JP2024072788A - Body clock regulator - Google Patents

Abstract

To provide an agent or the like capable of advancing the phase of a clock gene.SOLUTION: A biological clock regulator contains, as an active ingredient, at least one among bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene, and advances the phase of the expression rhythm of a clock gene.SELECTED DRAWING: Figure 2

Description

This disclosure relates to an agent for regulating the biological clock.

It is known that the circadian clock is controlled by the expression regulation of various clock genes (e.g., Per, Cry, Bmal, Clock, etc.) that an organism possesses. Specifically, for example, in mammals, a complex of the BMAL1 protein, which is a product of the Bmal gene, and the CLOCK protein, which is a product of the Clock gene, activates the transcription of the Per gene and the Cry gene. Then, a complex of the PER protein and the CRY protein, which are products of the Per gene and the Cry gene, suppresses the activation of transcription by BMAL1 and CLOCK, forming a feedback loop that oscillates with a 24-hour period. Such circadian clocks include a central clock in the hypothalamic suprachiasmatic nucleus of the brain and a peripheral clock present in various peripheral tissues, and it is known that the expression of clock genes is controlled by a similar mechanism, and clock genes in the peripheral clock also keep rhythm in sync with the central clock. In this way, the circadian rhythm formed by the feedback mechanism of clock genes regulates various biological activities such as the sleep-wake rhythm, the autonomic nervous system (body temperature), the endocrine system (blood hormone concentration), and the immune and metabolic systems. As a result, it is known that disruption of the circadian rhythm can cause various physical and mental symptoms, such as sleep disorders related to falling asleep and waking up, lifestyle-related diseases including obesity and diabetes, and neuropsychiatric disorders including depression.

In order to regulate such circadian rhythms, a technique for regulating the expression of clock genes has been proposed. For example, Patent Document 1 discloses a composition for regulating circadian rhythms that contains black ginger and promotes the expression of Per2, Cry1, Bmal1 and Clock. Patent Document 2 discloses an agent that contains lavender oil or eucalyptus oil as an active ingredient and promotes the expression of clock genes and hyaluronic acid synthase genes. Patent Document 3 discloses a circadian rhythm regulator that contains elemi oil or the like as an active ingredient and induces the expression rhythm of the clock gene Bmal1. Patent Document 4 discloses a circadian rhythm regulator that contains valerian oil as an active ingredient and regulates the circadian rhythm via inhalation stimulation. Patent Document 5 discloses a circadian rhythm regulator that contains Japanese pepper extract as an active ingredient and induces the expression rhythm of the clock gene Period.

JP 2019-94357 A JP 2016-180007 A International Publication No. 2011/122040 JP 2010-215561 A Patent No. 5868313

Known circadian rhythm disorders include jet lag syndrome, shift work sleep disorder, delayed sleep phase syndrome, advanced sleep phase syndrome, and non-24-hour sleep-wake syndrome. For example, jet lag syndrome is caused by traveling long distances in a short time by airplane, shift work sleep disorder is caused by shift work with fluctuating working hours, and delayed sleep phase syndrome can be caused by a nocturnal lifestyle. In particular, in recent years, the problem of "social jet lag" caused by the difference between the sleep pattern on weekdays, which are subject to social constraints, and the sleep pattern on holidays, which are free of constraints (the difference between the sleep and wake-up rhythms on weekdays and holidays), has become prominent. In other words, the number of people who complain of various mental and physical symptoms such as sleep disorders, drowsiness, fatigue, and gastrointestinal symptoms due to the discrepancy between the biological clock and the social clock (due to social constraints such as work and school) is increasing. Such social jet lag is a chronic phenomenon that continues throughout the period when people are required to live according to a social clock that does not match their biological clock due to employment, etc. Among the above-mentioned circadian rhythm disorders, at least some of them, including social jet lag, are often caused by a delay in the phase of sleep onset and awakening compared to the natural rhythm of the body clock. Therefore, there has been a demand for technology that can advance the phase of the circadian rhythm.

The present disclosure can be realized in the following forms.
(1) According to one embodiment of the present disclosure, there is provided a biological clock regulating agent, the biological clock regulating agent comprising at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient, and regulating the biological clock by advancing the phase of the expression rhythm of clock genes.
According to this form of biological clock regulating agent, the biological clock is adjusted by advancing the phase of the expression rhythm of clock genes by containing at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient, and therefore it becomes possible to treat, improve, or prevent circadian rhythm disorders accompanied by a delay in the phase of the expression rhythm of clock genes and symptoms associated with such circadian rhythm disorders.
(2) The biological clock regulating agent of the above-mentioned embodiment may contain at least one of bergamot oil, cedarwood virginia oil, and citral as an active ingredient, and may increase the amplitude of the expression rhythm of the clock gene. With this configuration, the amplitude of the expression rhythm of the clock gene is increased, and the amplitude of the circadian rhythm can be increased, and the circadian rhythm can be made a strong rhythm with a good rhythm. Therefore, the biological clock regulating agent can enhance the effect of treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders.
(3) The biological clock regulating agent of the above embodiment may contain both bergamot oil and citral as active ingredients, which can provide a phase advance effect and an additive amplitude enhancement effect.
(4) The biological clock regulating agent of the above embodiment may be an biological clock regulating agent for advancing the phase of the circadian rhythm. With such a configuration, by advancing the phase of the circadian rhythm, it is possible to bring the phase that has been delayed due to a circadian rhythm disorder or the like closer to a desired (original) phase.
(5) The biological clock regulating agent of the above embodiment may be a biological clock regulating agent for treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders. With such a configuration, it becomes possible to treat, improve, or prevent circadian rhythm disorders caused by jet lag, delayed sleep phase syndrome, social jet lag, etc., and symptoms associated with circadian rhythm disorders (such as various mental and physical symptoms, including sleep disorders, drowsiness, fatigue, and gastrointestinal symptoms).
(6) According to another aspect of the present disclosure, there is provided a transdermal preparation for treating a circadian rhythm disorder, comprising the biological clock regulating agent of the above aspect. With such a configuration, it is possible to treat, improve, or prevent a circadian rhythm disorder or a symptom associated with a circadian rhythm disorder by introducing the biological clock regulating agent into the body through transdermal administration.
(7) According to yet another aspect of the present disclosure, there is provided an orally administered preparation for treating a circadian rhythm disorder, comprising the biological clock regulating agent of the above aspect. With such a configuration, it becomes possible to treat, improve, or prevent a circadian rhythm disorder or a symptom associated with a circadian rhythm disorder by introducing the biological clock regulating agent into the body through oral administration.
(8) According to yet another aspect of the present disclosure, there is provided an aromatic containing the biological clock regulating agent of the above aspect. With such a configuration, it becomes possible to treat, improve, or prevent circadian rhythm disorders or symptoms associated with circadian rhythm disorders by simply installing the aromatic, thereby allowing the biological clock regulating agent to be taken into the body without the need for active ingestion.
(9) According to yet another aspect of the present disclosure, there is provided a bath additive containing the biological clock regulating agent of the above aspect. With such a configuration, it is possible to treat, improve, or prevent circadian rhythm disorders or symptoms associated with circadian rhythm disorders by taking a bath using the bath additive in a simple manner, without the need for active intake, and by introducing the biological clock regulating agent into the body.
(10) According to yet another aspect of the present disclosure, there is provided an inhalant containing the biological clock regulating agent of the above aspect. With such a configuration, by inhaling the inhalant, the biological clock regulating agent is taken into the body, making it possible to treat, ameliorate, or prevent circadian rhythm disorders or symptoms associated with circadian rhythm disorders.
(11) According to yet another aspect of the present disclosure, there is provided a food or drink containing the biological clock regulating agent of the above aspect. With such a configuration, it becomes possible to take in the biological clock regulating agent into the body by ingestion of the food or drink, thereby treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders.
(12) According to yet another aspect of the present disclosure, there is provided a method for adjusting a biological clock, comprising diffusing the biological clock regulating agent according to any one of (1) to (3) above in a space in which a subject suffering from a circadian rhythm disorder is present, and having the subject inhale the biological clock regulating agent.
According to this form of the method for adjusting the body clock, the body clock adjustment agent is diffused in the space in which the subject is present and the subject inhales the body clock adjustment agent, allowing the body to take in the body clock adjustment agent without the subject having to actively ingest it, making it possible to treat, ameliorate, or prevent circadian rhythm disorders or symptoms associated with circadian rhythm disorders.
The present disclosure can be realized in various forms other than those described above, for example, a method for adjusting the body clock that advances the phase of the expression rhythm of a clock gene, or a method for adjusting the body clock that advances the phase of a circadian rhythm.

