JP6976515B2 - Sleep improver - Google Patents

Description

The present invention relates to a sleep improving agent containing an amino acid having a sulfinyl group.

Human sleep is divided into REM sleep with rapid eye movement (REM) and non-REM sleep without it, and humans have REM sleep and non-REM sleep during sleep. It repeats at regular intervals. In REM sleep, the body is in a state where skeletal muscles are relaxed and rested, but the brain is in a state of being awake and active, and humans dream during REM sleep. On the other hand, non-rem sleep is a state of sound sleep, and the brain also stops its activity, and it does not wake up with some noise or light shaking. It is said that there are 1 to 4 stages in non-rem sleep, and low-frequency brain waves called delta waves are frequently observed when the sleep is in deep stages (stages 3 to 4). Usually, non-REM sleep is reached shortly after falling asleep, and then the sleep gradually becomes lighter and becomes REM sleep, and after that, non-REM sleep and REM sleep appear alternately. It is said that 4 to 6 times of REM sleep appear in the average sleep time of one night, and there is a tendency that non-REM sleep is more frequent in the first half of the night and REM sleep is more frequent in the latter half (near dawn). It has also been reported that older people tend to have less time for non-rem sleep, resulting in lower sleep quality.

Good sleep is not only indispensable for mental and physical rest, but also deeply involved in memory reconstruction. For good sleep, the proportion of time from bedtime to waking up that is awake both physically and mentally is small, non-rem sleep time is long, and delta waves are observed among them. It is important to have a deep sleep time. Furthermore, it is said that the time of the first sleep cycle from non-REM sleep to REM sleep after falling asleep is responsible for the repair of brain nerve cells, etc., and it is also said that this time is sufficiently secured, sleep rhythm, etc. It is important from the point of view.

The average sleep time of Japanese people has been decreasing in recent years, and some people complain of sleep dissatisfaction, such as poor sleep, light sleep and waking up immediately, and feeling tired even when waking up in the morning without feeling a good night's sleep. is increasing. These lack of sleep and the resulting fatigue can lead to inefficiencies in work and even lead to accidents.

In order to avoid such symptoms, medicines such as sleeping pills and sleep-inducing agents may be used, but these medicines require a doctor's prescription and there are concerns about side effects, so they can be used easily and safely. There is a difficult side. In addition, some medicines reduce non-rem sleep, and depending on how they are used, the quality of sleep may actually deteriorate.

Therefore, research is being conducted to utilize sleep-improving ingredients of foods that can be ingested on a daily basis instead of pharmaceuticals, and the effects of yeast-derived adenosine derivatives on sleep induction and sleep quality improvement have been evaluated so far. There is. Patent Document 1 relates to a sleep improving agent containing S-adenosylmethionine-containing yeast as an active ingredient. S-adenosylmethionine is a compound formed by linking adenosine and methionine in a methylsulfonium bond, and acts as a methyl group donor in a methylation reaction in vivo, promotes phospholipid metabolism, and promotes proteins and nucleic acids. It plays an important role in the methylation of methionine. Patent Document 1 describes that S-adenosylmethionine-containing yeast exhibits a sleep-inducing effect even though S-adenosylmethionine itself does not show a sleep-inducing effect. Patent Document 2 relates to a sleep improving agent containing methylthioadenosine (MTA) or a salt thereof as an active ingredient, and particularly focuses on promotion of non-rem sleep and deep sleep and induction of natural sleep by MTA-containing yeast.

On the other hand, regarding sulfur-containing amino acids contained in onion extracts and the like, many studies have focused on the effects on human health, especially the effect of increasing male hormones, but they have been used to induce sleep and improve sleep. The effect has not been confirmed. Patent Document 3 relates to a steroid hormone increasing agent containing an onion or a processed product thereof, and increases steroid hormones in plasma, particularly testosterone, which is a male hormone, and is effective in enhancing male gonad function and male hormones. It is expected to be effective in the preventive treatment of breast cancer, uterine body cancer, ovarian cancer, thyroid cancer, etc. Patent Document 4 relates to a male hormone-increasing agent containing S-1-propenyl-L-cysteine sulfoxide and zinc, and is intended for prevention or improvement of symptoms associated with androgen decline such as male menopausal syndrome. It is said to be valid.

