JP2018118910A - Oyster meat extract that has the effect of suppressing the deterioration of sleep efficiency and the effect of improving sleep difficulty, the improvement of sleep based on the item of sleep of Quality of life (hereinafter referred to as QOL), and the suppression of the increase in mid arousal by maintaining the total number of arousals - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oyster meat extract having an action of suppressing deterioration of sleep efficiency, an action of improving insomnia, an action of improving sleep based on an item of QOL of sleep, and an action of suppressing an increase in midway awakening by maintaining the total number of awakenings. SOLUTION: An oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol contained in an extract extracted from oyster meat as an active ingredient. An oyster meat extract containing the compound produced by heating raw oyster meat in which the active ingredient is not detected in the raw state. An oyster meat extract obtained by heat-treating raw oyster meat at 80 ° C. or higher for 3 hours or longer, preferably 98 to 100 ° C. for 6 hours or longer. [Selection diagram] None

Description

The present invention has an effect of suppressing the deterioration of sleep efficiency using 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as E6) extracted from oyster meat as an active ingredient. The present invention relates to a oyster meat extract having an effect of improving sleep difficulty, improvement of sleep based on sleep items of QOL, and suppression of increase in mid-wakefulness by maintaining the total number of awakenings.

In recent years, it is said that an increasing number of modern people are constantly feeling tired and have problems sleeping.
In other words, it is said that the quality of sleep, sleep time, sleep time, and difficulty in waking up during the day are getting worse.

By the way, it is generally known that the generation of active oxygen is caused by aerobic life and causes oxidation of lipids, proteins, and nucleic acids and damages cells.
Normally, the level of oxidation in the living body is almost constant by the balance between the active oxygen production system and the elimination system by antioxidants, but this balance is lost due to various factors such as drugs, radiation, and ischemia, and active oxygen production Leaning toward the system is said to be oxidative stress.

  This accumulation of oxidative stress is thought to contribute to various diseases and aging such as cancer, arteriosclerotic disease, ischemia / reperfusion injury, rheumatoid arthritis, diabetes, Alzheimer's disease and Parkinson's disease neuropathy It is.

  So-called antioxidants are roughly classified into two groups based on their structure. Examples of enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (glutathionereductase), Examples include peroxiredoxin (Prx). On the other hand, non-enzymatic antioxidants include ascorbic acid, α-tocopherol, α-tocopherol, glutathione, GSH, carotenoids, flavonoids, metallothionein, etc. .

  Here, oysters, such as oysters (Crassostrea gigas), are bivalves belonging to the order of the genus Coleoptera, and their habitat extends throughout Japan and other parts of East Asia. In recent years, oysters have been cultivated in France and Australia and are famous as the most edible oysters in the world.

  Oysters have been edible since ancient times due to their high nutritional value. As described above, oysters are said to contain a large amount of minerals such as calcium, zinc, selenium, copper, and manganese in addition to glycogen and protein.

In addition, SOD, CAT, GPx, and Prx6 are reported as antioxidant substances derived from oysters, and metallothionein, uncouplingprotein5 (UCP5), ascorbic acid are used as non-enzymatic antioxidants. , Α-tocopherol, and β-carotene.

JP 2010-193756 A

  Thus, the inventors of the present invention have already succeeded in finding an excellent novel antioxidant substance from oysters, in particular, oyster soft body, further determining its chemical structure, and chemical synthesis of the antioxidant substance. In addition, the present invention has succeeded in providing excellent so-called novel antioxidants and antioxidant compositions both when they are not derived from oysters or when they are derived from oysters.

  Furthermore, the antioxidant ability of the substance in human low-density lipoproteins (LDL) oxidation experiments and liver cell established cell oxidation experiments has also been confirmed.

  Thus, this time, to the oyster meat extract extracted from the oyster soft body part, the sleep efficiency deterioration suppressing action and the sleep difficulty improving action using E6 as an active ingredient, the sleep improvement based on the QOL sleep items, the total number of awakenings It has succeeded in finding the suppression of the increase in arousal during the maintenance.

Therefore, the present invention, as described above, from the oyster meat extract extracted from the oyster soft body part, the sleep efficiency deterioration effect and the sleep difficulty improvement effect containing E6 as an active ingredient, the sleep of the item based on QOL sleep items An object of the present invention is to produce and provide an oyster meat extract having an improvement and suppression of an increase in midway arousal by maintaining the total number of arousals.