FIG. 1 is an explanatory diagram showing indicators for evaluating the action of a test substance. An explanatory diagram showing the luminescence rhythm when geranium oil is added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of geranium oil. FIG. 1 is an explanatory diagram showing the change in the relative expression level of Bmal1 upon addition of geranium oil. FIG. 1 is an explanatory diagram showing the luminescence rhythm when bergamot oil is added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of bergamot oil. FIG. 13 is an explanatory diagram showing the change in amplitude due to the addition of bergamot oil. FIG. 1 is an explanatory diagram showing the change in the relative expression level of Bmal1 upon addition of bergamot oil. FIG. 1 is an explanatory diagram showing the change in the relative expression level of Per2 upon addition of bergamot oil. An explanatory diagram showing the luminescence rhythm when cedarwood virginia oil is added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase when cedarwood virginia oil is added. FIG. 2 is an explanatory diagram showing the change in amplitude due to the addition of cedarwood virginia oil. FIG. 1 is an explanatory diagram showing the luminescence rhythm upon addition of β-caryophyllene. An explanatory diagram showing the amount of change in peak phase when β-caryophyllene is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when citral is added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of citral. FIG. 1 is an explanatory diagram showing the change in amplitude due to the addition of citral. FIG. 1 is an explanatory diagram showing the luminescence rhythm when geraniol is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when isomenthone is added. An explanatory diagram showing the luminescence rhythm when linalool is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when d-limonene is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when γ-terpinene is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when α-cedrene is added. An explanatory diagram showing the luminescence rhythm when lavender oil is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when palmarosa oil is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when rosemary oil is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when ylang-ylang oil is added. An explanatory diagram showing the luminescence rhythm when petitgrain oil is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when eucalyptus oil is added. An explanatory diagram showing the luminescence rhythm when Cedarwood Atlas Oil is added. FIG. 1 is an explanatory diagram showing the luminescence rhythm when caffeine is added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of caffeine. FIG. 13 is an explanatory diagram showing the change in amplitude due to the addition of caffeine. FIG. 1 is an explanatory diagram showing the luminescence rhythm when bergamot oil and citral are added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of bergamot oil and citral. An explanatory diagram showing the change in amplitude due to the addition of bergamot oil and citral. FIG. 1 is an explanatory diagram showing the luminescence rhythm when bergamot oil and β-caryophyllene are added. An explanatory diagram showing the change in amplitude due to the addition of bergamot oil and β-caryophyllene. FIG. 1 is an explanatory diagram showing the luminescence rhythm when bergamot oil and caffeine are added. FIG. 2 is an explanatory diagram showing the amount of change in peak phase upon addition of bergamot oil and caffeine. An explanatory diagram showing the change in amplitude due to the addition of bergamot oil and caffeine. An explanatory diagram showing the luminescence rhythm when citral and β-caryophyllene are added. An explanatory diagram showing the amount of change in peak phase upon addition of citral and β-caryophyllene. An explanatory diagram showing the change in amplitude due to the addition of citral and β-caryophyllene.

The biological clock regulating agent of this embodiment is an biological clock regulating agent that contains at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient and advances the phase of the circadian rhythm. The biological clock regulating agent of this embodiment may contain any one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene alone as an active ingredient, or may contain a combination of two or more of the above-mentioned active ingredients. Bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene are all readily available commercially.

(1) Bergamot oil Bergamot oil is an essential oil obtained from bergamot (Citrus bergamia) of the Rutaceae family. Bergamot oil mainly contains limonene, linalyl acetate, linalool, β-pinene, γ-terpinene, β-myrcene, p-cymene, etc. Bergamot oil can be extracted from the peel of bergamot by pressing.

(2) Geranium oil Geranium oil is an essential oil obtained from geranium (Pelargonium graveolens) of the family Geranium family. Geranium oil mainly contains citronellol, geraniol, citronellyl formate, isomenthone, linalool, geranyl formate, β-caryophyllene, etc. Geranium oil can be extracted from geranium flowers and leaves by steam distillation.

(3) Cedarwood Virginia Oil Cedarwood Virginia oil is an essential oil obtained from Juniperus Virginiana, a member of the Cupressaceae family. Cedarwood Virginia oil mainly contains α-cedrene, thujopsene, cedrol, β-cedrene, β-funebrene, widrol, β-himachalene, β-caryophyllene, etc. Cedarwood Virginia oil can be extracted from the wood of Cedarwood Virginia by steam distillation.

(4) Citral Citral is a general term for geranial (trans (E) form) and neral (cis (Z) form), which are chain monoterpene aldehydes and are cis-trans isomers. Citral is a component of essential oils such as lemongrass, lemon balm, lemon verbena, lemon myrtle, lemon, and orange. However, when the biological clock regulating agent of this embodiment contains citral, it is desirable that the citral is contained in the biological clock regulating agent in a form different from that of the essential oil, for example, in the form of refined citral or synthetic citral.