Japanese Unexamined Patent Publication No. 2013-82641 Republished WO2014 / 163152 Gazette Japanese Unexamined Patent Publication No. 9-169661 Japanese Unexamined Patent Publication No. 2016-150911

An object of the present invention is to provide a sleep improving agent which is low in cost, safe and easy to ingest.

The above problem can be solved by a sleep improving agent containing a sulfur-containing amino acid having a sulfinyl group, that is, (-S (= O)-). More specifically, the sleep improving agent is a sleep quality improving agent, a non-rem sleep promoting agent, and a sleeping rhythm improving agent.

The sulfur-containing amino acid having a sulfinyl group of the present invention has an effect of improving sleep quality and sleep rhythm and promoting non-rem sleep. Further, according to the present invention, since the effect can be obtained by the sulfur-containing amino acid having a sulfinyl group derived from a safe plant of the genus Allium, it is simple and highly safe, so that it can be continuously ingested.

The present invention includes the following forms.
(1) A sleep improving agent containing a sulfur-containing amino acid having a sulfinyl group.
(2) Sulfur-containing amino acids having a sulfinyl group are cycloalliin, S-methylcysteine sulfoxide, S-propylcysteine sulfoxide, S-allyl-L-cysteine sulfoxide, S-1-propenyl-L. -A sleep improving agent according to (1), which is one or more selected from cysteine sulfoxide.
(3) The sleep improving agent according to (1) or (2), which contains 5 to 1000 mg of a sulfur-containing amino acid having a sulfinyl group as a daily intake.
(4) The sleep improving agent according to any one of claims (1) to (3), wherein a sulfur-containing amino acid having a sulfinyl group is contained in the onion extract.
(5) The sleep improving agent according to any one of (1) to (4), wherein sleep improvement is improvement of sleep quality.
(6) The sleep improving agent according to any one of (1) to (4), wherein sleep improving is promotion of non-rem sleep.
(7) The sleep improving agent according to any one of (1) to (4), wherein sleep improvement is improvement of sleep rhythm.
(8) The sleep improving agent according to any one of (1) to (7), which is characterized by targeting females.

The sulfur-containing amino acid having a sulfinyl group, which is the active ingredient of the present invention (hereinafter, simply referred to as “sulfate-containing amino acid”), is the above-mentioned cycloalliin, cysteine sulfoxide, and analogs thereof. For these, synthetic products may be used, or commercially available reagents may be used. Alternatively, a composition containing a sulfur-containing amino acid produced by extraction, purification or the like from a natural product such as an animal or plant containing the sulfur-containing amino acid can also be used. Preferably, cycloallyin and S-methylcysteine sulfoxide (MCSO), S-propylcysteine sulfoxide, S-allyl-L-cysteine sulfoxide, S-1 extracted from Allium plants such as onions. Examples thereof include compositions containing sulfur-containing amino acids selected from -propenyl-L-cysteine sulfoxide (PeCSO).

Among them, since PeCSO and MCSO, which are abundantly contained in onions, are highly effective, onion extracts, which are sulfur-containing amino acid compositions derived from onions, are most preferable. However, since onions contain CS lyase (alliinase), which is an enzyme that converts PeCSO and MCSO to sulfenic acid, when plant tissue is destroyed by operations such as extraction and cooking, the action of the enzyme causes it. PeCSO and MCSO in the plant are lost. Therefore, PeCSO and MCSO are hardly contained in the extract of the above-mentioned plant and the cooked food containing the above-mentioned plant obtained by the conventional usual method.
Therefore, when preparing PeCSO and MCSO from onions, it is preferable to heat-treat the plant before cutting to inactivate the CS lyase and then perform the extraction treatment. As such a method, for example, the method for preparing cysteine sulfoxide from onion, which is described in Patent Document 4, can be adopted. More specifically, the uncut plants of the genus Allium are heat-treated at a pressure of 1 to 5 atm and a temperature of 40 to 150 ° C. for 5 to 120 minutes to remove sulfur-containing amino acids contained in the plants. Inactivates the degrading CS lyase. Then, the heat-treated onion obtained is extracted with alcohol and concentrated under reduced pressure to obtain a heat-treated extract containing an L-cysteine sulfoxide derivative.