The oyster meat extract having the inhibitory effect on sleep efficiency deterioration and the sleep difficulty improving effect according to the present invention, the improvement of sleep based on the sleep item of QOL, and the suppression of the increase in awakening due to the maintenance of the total number of awakenings,
E6 contained in the extract extracted from oyster meat as an active ingredient,
It is characterized by
Or
By heating raw oyster meat in which E6 is not detected in the raw state, E6 is generated from the heat-treated oyster meat liquid, and the generated E6 is used as an active ingredient.
It is characterized by
Or
The raw oyster meat in which E6 is not detected in the raw state is crushed, and the crushed material is heated at 80 ° C. or higher for 3 hours or more to produce E6 from the heat-treated oyster meat liquid, and the generated E6 as an active ingredient,
It is characterized by
Or
Raw oyster meat in which E6 is not detected in the raw state is heated at 98 ° C. to 100 ° C. for 6 hours or longer to produce E6 from the heat-treated oyster meat liquid, and the produced E6 is used as an active ingredient. ,
It is characterized by
Or
By heating raw oyster meat in which E6 is not detected in a raw state at 90 ° C. or more for at least 9 hours, E6 is produced from the heat-treated oyster meat liquid, and the produced E6 is used as an active ingredient.
It is characterized by
Or
Raw oyster meat in which E6 is not detected in the raw state is put into an extraction solution to extract the oyster meat extract, and the extracted oyster meat extract solution is heated at 90 ° C. or higher for at least 10 hours or more at 1 atm. E6 is produced from the heated oyster meat extract, and the produced E6 is an active ingredient.
It is characterized by
Or
By heating raw oyster meat in which E6 is not detected in a raw state in a pressurized state of 1 atm or higher, E6 is generated from the oyster meat liquid heated in the pressurized state, and the generated E6 is used as an active ingredient. To
It is characterized by
Or
By heating raw oyster meat in which E6 is not detected in a raw state at 90 ° C or higher for at least 1 hour in a pressurized state of 3 atmospheres or more, E6 is generated from the oyster meat liquid heated in the pressurized state, The generated E6 as an active ingredient,
It is characterized by
Or
E6 fraction is collected from oyster meat using ethyl acetate, and the collected E6 is used as an active ingredient.
It is characterized by
Or
E6 fraction is collected from oyster meat using ethyl acetate and ethanol, and the collected E6 is used as an active ingredient.
It is characterized by
Or
The oyster meat was stored in the extraction container in which the extraction solution was stored, and the solution containing the oyster meat extract was collected. Ethyl acetate was added to the collected solution containing the oyster meat extract, and the E6 fraction was collected and collected. E6 as an active ingredient,
It is characterized by
Or
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethyl acetate and ethanol are added to the collected solution containing the oyster meat extract, and the E6 fraction is collected, The recovered E6 is used as an active ingredient.
It is characterized by
Or
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethanol is added to the collected solution containing the oyster meat extract, and the supernatant liquid and the precipitate are separated. The separated supernatant is taken out, and ethyl acetate is added to the supernatant to separate the ethyl acetate layer and the aqueous layer,
The separated solution of the ethyl acetate layer is volatilized and concentrated to recover the E6 fraction, and the recovered E6 is used as an active ingredient.
It is characterized by this.

ADVANTAGE OF THE INVENTION According to this invention, the sleep efficiency deterioration inhibitory effect and sleep difficulty improvement effect which use E6 extracted from the extract of oyster meat as an active ingredient, sleep improvement based on the item of sleep of QOL, and the middle by maintenance of the total number of awakenings There is an excellent effect that an oyster meat extract having suppression of arousal increase can be provided.

It is a table | surface explaining the classification | category and the breakdown of a test subject by Experiment 1 of this invention. It is a table | surface explaining the background of the effectiveness analysis object person by Experiment 1 of this invention. It is a table | surface explaining the total score of AIS by Experiment 1 of this invention. It is a table | surface explaining the numerical value of PSQI-J by Experiment 1 of this invention. It is a table | surface explaining the numerical value (subject group under 50 years old) of PSQI-J by Experiment 1 of this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is schematic structure explanatory drawing (1) which shows schematic structure of this invention. It is schematic structure explanatory drawing (2) which shows schematic structure of this invention. It is explanatory drawing explaining the flowchart of this invention. It is explanatory drawing explaining the state which raises the polarity of the organic solvent for E6 extraction in steps. It is explanatory drawing which shows the result of the antioxidant activity test of each extract. It is explanatory drawing explaining extraction by a silica open column. It is explanatory drawing explaining ethyl acetate fraction extraction. It is explanatory drawing explaining the structure of E6 extracted by this invention. It is explanatory drawing explaining the measurement principle of ORAC method. It is explanatory drawing explaining the antioxidant ability of E6 by this invention. It is explanatory drawing (1) explaining the structural analysis of E6 by this invention. It is explanatory drawing (2) explaining the structural analysis of E6 by this invention. It is a table | surface explaining the background of the test subject of effectiveness analysis by Experiment 2 of this invention. It is a table | surface explaining the background of the test subject by the brain wave and OSA-MA effectiveness analysis by Experiment 2 of this invention. It is a table | surface explaining the QOL survey form by Experiment 2 of this invention. It is a table | surface explaining the electroencephalogram (halfway awakening and sleep efficiency) by Experiment 2 of this invention.