(5) β-caryophyllene β-caryophyllene is a bicyclic sesquiterpene and is a component of essential oils such as ylang-ylang oil, lemon balm oil, rosemary oil, lavender oil, palmarosa oil, geranium oil, and cedarwood virginia oil. When the biological clock regulating agent of this embodiment contains β-caryophyllene, it is preferable that it is contained in the form of, for example, geranium oil or cedarwood virginia oil among the above-mentioned essential oils. Furthermore, when the biological clock regulating agent of this embodiment contains β-caryophyllene, it is preferable that β-caryophyllene is contained in the biological clock regulating agent in a form different from the form of the essential oil, for example, in the form of refined β-caryophyllene or synthetic β-caryophyllene.

(6) Biological clock regulating agent As described above, there are various clock genes such as Per, Cry, Bmal, Clock, and the clock gene whose phase of expression rhythm is advanced by the biological clock regulating agent of the present embodiment is not particularly limited. The biological clock regulating agent of the present embodiment preferably advances the phase of the expression rhythm of the Bmal1 gene, but the clock gene whose phase of expression rhythm is advanced by the biological clock regulating agent of the present embodiment may be, for example, the Per gene or the Cry gene whose transcription is activated by the BMAL1 protein, which is a product of the Bmal gene. Since the circadian rhythm is formed based on the feedback loop of transcription and translation of the clock gene, the biological clock regulating agent of the present embodiment advances the phase of the expression rhythm of the clock gene as described above, thereby making it possible to advance the phase of the entire circadian rhythm.

The biological clock regulating agent of this embodiment has the effect of shortening the period of the expression rhythm of clock genes. Specifically, the biological clock regulating agent of this embodiment advances at least the phase of the expression rhythm of clock genes by administration of the biological clock regulating agent, thereby shortening the period of the expression rhythm of clock genes.

The biological clock regulating agent of this embodiment may further contain, in addition to the active ingredients already described, other ingredients that advance the phase of the expression rhythm of any of the clock genes. For example, the biological clock regulating agent may be configured by combining the active ingredients described above with an ingredient that advances the phase of the expression rhythm of the Bmal1 gene, which is different from the active ingredients of this embodiment described above, an ingredient that advances the phase of the expression rhythm of the Clock gene that encodes the CLOCK protein that forms a complex with the BMAL1 protein, or an ingredient that advances the phase of the expression rhythm of the Per gene or the Cry gene that are expressed with an increase/decrease rhythm opposite to that of Bmal1.

The biological clock regulating agent of this embodiment may contain other ingredients in addition to the active ingredients already described, so long as the effect of advancing the phase of the expression rhythm of clock genes is not impaired. Examples of other ingredients contained in the biological clock regulating agent include excipients, bulking agents, diluents, solubilizers, binders, thickeners, emulsifiers, lubricants, buffers, surfactants, antioxidants, colorants, and fragrances, and may be appropriately selected depending on the product form of the biological clock regulating agent as described below.

The biological clock regulating agent of this embodiment can be used in various product forms, for example, as an oral composition or a parenteral composition. The active ingredient in the biological clock regulating agent taken into the body orally or parenterally is taken into the blood, for example, and is supplied to cells in various parts of the body and acts there.

When the biological clock regulating agent of this embodiment is used as an oral composition, the product form of the oral composition can be, for example, a pharmaceutical product such as an oral preparation, a quasi-drug, a food or drink including health foods, supplements, and beverages. The dosage form of the oral composition can be, for example, a solid preparation such as a tablet, capsule, granules, fine granules, or powder, or a liquid preparation such as a syrup or suspension.

When the biological clock regulating agent of this embodiment is used as a non-oral composition, the product form of the non-oral composition can be, for example, a pharmaceutical product such as a transdermal formulation, a nasal formulation, or a pulmonary (inhalation, etc.) formulation. In addition, other product forms of the non-oral composition can include cosmetics and miscellaneous goods for ingesting the active ingredient of this embodiment into the body.

Among pharmaceuticals, the dosage form of transdermal and nasal administration preparations can be, for example, liquids, emulsions such as creams and milky lotions, gels, or patches. Furthermore, the dosage form of pulmonary administration preparations can be, for example, inhalation preparations in which the biological clock regulating agent is sprayed.

The cosmetic product may be in the form of, for example, a basic cosmetic product including cream or lotion, a makeup cosmetic product, a cosmetic product for hair or scalp, a perfume including eau de toilette, a treatment agent used for massage or the like, or a cleanser including soap. When the biological clock regulating agent of this embodiment is used in the form of a cosmetic product, the active ingredient in the biological clock regulating agent can be taken transdermally and via the lungs.

The miscellaneous goods for ingesting the active ingredient into the body can be, for example, fragrances that are used by diffusing the active ingredient in the biological clock regulating agent into the air, bath additives, and inhalants that volatilize or spray the active ingredient in the biological clock regulating agent and then inhale it. The fragrances include fragrances that are used by storing the biological clock regulating agent in a container or the like and gradually volatilizing the active ingredient into the air, as well as fragrances that are used by using a diffuser such as a heating type, ultrasonic type, or nebulizer type to diffuse the active ingredient into the air, as well as aroma candles and incense. When the biological clock regulating agent of this embodiment is used as a fragrance, bath additive, or inhalant, the active ingredient in the biological clock regulating agent can be ingested via the lungs. When the biological clock regulating agent of this embodiment is used as a bath additive, the active ingredient in the biological clock regulating agent can be ingested via the skin.

The content of the active ingredient contained in the biological clock regulating agent of this embodiment is not particularly limited as long as the phase advance effect of the expression rhythm of the clock gene is obtained, but the ratio to the entire composition of the biological clock regulating agent is preferably 0.00001% by mass or more, and more preferably 0.0001% by mass or more. The content of such active ingredients may be appropriately set, for example, taking into consideration the efficiency of absorption into the body determined by the mode of administration (ingestion) of the active ingredient of the biological clock regulating agent, the irritation effect of the active ingredient on the skin, mucous membranes, etc., etc. For example, when the active ingredient is diluted with a carrier oil and used for oil treatment, the concentration of the active ingredient is preferably 1% or less with respect to the carrier oil. In addition, when the active ingredient is used by diffusing it in a space such as a room, the concentration of the diffused active ingredient is preferably 0.01 ppb to 100 ppm.

According to the biological clock regulating agent of the present embodiment configured as described above, by containing at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient, it is possible to advance the phase of the expression rhythm of clock genes. Therefore, the biological clock regulating agent of the present embodiment can be used to advance the phase of the circadian rhythm, and it is possible to bring the phase that has been delayed due to circadian rhythm disorders, etc. closer to the desired (original) phase. As described above, the biological clock regulating agent of the present embodiment can be used to treat, improve, or prevent circadian rhythm disorders caused by, for example, jet lag syndrome, delayed sleep phase syndrome, social jet lag, etc., and symptoms associated with circadian rhythm disorders (such as various mental and physical symptoms such as sleep disorders, drowsiness, fatigue, and digestive symptoms).