The detailed procedure of this method will be described below by taking an onion as an example.
Preferably, the cysteine sulfoxides used in the present invention, especially PeCSO and MCSO, are (i) heated onions and (ii) treated with γ-glutamyl bond cleaving enzyme and then (iii). It can be prepared by subjecting the obtained enzyme-treated product to ion exchange chromatography. The steps (i) to (iii) are preferably carried out under acidic pH conditions in order to prevent deterioration of PeCSO and MCSO, unless required for an enzymatic reaction or the like. The preferred pH is pH 5.5 or less, more preferably pH 4.5 or less.

In step (i), the onions are heated. The heating inactivates the enzyme CS lyase, which decomposes PeCSO and MCSO contained in onions. The heating conditions are not particularly limited as long as they can inactivate the CS lyase without deteriorating the target PeCSO and MCSO, but for example, the pressure is 1 to 5 atm and the temperature is 40 to 150 ° C. It is preferably 5 to 120 minutes, and more preferably 15 to 40 minutes at a pressure of 1 to 2 atm and a temperature of 80 to 120 ° C.

When the inside of an onion is exposed to the air by cutting, crushing, perforating or the like, the contained PeCSO and MCSO are decomposed and the content thereof is reduced. Therefore, the heating is preferably performed on undivided onions. Here, "undivided" onions are either uncut, fragmented, crushed, perforated, scratched, etc., or have been processed but have a sulfur-containing amino acid content due to the processing. Those that are not significantly reduced, for example, those that can achieve a final yield of sulfur-containing amino acids of 80% or more with respect to intact onions.

Step (ii) is a step of treating the onion heated in the above step (i) with a γ-glutamyl bond-cleaving enzyme. Since a part of PeCSO and MCSO in the onion exists as a glutamil form, glutamic acid is cleaved from the glutamil form by enzymatic treatment to release PeCSO and MCSO. In order to sufficiently proceed the enzymatic reaction, it is preferable to cut, crush, shred, etc. the onion heated in the above step (i) before the enzymatic treatment. The subdivided onions are further diluted with an aqueous liquid such as water, acidic water, and alkaline water about 2 to 20 times. The pH of the aqueous liquid is adjusted to the optimum pH of the γ-glutamyl bond-cleaving enzyme to be used later or its vicinity.

Examples of the γ-glutamyl bond-cleaving enzyme used for the enzyme treatment include γ-glutaminase, γ-glutamyl transferase, γ-glutamyl transpeptidase, and γ-glutamyl peptidase. These enzymes may be extracted from animals, plants, microorganisms, etc., or may be commercially available products. Examples of commercially available products include glutaminase SD-C100S manufactured by Amano Enzyme. The conditions for the enzyme treatment may be appropriately set according to the optimum conditions for the enzyme, the type of Allium plant to be used, the site to be used, the size, the state of subdivision, and the like. Generally, the amount of the enzyme added is 0.001 to 1% by mass, preferably 0.01 to 0.1% by mass, based on the total amount of the onion. The reaction conditions may be the optimum pH of the enzyme, about 1 to 24 hours at a temperature of 15 to 65 ° C., preferably about 2 to 6 hours at 35 to 60 ° C. After the completion of the above enzyme treatment, it is preferable to inactivate the γ-glutamyl bond-cleaving enzyme by heating, pH adjustment, or the like. If necessary, the reaction product obtained by the above enzyme treatment may be filtered, centrifuged, squeezed or the like to separate the solution containing PeCSO and MCSO. Further, the obtained solution may be concentrated.