  Hereinafter, the present invention will be described based on an embodiment shown in the drawings.

  6 and 7, reference numeral 1 denotes an extraction container. In the extraction container 1, an extraction solution 2 for extracting an extract from oyster meat is stored. And the raw oyster meat 3 is accommodated in the extraction container 1 in which this extraction solution 2 is stored, and the process of extracting the extract containing various active ingredients of oyster meat is performed.

  By the way, conventionally, when extracting the oyster meat extract, the extraction solution 2 containing the oyster meat 3 in the extraction container 1 was agitated to make the extraction more efficient. The oyster meat 3 itself may be damaged, and it is preferable not to perform the stirring operation at the time of the extraction process.

  The extraction solution 2 from which the oyster meat extract has been extracted as described above is then concentrated in the concentration step.

Next, the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70%. Thereafter, the mixture is stirred and separated into a precipitate 7 and a supernatant 8.
Then, as can be understood from FIG. 6, the precipitate 7 is dried, tableted, and finally used for health food or the like.

  By the way, conventionally, the supernatant liquid 8 may be discarded because it does not contain any active ingredient of oyster meat extract or is contained in a very small amount. However, after that, as a result of experiments and researches, it became clear that many active ingredients relating to the oyster meat extract were also present in this supernatant liquid 8, and now this supernatant liquid 8 is used without being discarded. Yes.

  In recent years, the supernatant 8 is concentrated again and the concentrated solution is finally dried. The dried product does not become a complete solid, but can be formed into a paste, and is manufactured as a paste-like health food. And this paste-form health food is melt | dissolved with a white hot water etc. in the consumer side, and is provided to the health food for drinks.

  First, in this example, the supernatant liquid 8 is used to recover the oyster meat extract containing E6, which will be described later.

That is, after separating into the precipitate 7 and the supernatant liquid 8 as described above, the supernatant liquid 8 is first removed by removing the ethanol content of the supernatant liquid 8 with an evaporator or the like and concentrated to about half the amount.
For example, the supernatant liquid 8 of 40 mL is concentrated to ensure the concentrated liquid 9 of the 20 mL supernatant liquid 8.

  Next, the diluted solution 10 is produced by diluting the 20 mL concentrated solution to about 5 times. For example, the amount of the diluted solution 10 is 100 mL. This process is performed in order to remove impurities as much as possible.

  Thereafter, for example, about 200 mL of ethyl acetate 5 is added to the 100 mL of the diluted solution 10. Then, the aqueous layer 10a and the ethyl acetate layer 11 are separated by stirring or using a separator. Then, with the passage of time, this mixed solution is formed separately into the aqueous layer 10a and the ethyl acetate layer 11.

  Then, when the separated solution of the ethyl acetate layer 11 was volatilized and concentrated, it was confirmed that E6 described later was present in the ethyl acetate layer 11.

  Here, although it was the amount of E6 that could be confirmed, specifically, it was confirmed that there was about 3 mg of E6 from the ethyl acetate layer 11 collected for about 2 L.

  Next, in what process the E6 can be separated and purified from the oyster meat extract, the presence of the E6 in the oyster meat extract has been confirmed, what kind of structure the E6 is composed of, The following explains how the antioxidant action of E6 was confirmed.

First, a description will be given according to the flowchart shown in FIG.
For example, the oyster meat 3 is put into the extraction solution 2 containing the ethanol solution 4 to extract the oyster meat active ingredient extract (step 100, step 102).

  After extraction, the extract is concentrated (step 104). Then, for example, the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70% (step 106). Then, it stirs and isolate | separates into the precipitate 7 and the supernatant liquid 8 (step 108).