When the biological clock regulating agent of this embodiment contains at least one of bergamot oil, cedarwood Virginia oil, and citral as an active ingredient, in addition to the effect of advancing the phase of the expression rhythm of the clock gene, the effect of increasing the amplitude of the expression rhythm of the clock gene can be obtained. Therefore, the biological clock regulating agent of this embodiment containing at least one of bergamot oil, cedarwood Virginia oil, and citral as an active ingredient can be used to increase the amplitude of the circadian rhythm. In particular, when both bergamot oil and citral are contained as active ingredients, an additive effect can be obtained in terms of the amplitude enhancement effect. By increasing the amplitude, the circadian rhythm becomes a strong rhythm with a sharp rhythm. Therefore, the biological clock regulating agent of this embodiment can enhance the above-mentioned effect of treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders. In addition, it is known that, for example, in the elderly, the amplitude of the circadian rhythm generally tends to become smaller and less sharp. Therefore, by using the biological clock regulating agent of this embodiment, which contains at least one of bergamot oil, cedarwood virginia oil, and citral as an active ingredient, it is possible to improve various disorders caused by a decrease in the ability to maintain circadian rhythms in elderly people and the like.

The active ingredients contained in the biological clock regulating agent of this embodiment, bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene, are all volatile aromatic ingredients. Therefore, the biological clock regulating agent of this embodiment can be easily administered to the subject by diffusing the biological clock regulating agent in a space in which a subject with a circadian rhythm disorder is present and having the subject inhale the biological clock regulating agent. In other words, by filling a space such as a room in which the subject is present with the above-mentioned active ingredients to create a well-being space, it becomes possible to naturally regulate the rhythm of the biological clock without requiring special actions such as actively taking medicines.

<Method of evaluating the effect of test substances on circadian rhythm>
Using a system using mouse embryonic fibroblast cells, the effects of various test substances (essential oils or essential oil components, etc.) including bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene on circadian rhythms were evaluated. Specifically, mouse embryonic fibroblast cells into which the gene for Emerald Luc (Eluc), a luciferase derived from click beetles, was introduced as a reporter gene downstream of the promoter of the clock gene Bmal1 or Per2 were used as evaluation cells. The evaluation cells were cultured in D-MEM medium (high glucose) containing 10% FBS and 100 U/mL Penicillin/Streptomycin at 37°C under a 5% _CO2 atmosphere. Next, the evaluation cells were seeded on a 96-well plate, a 24-well plate, or a 35 mm dish, and 24 hours later, the cells were treated with 100 nM dexamethasone for 2 hours to reset and synchronize the cycle of the circadian rhythm of the evaluation cells. Thereafter, the medium in the plate or dish was replaced with a medium containing 400 μM D-Luciferin (medium containing a luminescence substrate), and the evaluation cells were placed in a luminometer with a culture function (Kronos HT or Kronos Dio, manufactured by ATTO Corporation), which is a measuring device, and the luminescence (expression level of the reporter gene) was measured over time. After the start of the measurement, the time at which the luminescence value reached its peak was confirmed. Three hours after the luminescence value reached its peak, the medium was replaced with a medium containing a luminescence substrate to which the test substance diluted with 0.3% DMSO (dimethyl sulfoxide) solution was added to various concentrations, and the plate or dish was returned to the measuring device to resume the measurement. The evaluation cells were used as a control, in which the medium was replaced with a luminescence substrate-containing medium containing 0.3% DMSO solution not containing the test substance at the same timing as the replacement with the medium containing the test substance described above. Based on the obtained data, the period and amplitude of the measured luminescence intensity were compared with those of the control to evaluate the effect of the test substance.

Figure 1 is an explanatory diagram showing the index of evaluation of the action of a test substance. In Figure 1, the horizontal axis shows time, and the vertical axis shows luminescence intensity, which represents the expression level. Figure 1 shows a schematic diagram of the fluctuation (expression rhythm) of the expression level of a reporter gene after detrending. The reporter gene incorporated downstream of the promoter of a clock gene is expressed according to the expression rhythm of the clock gene. The peaks of the expression rhythm are called peak1, peak2, peak3, etc., in order from the first peak after the medium is replaced with the medium containing the test substance. The length between adjacent peaks, that is, the length of one cycle, is shown as the "cycle length", and the cycle length is about 24 hours. In addition, the magnitude of the fluctuation of the luminescence amount (expression level of the clock gene) in one cycle (the difference between the peak at the beginning of the cycle and the bottom) is called the "amplitude". For example, the amplitude in the cycle between peak1 and peak2 is called amplitude 1, and the amplitude in the cycle between peak2 and peak3 is called amplitude 2. In Figure 1, the magnitude of amplitude 1 is shown by a double arrow. Additionally, changes in the "phase" of the expression rhythm were evaluated based on the timing (time) at which each peak in the expression rhythm appeared. In the expression rhythm shown in Figure 1, when the waveform shifts to the left along the time axis, it is said that the "phase advances," and when the waveform shifts to the right, it is said that the "phase retreats."

<Method for analyzing the relative expression levels of clock genes>
The evaluation cells were prepared in the same manner as described in the above "Method of evaluating the effect of a test substance on circadian rhythm", and the evaluation cells were seeded on a 6-well plate. After 24 hours, the cells were treated with 100 nM dexamethasone for 2 hours to reset and synchronize the cycle of the circadian rhythm of the evaluation cells. After that, the medium in the plate was replaced with the medium containing the luminescent substrate described above, and the evaluation cells were placed in the measurement device with the culture function described above. Then, 3 hours after the luminescence value reached its peak, the medium was replaced with the medium containing the luminescent substrate to which the test substance diluted with 0.3% DMSO (dimethyl sulfoxide) solution was added to various concentrations, and the culture was continued. The cells were collected 0, 4, 8, 12, 16, 20, and 24 hours after the addition of the test substance, and RNA was extracted from 3 wells at each of the above-mentioned collection times using FastGene RNA Basic Kit (manufactured by Nippon Genetics Co., Ltd.). Using 300 ng of the extracted RNA, reverse transcription reaction was performed using PrimeScript (registered trademark) RT Master Mix (Perfect Real Time) (manufactured by Takara Bio Inc.). Then, quantitative PCR (qPCR; quantitative PCR) was performed using Power SYBR Green PCR Master Mix (manufactured by Thermo Fisher Scientific) to analyze the relative expression level of Bmal1 or Per2. The evaluation cells were replaced with a luminescence substrate-containing medium containing 0.3% DMSO solution not containing the test substance at the same timing as the replacement with the medium containing the test substance described above, and the same analysis was performed using them as a control.

