjects (5 males, 4 females, 9 in total)
Ingestion: 100 mg of dry powder prepared as described above in a capsule (gelatin capsule manufactured by Hill Half Research Institute Co., Ltd.) is orally watered between 8:30 and 9:00 am for 10 days. I had it taken with.
Sleep test: The following KSS and VAS were performed on the day before ingestion of the water extract of Albizia bark and at the timing of 13:00 to 16:00 on the 10th day to evaluate sleepiness. In addition, on the day before and on the 10th day before ingestion of the water extract of Albizia bark, the tendency of morning type and night type in the following MEQ was investigated.

Kwansei Gakuin University Sleeping Scale (KSS; Kwansei-Gakuin Sleeping Scale)
The KSS is a drowsiness questionnaire prepared as a Japanese version with reference to the Stanford Drowsiness Scale, and consists of 22 items. Each item is given a scale value of 0 to 7, and the larger the number, the stronger the drowsiness. Calculate the average scale value of the selected item (multiple selections allowed) as the drowsiness score (Kaneyoshi Ishihara et al., "Sleep Scale and Its Experimental Study", Psychology Research, 1982, Vol. 52, pp. 362-365. reference). The obtained results (mean values of a total of 9 subjects) are shown in FIG.

Visual analog scale (VAS)
A visual analog scale (VAS) is used to assess a subject's subjective sleep. More specifically, by writing words such as "not sleepy" or "very sleepy" on the left and right ends of a 100 mm straight line, and having them draw a vertical line at a position that seems to be close to the current state. be. The distance from the point 0 to the vertical line is measured in millimeters, and the value is used as the drowsiness score. The score is 1 point per 1 mm, and the score is 0 to 100 points. This time, in addition to the degree of arousal, the survey was conducted on a total of eight items: motivation, mood, feelings, physical fatigue, concentration, appetite, and self-confidence. The obtained results (mean values of a total of 9 subjects) are shown in FIGS. 9 to 12.

Morning / Night Questionnaire (MEQ; Morningness-Eveningness Questionnaire)
MEQ is a questionnaire that examines individual differences in sleep / wake rhythms and lifestyle rhythm orientation, and consists of 19 questions. Scores are given according to the answers to each item, and the higher the total score, the more likely it is to be a morning type (Kaneyoshi Ishihara et al., "Japanese version of morning type-eveningness"). Questionnaire Survey Results ”, Psychology Research, 1986, Vol. 57, pp. 87-91). The obtained results are shown in FIG. Regarding MEQ, we also prepared subjects who had the water extract of Albizia bark taken at dinner instead of taking it in the morning (8:30 to 9:00), and had them undergo this test. The obtained results (mean values of a total of 9 subjects) are shown in FIG.

As is clear from the results shown in FIG. 2, by adding a water extract of Nemunoki bark to mouse embryonic fibroblasts, the amplitude of the clock gene (Per2 gene) of the cells is changed over time. Suppressed attenuation (maintained Per2 gene expression levels). Furthermore, as is clear from the results shown in Table 1, when the water extract of Albizia bark is added to the cells in the process of tapering the Per2 expression rhythm, the peak (phase) of the expression rhythm is concentration-dependent. Advance to. On the other hand, although not shown in the figure, it was clarified that when the extract was added to cells in the process of gradually increasing the Per2 expression rhythm, the peak (phase) of the expression rhythm receded in a concentration-dependent manner. rice field.

Therefore, it was clarified that the water extract of Albizia bark can be used to regulate the expression level and phase of the clock gene because the phase can be moved back and forth depending on the timing of administration.

In addition, as shown in FIGS. 4 and 5, as a result of oral administration of a water extract of Albizia japonica bark to mice, the expression levels of the Per1 gene and the Per2 gene in peripheral tissues (liver) decreased in a dose-dependent manner. On the other hand, as shown in FIG. 6, the expression level of the Bmal1 gene in peripheral tissues was increased by the administration of a water extract of Albizia japonica bark.

In mammals, the BMAL1 protein forms a heterodimer with the CLOCK protein and promotes transcription of the Period genes (Per1, Per2). On the other hand, the PER protein forms a heterodimer with the CRYPTOCHROME protein and suppresses the activities of the BMAL1 protein and the CLOCK protein. It is known that the circadian rhythm is adjusted by such a feedback loop, and the expression rhythms of the Period gene and the Bmal1 gene are shifted by about 12 hours. Therefore, the discrepancy in the expression level of the clock genes (Per1 and Per2 and Bmal1) shown in FIGS. 4 to 6 reflects that Per1 and Per2 and Bmal1 have an opposite phase relationship in the expression rhythm. It is thought that it is.

Furthermore, in consideration of the change in Per1 gene expression level every 3 hours from ZT0 to 24 in mice (without administration of water extract of Albizia japonica) (see FIG. 7), ZT5. Since the expression level of the Per1 gene of No. 5 was decreased, it is suggested that the phase of the expression rhythm of the gene is receded.

From the above results in mice, it was also clarified that oral ingestion of a water extract of Albizia japonica can be used to regulate the expression level and phase of clock genes in peripheral tissues.

In addition, as shown in FIGS. 8 to 12, as a result of oral administration of a water extract of Albizia bark to humans, the extract improved the drowsiness in the afternoon (decreased KSS score), and also arousal, motivation, mood, and concentration. It was revealed that the force was also significantly improved (increased VAS score).

Furthermore, as shown in FIG. 13, as a result of oral administration of a water extract of Albizia bark to humans in the morning, it was revealed that the morning-type tendency became stronger (increase in MEQ score). On the other hand, as shown in FIG. 14, no significant change in the MQ score was observed even when the extract was administered in the afternoon (at the time of dinner).

As described above, oral administration of a water extract of Albizia bark reduced afternoon drowsiness, increased arousal, motivation, mood and concentration, and strengthened the morning tendency. From this, the ingestion of the extract changed the expression rhythm of the clock gene, and as a result of the change in the circadian rhythm, sleep onset was quicker, it became easier to get up early in the morning, and drowsiness in the afternoon was reduced. It is presumed that the sex has improved.

As described above, according to the present invention, the expression of the clock gene can be changed, and the circadian rhythm can be changed by the change in the expression. Further, since the abnormality in the circadian rhythm can be eliminated by causing such a change, the composition of the present invention treats and improves (alleviates) a disease (circadian rhythm disorder, etc.) caused by the abnormality. Alternatively, it is useful as a medicine, food, etc. for prevention. Furthermore, the composition of the present invention is also useful as a reagent for changing the expression of a clock gene and for changing the circadian rhythm due to the change in expression.

Claims (5)

It contains only a water extract of Nemunoki bark as an active ingredient, and the water extract advances or advances the phase of the expression rhythm of the clock gene of mammals, which is an extract of Nemunoki bark extracted with water at a temperature of 70 to 100 ° C. A composition for retreating. The composition according to claim 1, which is a composition for advancing or retreating the phase of a circadian rhythm. It contains only a water extract of Nemunoki bark as an active ingredient, and the water extract is an extract of Nemunoki bark extracted with water at a temperature of 70 to 100 ° C. Composition for suppression. The composition according to any one of claims 1 to 3, wherein the clock gene is at least one gene selected from the group consisting of the Per1 gene, the Per2 gene and the Bmal1 gene of mammals. The composition according to claim 1 to 4, which is a composition for treating, ameliorating or preventing a circadian rhythm disorder and a symptom associated with the circadian rhythm disorder.

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