  Then, using the supernatant 8, an extraction operation using ethyl acetate for E6 extraction is performed.

As can be seen from FIG. 8, the ethanol content is removed (step 110), and chloroform, ethyl acetate, and butanol are added to each supernatant solution 8 diluted about 5 times to produce each fraction. To do.
For example, it concentrates to 100 mL with a rotary evaporator etc., 80 mL of distilled water is added to 20 mL of this concentrate, and it transfers to a separatory funnel, and hexane extraction is performed.

  After removing the hexane layer (200 mL), the aqueous layer is extracted stepwise in the order of 200 mL chloroform, 200 mL ethyl acetate, and 200 mL butanol.

  That is, the polarity of the organic solvent for E6 extraction is increased stepwise, and fractions into which these are respectively added are generated, and it is confirmed whether E6 is extracted in each fraction (see FIG. 9).

  Next, the fractions charged with the respective organic solvents are concentrated by, for example, an evaporator, and then observed by thin-layer-chromatography (hereinafter referred to as TLC; TLC: thin layer chromatography), and the so-called ORAC method (Oxygen Radial Absorbance Capacity method) ) To search for antioxidant power.

  As a result, in the TLC image, it was confirmed that elution was performed from hexane to chloroform, ethyl acetate, and butanol from low polarity to high polarity.

  Further, a plateau portion was observed in the ethyl acetate fraction by the ORAC method, and a high antioxidant capacity was observed in the ethyl acetate fraction. Therefore, it was judged that E6 was present in this ethyl acetate fraction. (See FIG. 10, step 112 in FIG. 8).

  Next, the ethyl acetate fraction is concentrated by an evaporator and then extracted by a so-called silica open column (step 116 in FIGS. 11 and 8), and the ethyl acetate: chloroform is extracted at a ratio of 3: 2. It was selected (see FIG. 12), and finally, the fraction could be separated and purified by HPLC (reverse phase column) (step 116).

  In this way, E6 could be separated and purified from the supernatant 8 from which the oyster meat extract was extracted.

  E6 can also be separated and purified by the following operation.

First, Trolox dissolved in a mixed solution of 0.075 mol / L phosphate buffer 2.35 mL, 6.3 x 10 ^-7 mol / L Fluorescein (fluorescent probe) 0.3 mL, 7% (w / v) methylated β-cyclodextrin (Wako) ( Wako) or 0.05 mL of the test sample is heated at 37 ° C. for 10 minutes.

1.28x10 ^-1 mol / L preheated to 37 ° C
2, Add 0.3 mL of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH, Wako) and stir with a stirrer, for example, with a spectrofluorometer (FP-6500, JASCO, Tokyo) every 10 seconds for 5,000 seconds The fluorescence intensity (excitation wavelength: 493 nm, fluorescence wavelength: 515 nm) is measured.

  The antioxidant activity is indicated by the length of time (horizontal axis) during which the fluorescence measurement value at the start of measurement (for example, the vertical axis in FIG. 10) is maintained, and the longer the time, the stronger the antioxidant activity. To do.

  Then, the antioxidant activity was confirmed in the ethyl acetate extract fraction among the four kinds of extract fractions.

  Subsequently, normal phase silica gel thin layer preparative chromatography is carried out on the ethyl acetate extract showing the antioxidant activity. Silica gel thin layer plates (200 × 200 mm, thickness 0.5 mm, Merck, Darmstadt) were used, and ethyl acetate-chloroform (2: 1, v / v) was used as the mobile phase. The developed plate was irradiated with a UV lamp (254 nm) to obtain 11 UV-absorbing fractions. A sample of each fraction was separated together with the gel carrier, and when the antioxidant activity was measured after elution with, for example, methanol, the antioxidant activity was observed in the sixth fraction from the low polarity side.

  Further, the fraction showing the antioxidant activity by the thin layer chromatography is purified by high performance liquid chromatography (HPLC). HPLC system (pump: L-2130, UV detector: L-2420, HITACHI, Tokyo), reverse phase column (APCELLPACC18, 250 x 4.6 mm I.D., SHISEIDO, Tokyo), mobile phase 5% acetonitrile aqueous solution (flow rate) 1.0 mL / min) at room temperature.

  Thus, even by this operation, 3.0 mg of E6 was finally obtained from the raw material 160 mL ethanol extract.