In the step (iii), the reaction product obtained by the enzymatic treatment of the above step (ii) is subjected to ion exchange chromatography. The ion exchange resin for the ion exchange chromatography may be a cation exchange resin, but a strongly acidic cation exchange resin is preferable, and a sulfonic acid type strongly acidic cation exchange resin is more preferable. Commercially available products can be used as the ion exchange resin, for example, Diaion (registered trademark) UBK-550, Diaion (registered trademark) SK1B (manufactured by Mitsubishi Chemical Corporation), Amberlite (registered trademark) IR120B, Amber. Light (registered trademark) 200C, Dowex (registered trademark) MSC-1 (The Dow Chemical Company), Duolite C26 (Rohm and Haas), LEWATTI (registered trademark) SP-112 (LANXESS Distribution GmbH), etc. are preferably used. Can be done. Ion exchange chromatography may be performed according to a usual procedure. The eluate containing PeCSO and MCSO obtained by ion exchange chromatography may be used as it is in the present invention, but concentration or further desalting treatment is preferable because the purity of PeCSO and MCSO is increased.

By the above-mentioned method, a fraction containing a high amount of PeCSO and MCSO can be obtained from the onion. The obtained fraction can be dried as it is and contained in the sleep improving agent of the present invention. Further, if necessary, in addition to or in place of the above-mentioned drying, treatments such as freeze-drying, solidification, granulation, powdering, granulation, and tableting may be performed.

The content of sulfur-containing amino acids in the sleep improving agent of the present invention is preferably 5 to 1000 mg, more preferably 6 to 200 mg, still more preferably 6 to 200 mg, based on the daily intake of an adult as the content of PeCSO and MCSO. Is 7 to 50 mg, most preferably 9 to 30 mg. Specifically, when the sleep improving agent of the present invention is in the form of granules or tablets, the content of sulfur-containing amino acids is, for example, 0.2 to 10% by mass as the content of PeCSO and MCSO, preferably 0. It is 5 to 5% by mass.

The sleep improving agent of the present invention may be a medicine, a food or drink, a feed or the like for improving sleep. The sleep improver of the present invention may be applied to human or non-human mammals. Examples of non-human mammals include pet animals, livestock animals, and animals bred in breeding facilities. The sleep improving agent of the present invention can also be contained in food or feed. When used as food or feed, it is preferable to contain PeCSO and MCSO so that the daily intake is within the above range, but in consideration of long-term intake, 1 The content may be such that the daily intake is less than 5 mg.

In the present invention, sleep was evaluated by measuring the brain waves of the subject during bedtime. Sleep quality is assessed by delta power values, which indicate the amount of low frequency brain waves (delta waves) that appear during non-rem sleep. Non-rem sleep volume is assessed by the length of non-rem sleep time during sleep. Sleep rhythm is assessed by the length of time in the first sleep cycle (sleep onset-non-REM sleep-REM sleep).