<Evaluation Results>
[Geranium oil]
Fig. 2 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when geranium oil was added as a test substance. Fig. 2 shows the results for the cases where the concentrations of geranium oil added to the luminescence substrate-containing medium were 100 μg/mL, 20 μg/mL, and a control. As shown in Fig. 2, when geranium oil was used as a test substance, the phase advanced in a concentration-dependent manner.

Figure 3 is an explanatory diagram showing the phase change of each peak (peak 1, peak 2, peak 3) compared to the control when the geranium oil concentration was 100 μg/mL from the results shown in Figure 2. In Figure 3, the vertical axis shows the phase change compared to the control, and the larger the absolute value of the negative value, the more advanced the phase is. As shown in Figure 3, the addition of geranium oil advanced the phase by a maximum of approximately 3.7 hours.

Figure 4 is an explanatory diagram showing the change in the relative expression level of the Bmal1 gene when geranium oil was added as a test substance. In Figure 4, the expression level of Bmal1 at each elapsed time is shown, with the expression level of Bmal1 at the timing of adding the test substance being 1.0. As shown in Figure 4, when geranium oil was used as the test substance, the phase of the expression rhythm of the Bmal1 gene advanced depending on the concentration of geranium oil. Specifically, in the control, the expression level of Bmal1 peaked after 16 hours, whereas when geranium oil was added at a concentration of 100 μg/mL, the expression level of Bmal1 peaked after 4 hours, and when geranium oil was added at a concentration of 20 μg/mL, the expression level of Bmal1 peaked after 8 hours.

[Bergamot oil]
Fig. 5 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when bergamot oil was added as a test substance. Fig. 5 shows the results for the cases where the concentrations of bergamot oil added to the luminescence substrate-containing medium were 100 μg/mL, 10 μg/mL, and 1 μg/mL, as well as the control. As shown in Fig. 5, when bergamot oil was used as the test substance, the phase advanced and the amplitude increased in a concentration-dependent manner.

Figure 6 is an explanatory diagram similar to Figure 3, showing the phase change of each peak (peak 1, peak 2, peak 3) compared to the control when the bergamot oil concentration was 100 μg/mL, from the results shown in Figure 5. As shown in Figure 6, the addition of bergamot oil advanced the phase by a maximum of approximately 2.7 hours.

Figure 7 is an explanatory diagram showing the results shown in Figure 5, in which the magnitude of amplitude 1 and amplitude 2 is compared with the control when the bergamot oil concentration is 100 μg/mL. As shown in Figure 7, the addition of bergamot oil increased the magnitude of both amplitude 1 and amplitude 2 by more than two-fold. Thus, it was confirmed that the addition of bergamot oil resulted in a strong and well-defined rhythm in the expression rhythm of Bmal1.

Figure 8 is an explanatory diagram showing the change in the relative expression level of the Bmal1 gene when bergamot oil was added as a test substance. In Figure 8, the expression level of Bmal1 at the timing of adding the test substance is set to 1.0, and the relative values of the expression level of Bmal1 at each elapsed time are shown. As shown in Figure 8, when bergamot oil was used as the test substance, the phase of the expression rhythm of the Bmal1 gene was advanced. Specifically, in the control, the expression level of Bmal1 peaked after 12 hours, whereas when bergamot oil was added at a concentration of 100 μg/mL, the expression level of Bmal1 peaked after 4 hours.

Figure 9 is an explanatory diagram showing the change in the relative expression level of the Per2 gene when bergamot oil was added as a test substance. In Figure 9, the expression level of Per2 at the timing of addition of the test substance is set to 1.0, and the relative values of the expression level of Per2 at each elapsed time are shown. As shown in Figure 9, when bergamot oil was used as the test substance, the phase of the expression rhythm of the Per2 gene was advanced. Specifically, in the control, the expression level of Per2 bottomed out after 16 hours, whereas when bergamot oil was added at a concentration of 100 μg/mL, the expression level of Per2 bottomed out after 12 hours.

[Cedarwood Virginia Oil]
Fig. 10 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when cedarwood Virginia oil was added as a test substance. Fig. 10 shows the results for the case where the concentration of cedarwood Virginia oil added to the luminescence substrate-containing medium was 100 μg/mL and the control. As shown in Fig. 10, when cedarwood Virginia oil was used as the test substance, the phase advanced and the amplitude increased.

Figure 11 is an explanatory diagram similar to Figure 3, showing the phase change of each peak (peak 1, peak 2) compared to the control when the cedarwood virginia oil concentration was 100 μg/mL for the results shown in Figure 10. As shown in Figure 11, the addition of cedarwood virginia oil advanced the phase by a maximum of about 1 hour.

Figure 12 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 when the cedarwood virginia oil concentration shown in Figure 10 was 100 μg/mL with the control. As shown in Figure 12, the addition of cedarwood virginia oil increased the magnitude of the amplitude by up to 1.6 times. In this way, it was confirmed that the addition of cedarwood virginia oil resulted in a strong and well-defined rhythm in the expression rhythm of Bmal1.

[β-caryophyllene]
Fig. 13 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when β-caryophyllene was added as a test substance. Fig. 13 shows the results when β-caryophyllene was added to the luminescence substrate-containing medium at concentrations of 500 μM and 100 μM, as well as the control. As shown in Fig. 13, when β-caryophyllene was used as the test substance, the phase advanced in a concentration-dependent manner.

Figure 14 is an explanatory diagram similar to Figure 3, showing the amount of phase change of each peak (peak 1, peak 2) compared to the control for β-caryophyllene concentrations of 500 μM and 100 μM, based on the results shown in Figure 13. As shown in Figure 14, the addition of β-caryophyllene advanced the phase in a concentration-dependent manner, and the phase was advanced by a maximum of approximately 2.3 hours by increasing the concentration of β-caryophyllene to 500 μM.

[Citral]
Fig. 15 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when citral was added as a test substance. Fig. 15 shows the results for the case where the concentration of citral added to the luminescence substrate-containing medium was 100 μM and the control. As shown in Fig. 15, when citral was used as the test substance, the phase advanced and the amplitude increased.

Figure 16 is an explanatory diagram similar to Figure 3, showing the phase change of each peak (peak 1, peak 2, peak 3) when the citral concentration was 100 μM, compared to the control, based on the results shown in Figure 15. As shown in Figure 16, the phase was advanced by the addition of citral, and the phase was advanced by a maximum of about 4 hours by increasing the citral concentration to 100 μM.