  By the way, the presence of E6 is measured by measuring ultraviolet absorption spectrum (V-530, JASCO), nuclear magnetic resonance spectrum (NMR: AMX-500, Bruker, Karlsruhe), mass spectrum (JMS-T100CS, JEOL, Tokyo). Then, structural analysis is performed (FIGS. 16 and 17), and as a result, the structure of the separated and purified product is estimated to be E6.

conditions
UV (EtOH), λ _max 270 nm; ¹ H-NMR (500 MHz, Acetone-d ₆ ) δ _H : 7.82 (2H, br.s, aromatic-OH), 6.40 (2H, s, H-2, 6), 4.42 (2H, s, H-1 ′), 3.94 (1H, br.s, —OH), 3.79 (3H, s, —OMe); ¹³ C-NMR (125 MHz, Acetone-d ₆ ) δ _C : 151.1 (C-3, 5), 139.4 (C-1), 13, 5.1 (C-4), 106.5 (C-2, 6), 64.5 (C-1 '), 60.6 (-OMe); ESI-TOFMS , M / z 153.05451 [M-OH] ⁺ (calc.forC ₈ H ₈ O ₃ , 153.05517), 171.06911 [M + H] ⁺ (calc.forC ₈ H ₁₁ O ₄ , 171.006573).

  Here, the properties of the separated and refined E6 will be described. The properties are yellowish pale powder, which shows fat solubility and water solubility.

  Further, it was confirmed that E6 is a phenolic compound as shown in FIG.

  In this embodiment, the antioxidant power is measured by the ORAC method.

By the way, in Japan, there is already a study group (AntioxidantUnit Study Group) that conducts research on the officialization of the ORAC method. The advantage of the ORAC method is that both water-soluble and fat-soluble samples can be measured, and any organic solvent fraction described above can be measured.
In addition, the duration of the antioxidant action and its titer can be evaluated in one measurement, and the experimental operation is easy, so it is considered advantageous for the measurement in this example.

  Here, the measurement principle of the ORAC method will be described briefly. First, when a certain reactive oxygen species is generated, the fluorescence intensity decomposed thereby is measured, and a fluorescence intensity curve that decreases with time is drawn, if an antioxidant coexists in this reaction system, the fluorescence of the fluorescent substance The rate of decrease in intensity is delayed. Therefore, the existence of the antioxidant substance can be confirmed by this principle (see FIG. 14).

  Thus, when the antioxidant ability of E6 in the present invention was observed by the ORAC method, a delay period was present as in the so-called standard substance (Trolox), and a strong antioxidant activity could be observed (FIG. 15). reference).

  In this example, in order to search from the supernatant solution 8 described above, E6 having a high antioxidant power in the ethyl acetate fraction could be found using the ORAC method.

  Next, Example 2 will be described.

  Conventionally, E6 has not been found in raw oyster meat, and it has not been confirmed by any production how the active ingredient E6 is produced from raw oyster meat.

However, when the present inventors produced a oyster meat extract containing many active ingredients from raw oyster meat, 3,5-dihydroxy-4-methoxybenzyl, which was not found at all from the original raw oyster meat. They succeeded in finding a production method capable of producing alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol: hereinafter may be referred to as E6) at the extraction stage of oyster meat extract.
And it came to patent application as Japanese Patent Application No. 2014-169316.

(Heating experiment)
In a heating experiment (without pressurization), 1 L of pure water was added to 1 kg of raw oyster, heated at standard pressure for 1 hour, for example, at 90 ° C. or higher, and then the oyster extract (extract) from which solid content (boiled oyster) was removed. For example, it was heated at 90 ° C. or higher for a long time.

  Then, in the heating state, sampling is performed every two hours (every hour from 16 hours to 19 hours), and the sampled extract is 100% so that the ethanol concentration of the extract becomes, for example, 70%. % Ethanol was added, and the extract was centrifuged (8100 G, 10 minutes) to obtain a supernatant.

The supernatant was diluted about 100 times, and the E6 concentration in the supernatant diluted about 100 times was measured by MRM.
However, E6 was not detected when the extract after extracting the oyster extract was heated at standard pressure for 2 hours.

  Next, referring to the MRM chromatogram of E6 in a sample heated at standard pressure for 18 hours, a single peak was confirmed at a retention time of 5.2 to 5.5 minutes, and it was understood that E6 was detected.

The E6 concentration was 4.86 ± 0.10 μg / mL.
Furthermore, since the liquid amount when heated for 18 hours was 125.38 g, the total amount of E6 produced from 1 kg of fresh oysters was 609 ± 13 μg.