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

(Production Example) Production of onion extract After washing and dehulling 5000 g of onion (Kita Momiji 2000), the whole onion was heat-treated in a hot water bath at 95 ° C. for 20 minutes without cutting. The heat-treated onions were crushed using a mixer (manufactured by Oster), and 1 mL of water was added and dispersed per 1 g of the onions. Glutaminase (glutaminase SD-C100S; manufactured by Amano Enzyme) was added to the obtained dispersion in an amount of 0.025% by mass based on the total amount of onions in the solution, and the reaction was carried out at 60 ° C. for 2 hours to complete the reaction. After that, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. The obtained reaction solution was centrifuged at 6000 rpm for 30 minutes, suction-filtered, and then freeze-dried to obtain about 500 g of a crude onion heat-treated extract containing about 3% by mass of sulfur-containing amino acids.
Distilled water was added to the above-mentioned crude onion heat-treated extract to obtain a 30% (w / v) aqueous solution. 1000 mL of this aqueous solution was used as a sample solution and passed through 500 mL of a strongly acidic cation exchange resin (Diaion SK1B, manufactured by Mitsubishi Chemical Corporation) regenerated with hydrochloric acid. Then, the sample solution remaining in the column was washed out with 3000 mL of distilled water. Then, 1000 mL of a 5% sodium hydroxide solution (pH = 14) was passed through the column to elute the sulfur-containing amino acids adsorbed on the ion exchange resin. Further, 2000 mL of distilled water was added to elute the liquid remaining in the column. The eluate eluted with the sodium hydroxide solution and the eluate eluted with distilled water are combined, concentrated with an evaporator (manufactured by Tokyo Rika Kikai), desalted, and then freeze-dried to obtain an excipient. The mixture was added to obtain a powdered onion extract containing a sulfur-containing amino acid. This powder contained about 2.2% by mass of PeCSO, about 0.72% by mass of MCSO, and about 3.8% by mass of cycloalliin.

(Example 1)
The onion extract of the production example was blended so as to contain 30 mg of sulfur-containing amino acids per 3 daily intakes, excipients and the like were added, and then tablets were tableted using a tableting machine to produce tablets. After that, it was confirmed that the appearance, flavor, and properties were indistinguishable by coating, and the test was blinded and used for the test.

(Comparative Example 1)
Excipients were blended in place of the onion extract, and tablets with the same raw materials were produced as placebo tablets. Further, in the same manner as in Example 1, it was confirmed that the appearance, flavor, and properties could not be distinguished by coating, and the cells were blinded and used in the test.

(Test Example 1) Measurement of Mouse Behavior A C57BL / 6 strain mouse (8-week-old male) was purchased from Nippon SLC Co., Ltd. All mice were bred in plastic cages under a light-dark cycle (7 o'clock on, 19 o'clock off) with a light period of 12 hours and a dark period of 12 hours. From 1 week before the start of the test, habituation was performed in individual cages and subjected to the test (n = 4). As the feed, MF (manufactured by Oriental Yeast Co., Ltd.) for mice, rats and hamsters was sterilized at 15 kGy and used.
The amount of activity was recorded every minute for 48 hours using a sensor (Biotex Japan) that detects infrared rays emitted from animals and software Biotex 16CH ActMonitor BAI2216 (Biotex Japan). On the test day, just before the electricity of the breeding cage was turned off, 250 μL of an aqueous solution prepared to be 2000 mg / kg with the onion extract (sulfur-containing amino acid, 6% by mass) obtained in the production example was forcibly orally administered to the mice. Then, the amount of activity was continuously measured by the locomotor. The percentage (%) of the cumulative activity amount from 19:00 to 7:00 after administration was calculated with respect to the cumulative activity amount for 12 hours from 19:00 to 7:00 on the day before the test.

(Test Example 2) Measurement of non-rem sleep amount (mouse)
C57BL / 6 strain mice (9-week-old male) were purchased from Nippon SLC Co., Ltd. All mice were bred in plastic cages under a light-dark cycle (7 o'clock on, 19 o'clock off) with a light period of 12 hours and a dark period of 12 hours. All mice were subjected to electrode implantation surgery for EEG measurement and acclimated for 4 days. Individuals for which it was confirmed that EEG could be measured stably were grouped and acclimated for 1 week in a dedicated individual cage (n = 5-7).
After grouping, the amount of activity, myoelectric potential, and brain wave are continuously measured, and the data is analyzed to calculate the length of non-REM sleep period (delta wave), REM sleep period (theta wave), and arousal period (myoelectric potential). did. SleepSign (manufactured by Kissei Comtech) was used for the analysis.
For the same mouse, 250 μL (50, 100, 200, 400 mg / kg) (sulfur-containing amino acid content, corresponding to 3, 6, 12, 24 mg / kg, respectively) of the onion extract solution obtained in the production example was forcibly applied. Oral administration was performed once, and electroencephalogram analysis was performed for a cumulative 12 hours after administration. It was evaluated whether non-rem sleep increased with respect to the administration of water.