Figure 17 is an explanatory diagram showing the results shown in Figure 15, in which the magnitude of amplitude 1 and amplitude 2 is compared with the control when the citral concentration is 100 μM. As shown in Figure 17, the addition of citral increased the magnitude of the amplitude by up to 1.4 times. In this way, it was confirmed that the addition of citral resulted in a strong and well-defined rhythm in the expression rhythm of Bmal1.

[Ingredients of geranium oil]
Fig. 18 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when geraniol was added as a test substance. Fig. 18 shows the results for the cases where the concentrations of geraniol added to the luminescence substrate-containing medium were 100 μM, 10 μM, and 1 μM, as well as the control. As shown in Fig. 18, regardless of the geraniol concentration or the presence or absence of geraniol addition, there was no significant difference in the expression rhythm of Bmal1, and no change in phase due to the addition of geraniol was observed.

Figure 19 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when isomenthone was added as a test substance. Figure 19 shows the results for the control and when the concentrations of isomenthone added to the luminescence substrate-containing medium were 100 μM and 10 μM. As shown in Figure 19, there was no significant difference in the expression rhythm of Bmal1, regardless of the isomenthone concentration or whether or not isomenthone was added, and no change in phase was observed due to the addition of isomenthone.

Figure 20 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when linalool was added as a test substance. Figure 20 shows the results for the cases where the concentrations of linalool added to the luminescence substrate-containing medium were 100 μM, 10 μM, and 1 μM, as well as the control. As shown in Figure 20, no phase change was observed in the expression rhythm of Bmal1 due to the addition of linalool.

The above-mentioned geraniol, isomenthone, and linalool are compounds contained in geranium oil. In addition, when other components of geranium oil, citronellol, citronellyl formate, and geranyl formate, were added to the evaluation cells as test substances, the luminescence rhythm was examined, and no phase change was observed due to the addition (data not shown). Thus, among the components of geranium oil, other than β-caryophyllene shown in Figure 13, no phase advance effect on the expression rhythm of Bmal1 was observed. In the example shown in Figure 2 using geranium oil, when the concentration of geranium oil added to the luminescence substrate-containing medium was 100 μg/mL, the concentration of β-caryophyllene was approximately 6.6 μM.

[Ingredients of bergamot oil]
Fig. 21 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when d-limonene was added as a test substance. Fig. 21 shows the results when the concentrations of d-limonene added to the luminescence substrate-containing medium were 100 μM, 10 μM, and 1 μM, as well as the control. As shown in Fig. 21, regardless of the d-limonene concentration or the presence or absence of d-limonene addition, there was no significant difference in the expression rhythm of Bmal1, and no change in phase due to the addition of d-limonene was observed.

Figure 22 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when γ-terpinene was added as a test substance. Figure 22 shows the results for when the concentrations of γ-terpinene added to the luminescence substrate-containing medium were 100 μM, 10 μM, and 1 μM, as well as a control. As shown in Figure 22, there was no significant difference in the expression rhythm of Bmal1, regardless of the concentration of γ-terpinene or whether or not γ-terpinene was added, and no change in phase was observed due to the addition of γ-terpinene.

The above-mentioned d-limonene and γ-terpinene are compounds contained in bergamot oil. As shown in FIG. 20, linalool, another component of bergamot oil, did not show any effect of advancing the phase of the expression rhythm of Bmal1. Furthermore, when β-pinene, β-myrcene, and p-cymene, which are further components of bergamot oil, were added to the evaluation cells as test substances, the luminescence rhythm was examined, and no change in phase was observed due to the addition (data not shown). Thus, none of the major components contained in bergamot oil showed any phase-advancing effect on the expression rhythm of Bmal1. Although the phase-advancing effect of the expression rhythm was observed when bergamot oil was used as the test substance, the phase-advancing effect was not observed when each component of bergamot oil was used as the test substance. Therefore, for example, it is considered that the phase-advancing effect may occur when multiple components in bergamot oil are combined. Alternatively, it is considered that the phase-advancing effect may occur due to other minor components contained in bergamot oil that are different from the above-mentioned components. Among the components of bergamot oil, linalyl acetate was not evaluated because it is broken down into acetic acid and linalool in the body.

[Ingredients of Cedarwood Virginia Oil]
Fig. 23 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when α-cedrene was added as a test substance. Fig. 23 shows the results for the case where α-cedrene was added to the luminescence substrate-containing medium at a concentration of 100 µM, and for the control. As shown in Fig. 23, a slight phase advance was observed by the addition of α-cedrene, but no amplitude enhancement effect was observed by the addition of α-cedrene. Among the components of cedarwood Virginia oil, β-caryophyllene also showed a phase advance effect as shown in Figs. 13 and 14, but no amplitude enhancement effect was observed.

[Essential oils containing β-caryophyllene]
Fig. 24 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when lavender oil was added as a test substance. Fig. 24 shows the results for the cases where the concentration of lavender oil added to the luminescence substrate-containing medium was 100 μg/mL, 10 μg/mL, and 1 μg/mL, and the control. As shown in Fig. 24, regardless of the lavender oil concentration or the presence or absence of addition of lavender oil, there was no significant difference in the expression rhythm of Bmal1, and no phase change was observed due to the addition of lavender oil.

Figure 25 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when palmarosa oil was added as a test substance. Figure 25 shows the results for palmarosa oil added to the luminescence substrate-containing medium at concentrations of 100 μg/mL, 10 μg/mL, and 1 μg/mL, as well as the control. As shown in Figure 25, there was no significant difference in the expression rhythm of Bmal1, regardless of the palmarosa oil concentration or whether or not palmarosa oil was added, and no change in phase was observed due to the addition of palmarosa oil.

Figure 26 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when rosemary oil was added as a test substance. Figure 26 shows the results when the rosemary oil concentrations added to the luminescence substrate-containing medium were 100 μg/mL, 10 μg/mL, and 1 μg/mL, as well as the control. As shown in Figure 26, there was no significant difference in the expression rhythm of Bmal1, regardless of the rosemary oil concentration or whether or not rosemary oil was added, and no change in phase was observed due to the addition of rosemary oil.

Figure 27 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when ylang-ylang oil was added as a test substance. Figure 27 shows the results for a control and a case where ylang-ylang oil was added to a medium containing a luminescence substrate at a concentration of 1 μg/mL. As shown in Figure 27, there was no significant difference in the expression rhythm of Bmal1 compared to the control, and no phase change was observed due to the addition of ylang-ylang oil. Note that cytotoxicity was observed when ylang-ylang oil was added to a medium containing a luminescence substrate at a concentration of 100 μg/mL and 10 μg/mL.

The above-mentioned lavender oil, palmarosa oil, rosemary oil, and ylang-ylang oil are essential oils that contain β-caryophyllene. In addition, when lemon balm oil, another essential oil that contains β-caryophyllene, was added to the evaluation cells as a test substance, the luminescence rhythm was examined, and no change in phase due to the addition was observed (data not shown). Thus, among the essential oils containing β-caryophyllene, the phase advance effect of the expression rhythm of Bmal1 was not observed except for the geranium oil shown in Figure 2. Although the phase advance effect of the expression rhythm was observed when β-caryophyllene was used as the test substance, the phase advance effect was not observed except for geranium oil when an essential oil containing β-caryophyllene was used as the test substance. Therefore, for example, it is considered that the above-mentioned essential oils other than geranium oil may contain components that inhibit the above-mentioned effect of β-caryophyllene.

[Essential oils containing citral]
Fig. 28 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when petitgrain oil was added as a test substance. Fig. 28 shows the results for the cases where the concentrations of petitgrain oil added to the luminescence substrate-containing medium were 100 μg/mL, 10 μg/mL, and 1 μg/mL, and for the control. As shown in Fig. 28, regardless of the petitgrain oil concentration or the presence or absence of addition of petitgrain oil, there was no significant difference in the expression rhythm of Bmal1, and no change in phase due to the addition of petitgrain oil was observed.

The above-mentioned petitgrain oil is an essential oil that contains citral. In addition, when lemon balm oil, another essential oil that contains citral, was added to the evaluation cells as a test substance to examine the luminescence rhythm, no change in phase was observed due to the addition (data not shown). Thus, essential oils containing citral did not show a phase-advance effect on the expression rhythm of Bmal1. Although a phase-advance effect on the expression rhythm was observed when citral was used as the test substance, no phase-advance effect was observed when an essential oil containing citral was used as the test substance. Therefore, it is considered that, for example, essential oils containing citral as mentioned above may contain components that inhibit the above-mentioned effect of citral.

[Other examples of essential oils]
Fig. 29 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when eucalyptus oil was added as a test substance. Fig. 29 shows the results for the cases where the concentrations of eucalyptus oil added to the luminescence substrate-containing medium were 100 μg/mL, 10 μg/mL, and 1 μg/mL, as well as the control. As shown in Fig. 29, regardless of the eucalyptus oil concentration or the presence or absence of addition of eucalyptus oil, there was no significant difference in the expression rhythm of Bmal1, and no change in phase due to the addition of eucalyptus oil was observed.

The above-mentioned eucalyptus oil and lavender oil shown in Figure 24 have been shown to have an effect of promoting the expression of clock genes such as the Bmal1 gene in, for example, the above-mentioned prior art document 2. However, even though eucalyptus oil and lavender oil have been reported to have an effect of promoting the expression of clock genes, no effect of changing the phase of the expression rhythm of the Bmal1 gene was observed.

Figure 30 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when Cedarwood Atlas (Atlas Cedar) oil was added as a test substance. Figure 30 shows the results for the cases where the concentration of Cedarwood Atlas oil added to the luminescence substrate-containing medium was 10 μg/mL, 1 μg/mL, and the control. As shown in Figure 30, regardless of the Cedarwood Atlas oil concentration or the presence or absence of Cedarwood Atlas oil addition, there was no significant difference in the expression rhythm of Bmal1, and no phase change was observed due to the addition of Cedarwood Atlas oil. When the concentration of Cedarwood Atlas oil was 100 μg/mL, cytotoxicity was observed. Cedarwood Atlas (Cedrus atlantica) is a plant of the Pinaceae family, which belongs to a different family from Cedarwood Virginia.

[Examples of components that exhibit phase delay effects]
Fig. 31 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when caffeine was added as a test substance. Fig. 31 shows the results for the cases where the concentrations of caffeine added to the luminescence substrate-containing medium were 1 mM and 0.3 mM, and for the control. As shown in Fig. 31, the addition of caffeine caused a peak phase delay effect and an amplitude enhancement effect.

Figure 32 is an explanatory diagram similar to Figure 3, showing the amount of phase change of each peak (peak 1, peak 2) compared to the control for caffeine concentrations of 0.3 mM and 1 mM, based on the results shown in Figure 31. As shown in Figure 32, it was confirmed that the addition of caffeine caused the phase to shift backward.

Figure 33 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 with the control for the results shown in Figure 31. As shown in Figure 33, the addition of caffeine increased the magnitude of amplitude 1 and amplitude 2 by approximately two to more than two times.

[Combination of Bergamot Oil and Citral]
FIG. 34 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when bergamot oil, citral, and a mixture of bergamot oil and citral were added as test substances. FIG. 34 shows the results for the case where the concentration of bergamot oil added to the luminescence substrate-containing medium was 100 μg/mL, the case where the concentration of citral was 100 μM, the case where bergamot oil at a concentration of 100 μg/mL and citral at a concentration of 100 μM were used in combination, and the control. As shown in FIG. 34, when bergamot oil and citral were used in combination as test substances, the phase advanced and the amplitude increased.

Figure 35 is an explanatory diagram similar to Figure 3, showing the phase change of each peak (peak 1, peak 2, peak 3) for each test substance shown in Figure 34 compared to the control. As shown in Figure 35, a phase advance effect was observed when bergamot oil, citral, or a combination of bergamot oil and citral was used as the test substance. However, in terms of the phase advance effect, no additive effect was observed by combining bergamot oil and citral.

Figure 36 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 for each of the test substances shown in Figure 34 with the control. As shown in Figure 36, an amplitude enhancement effect was observed when bergamot oil, citral, or a combination of bergamot oil and citral was used as the test substance. In this case, the magnitude of the amplitude increased by up to 2.7 times by combining bergamot oil and citral, and an additive effect was observed by combining bergamot oil and citral.

[Combination of Bergamot Oil and β-Caryophyllene]
FIG. 37 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when bergamot oil, β-caryophyllene, and a mixture of bergamot oil and β-caryophyllene were added as test substances. FIG. 37 shows the results for the case where bergamot oil was added to the luminescence substrate-containing medium at a concentration of 100 μg/mL, the case where β-caryophyllene was added at a concentration of 100 μM, the case where bergamot oil at a concentration of 100 μg/mL and β-caryophyllene at a concentration of 100 μM were used in combination, and the control. As shown in FIG. 37, when bergamot oil and β-caryophyllene were used in combination as test substances, a phase advance effect was observed, similar to the case where bergamot oil was used as the test substance and the case where β-caryophyllene was used as the test substance.

Figure 38 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 for each of the test substances shown in Figure 37 with the control. As shown in Figure 38, an amplitude enhancement effect was observed when bergamot oil, β-caryophyllene, and a combination of bergamot oil and β-caryophyllene were used as the test substance. Here, of bergamot oil and β-caryophyllene, bergamot oil has an amplitude enhancement effect on its own, but the amplitude enhancement effect of the combination of bergamot oil and β-caryophyllene was about the same as that of bergamot oil alone.