In addition, when the same test of Test Example 2 was carried out using the same histamine H1 receptor knockout mouse as the adenosine A1 receptor knockout mouse which is a sleep-related receptor, the 12-hour cumulative non-rem sleep amount at the time of solvent administration. No significant difference was observed between 12-hour cumulative non-rem sleep and onion extract. This suggests that the sleep-improving effect of the sulfur-containing amino acids of the present invention may be due to the action on the receptors related to sleep.

(Test Example 3) Human test A survey was conducted in advance using the Pittsburgh Sleep Questionnaire, and 10 healthy men and women between the ages of 30 and 50, who had relatively poor sleep conditions, were used as subjects. The study was a placebo-controlled randomized, double-blind crossover study, which was assigned and first ingested the Tablets of Example 1 (or Placebo Tablets of Comparative Example 1) for 5 days, followed by a 2-day rest period. The group that took the tablet of Example 1 took the placebo tablet (the group that took the placebo tablet first was the tablet of Example 1) for 5 days.
After dinner, the subject took one of the tablets three tablets daily with water or lukewarm water, and was equipped with a two-electrode portable electroencephalogram measuring device to measure the electroencephalogram during bedtime.
EEG was analyzed by requesting Sleepwell Co., Ltd. For the electroencephalogram analysis, a sleep scope sleep electroencephalogram analysis program (program for managed medical device electroencephalograph 11467032) was used. In this analysis, we focused on the delta power value, which is an index of sleep depth. When falling asleep, non-REM sleep first appears, and then REM sleep appears. The delta power value from immediately after falling asleep to the appearance of REM sleep was defined as the delta power value at the beginning of sleep. The delta power value was obtained by quantifying the amount of delta waves appearing in the brain waves during sleep.
Since there are individual differences in EEG, the average value of the measurement data for 3 days out of 5 days during the intake period was calculated, and the relative delta power value (%) for each subject was calculated by the following formula.
Relative delta power value (%) = (average value of sleep initial delta power value after ingestion of tablet of Example 1) / (average value of sleep initial delta power value after ingestion of placebo tablet) × 100
The non-rem sleep time was determined by measuring the brain waves during sleep with a sleep scope, analyzing them with a sleep scope sleep brain wave analysis program, and totaling the times determined to be non-rem sleep.
The first sleep cycle is the time from falling asleep to non-REM sleep after going to bed, and then reaching REM sleep and ending REM sleep (sleep onset-non-REM sleep-REM sleep). Generally, it is said that the closer this time is to 90 minutes, the better the sleep rhythm.

In the above endpoints, the subjects who took the tablets of Example 1 had improved sleep as compared with the subjects who took the placebo tablets of Comparative Example 1. Securing the time of the first sleep cycle, which contributes to the improvement of sleep rhythm, showed a more remarkable improvement, especially for females.

Claims (7)

Contains sulfur-containing amino acids with one or more sulfinyl groups selected from cycloallyin, S-methylcysteine sulfoxide, S-propylcysteine sulfoxide, S-1-propenyl-L-cysteine sulfoxide Sleep improver to do. The sleep improving agent according to claim 1, which contains 5 to 1000 mg of a sulfur-containing amino acid having a sulfinyl group as a daily intake. The sleep improving agent according to claim 1 or 2 , wherein a sulfur-containing amino acid having a sulfinyl group is contained in the onion extract. The sleep improving agent according to any one of claims 1 to 3 , wherein sleep improvement is an improvement in sleep quality. The sleep improving agent according to any one of claims 1 to 3 , wherein sleep improvement promotes non-rem sleep. The sleep improving agent according to any one of claims 1 to 3 , wherein sleep improvement is an improvement of sleep rhythm. The sleep improving agent according to any one of claims 1 to 6 , wherein the sleep improving agent is intended for females.