[Combination of bergamot oil and caffeine]
Fig. 39 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when bergamot oil and a mixture of bergamot oil and caffeine were added as test substances. Fig. 39 shows the results for the case where bergamot oil was added to the luminescence substrate-containing medium at a concentration of 100 μg/mL, the case where bergamot oil at a concentration of 100 μg/mL was used in combination with caffeine at a concentration of 0.3 mM, and the control.

Figure 40 is an explanatory diagram similar to Figure 3, showing the amount of phase change of each peak (peak 1, peak 2, peak 3) for each of the test substances shown in Figure 39 compared to the control. As shown in Figure 32, it was confirmed that caffeine exhibits a phase-delaying effect, but by combining caffeine with bergamot oil, which exhibits a phase-advancing effect, the phase-delaying effect of caffeine is suppressed, resulting in an overall phase-advancing effect.

Figure 41 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 for each of the test substances shown in Figure 39 with the control. As shown in Figure 41, bergamot oil alone exhibits an amplitude enhancing effect, and caffeine alone also exhibits an amplitude enhancing effect (see Figure 33). Also, as shown in Figure 41, when bergamot oil and caffeine were combined, the amplitude enhancing effect was higher at amplitude 1 than when bergamot oil was used alone, but at other amplitudes, the amplitude enhancing effect was about the same as when bergamot oil was used alone. In this way, it was confirmed that even if test substances that exhibit an amplitude enhancing effect are combined, the amplitude enhancing effect is not necessarily obtained additively.

[Combination of citral and β-caryophyllene]
Fig. 42 is an explanatory diagram showing the luminescence rhythm (expression rhythm of the Bmal1 gene) when citral, β-caryophyllene, and a mixture of citral and β-caryophyllene were added as test substances. Fig. 42 shows the results for the cases where the concentration of citral added to the luminescence substrate-containing medium was 100 μM, the concentration of β-caryophyllene was 100 μM, a combination of 100 μM citral and 100 μM β-caryophyllene, and a control.

Figure 43 is an explanatory diagram similar to Figure 3, showing the amount of phase change of each peak (peak 1, peak 2, peak 3) for each test substance shown in Figure 42 compared to the control. As shown in Figure 43, a phase advance effect was observed when citral, β-caryophyllene, and a combination of citral and β-caryophyllene were used as the test substance. However, in terms of the phase advance effect, no additive effect was observed by combining citral and β-caryophyllene.

Figure 44 is an explanatory diagram showing the results of comparing the magnitude of amplitude 1 and amplitude 2 for each of the test substances shown in Figure 42 with the control. As shown in Figure 44, an amplitude enhancement effect was observed both when citral was used as the test substance and when citral was combined with β-caryophyllene. Here, of citral and β-caryophyllene, citral alone has an amplitude enhancement effect, but the amplitude enhancement effect when citral and β-caryophyllene were combined was about the same as the amplitude enhancement effect when citral was used alone.

The present disclosure is not limited to the above-mentioned embodiments, etc., and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features in the embodiments corresponding to the technical features in each form described in the Summary of the Invention column can be replaced or combined as appropriate to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

The present disclosure can also be realized in the following forms.
[Application Example 1]
An agent for regulating a biological clock, comprising:
Contains at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient;
An agent for regulating the body clock that adjusts the body clock by advancing the phase of the expression rhythm of clock genes.
[Application Example 2]
The biological clock regulating agent according to Application Example 1,
Contains at least one of bergamot oil, cedarwood virginia oil, and citral as an active ingredient;
An agent for regulating the biological clock, which increases the amplitude of the expression rhythm of the clock gene.
[Application Example 3]
The biological clock regulating agent according to Application Example 2,
A biological clock regulating agent that contains both bergamot oil and citral as active ingredients.
[Application Example 4]
The biological clock regulating agent according to any one of Application Examples 1 to 3 or 2,
An agent for regulating the body clock to advance the phase of the circadian rhythm.
[Application Example 5]
The biological clock regulating agent according to any one of Application Examples 1 to 4,
An agent for regulating the body clock for treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders.
[Application Example 6]
A transdermal preparation for treating circadian rhythm disorders, comprising the biological clock regulating agent according to any one of Application Examples 1 to 5.
[Application Example 7]
An oral administration formulation for treating circadian rhythm disorders, comprising the biological clock regulating agent according to any one of Application Examples 1 to 5.
[Application Example 8]
An aromatic agent comprising the biological clock regulating agent according to any one of Application Examples 1 to 5.
[Application Example 9]
A bath additive comprising the biological clock regulating agent according to any one of Application Examples 1 to 5.
[Application Example 10]
An inhalant comprising the biological clock regulating agent according to any one of Application Examples 1 to 4.
[Application Example 11]
A food or drink containing the biological clock regulating agent according to any one of Application Examples 1 to 5.
[Application Example 12]
A method for adjusting a biological clock, comprising:
A method for adjusting a biological clock, comprising diffusing the biological clock adjustment agent described in any one of Application Examples 1 to 5 in a space in which a subject suffering from a circadian rhythm disorder is present, and having the subject inhale the biological clock adjustment agent.

Claims (12)

An agent for regulating a biological clock, comprising:
Contains at least one of bergamot oil, geranium oil, cedarwood virginia oil, citral, and β-caryophyllene as an active ingredient;
An agent for regulating the body clock that adjusts the body clock by advancing the phase of the expression rhythm of clock genes. The biological clock regulating agent according to claim 1,
Contains at least one of bergamot oil, cedarwood virginia oil, and citral as an active ingredient;
An agent for regulating the biological clock, which increases the amplitude of the expression rhythm of the clock gene. The biological clock regulating agent according to claim 2,
A biological clock regulating agent that contains both bergamot oil and citral as active ingredients. The biological clock regulating agent according to any one of claims 1 to 3,
An agent for regulating the body clock to advance the phase of the circadian rhythm. The biological clock regulating agent according to any one of claims 1 to 3,
An agent for regulating the body clock for treating, improving, or preventing circadian rhythm disorders and symptoms associated with circadian rhythm disorders. A transdermal preparation for treating circadian rhythm disorders, comprising the biological clock regulating agent according to any one of claims 1 to 3. An oral preparation for treating circadian rhythm disorders, comprising the biological clock regulating agent according to any one of claims 1 to 3. An air freshener containing the biological clock regulating agent according to any one of claims 1 to 3. A bath additive containing the biological clock regulating agent according to any one of claims 1 to 3. An inhalant containing the biological clock regulating agent according to any one of claims 1 to 3. A food or drink containing the biological clock regulating agent according to any one of claims 1 to 3. A method for adjusting a biological clock, comprising:
A method for adjusting a biological clock, comprising diffusing the biological clock regulating agent described in any one of claims 1 to 3 in a space in which a subject suffering from a circadian rhythm disorder is present, and having the subject inhale the biological clock regulating agent.

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