  In addition, in a heating experiment with pressure, 20 L of pure water was added to 20 kg of oyster, heated at standard pressure for 2 hours, solids (boiled oysters) were removed, and the pressure cooker (Kajiwara, OAMVPα-C -08EL) for 2 hours at 3 atm.

  Sampling was performed every hour, and 100% ethanol added so that the ethanol concentration became 70% was centrifuged (8100G, 10 minutes) to obtain a supernatant. Further, the supernatant was diluted about 100 times, and the E6 concentration in the sample was measured by MRM.

  Thus, the MRM chromatogram of E6 in the sample obtained by heating the extract after extracting oyster extract at 3 atm for 1 hour was confirmed, and a single peak was confirmed at a retention time of 5.2 to 5.5 minutes, and E6 was detected. The E6 concentration was 1.7 ± 0.10 μg / mL.

  Since the amount of liquid when heated at 3 atm for 1 hour was 21.2 kg, the total amount of E6 produced from 20 kg of raw oysters was 36040 ± 2120 μg. When converted to 1 kg of raw oysters, the produced E6 was 1802 ± 106 μg.

  At first glance, the E6 concentration is lower when pressure is applied (oyster extract extracted after heating for 1 hour at 3 atmospheres) compared to a heating experiment without pressure (oyster extract extracted for 18 hours at standard pressure). .

  However, when pressure is applied, the amount of water evaporation in the sample is small and the amount of liquid obtained is large. Therefore, the yield of E6 was about 3 times higher than that without pressure when compared with 1 kg of oyster. Understandable.

When referring to the MRM chromatogram of E6 in the sample heated for 2 hours at 3 atm with respect to the extract after the oyster extract was extracted, a single peak was confirmed at a retention time of 5.2 to 5.5 minutes, and E6 was detected.
The E6 concentration was 3.5 ± 0.39 μg / mL by analysis of the calibration curve of the E6 standard sample and the MRM chromatogram.

  Thus, since the liquid amount when heated at 3 atm for 1 hour was 19.1 kg, the total amount of E6 produced from 20 kg of raw oysters was 66850 ± 7449 μg. When converted to 1 kg of raw oysters, the produced E6 was 3343 ± 373 μg.

  Compared with the heating experiment without pressure (oyster extract extracted at standard pressure for 18 hours), the E6 concentration at first glance is lower when pressure is applied (oyster extract extracted at 3 atmospheres for 2 hours). .

  However, when pressure is applied, the amount of water evaporation in the sample is small, and the amount of liquid that can be obtained is large. Therefore, the yield of E6 is approximately 5.5 times higher than that without pressure when compared with 1 kg of oysters. .

  From these facts, it was confirmed that E6 was generated by heating for a long time and the collected amount increased by further pressurization.

Here, the present embodiment will be summarized based on the above analysis results.
First, raw oyster meat is pressed, crushed and liquefied.
Then, it was measured whether any concentration of E6 exists in the liquefied raw oyster.

  Thus, E6 was not detected at all in the liquid that was pressed, crushed and liquefied. That is, E6 is not found in the original cells of raw oyster meat.

  Next, as described above, raw oyster meat and extraction solution, for example, distilled water, are placed in an extraction container at a ratio of about 1: 1, and the extraction liquid containing the raw oyster meat is placed at 1 atm for 1 hour. For example, the heat extraction was performed at a high temperature of 92 ° C. to 94 ° C.

  This is a so-called heat extraction method for extracting oyster meat extract, and by this method, many active ingredients existing in raw oyster meat can be extracted into the extraction liquid. Here, a lot of active ingredients present in raw oyster meat are extracted, and the extraction liquid from which raw oyster meat is removed is referred to as an extract.

  Subsequently, the extract was then subjected to heat treatment at 1 atm for 2 hours (92 ° C. to 94 ° C. as described above). It is a heat treatment of 3 hours for convenience, including the initial extraction time.

  However, the presence of E6 was not observed in the extract subjected to the heat treatment for 3 hours.

  Furthermore, the extract is heated (92 ° C. to 94 ° C.) for 4 hours at 1 atm (a total of 5 hours including the extraction time), and whether or not E6 is present in the extract is measured. went.

  In this 5-hour heat treatment (92 ° C. to 94 ° C.), the presence of E6 in the extract is 0.09 (μg / ml), and the concentration measurement value is below the limit of quantification. The reliability of being surely present was not obtained.

  Subsequently, the extract is subjected to heat treatment (92 ° C. to 94 ° C.) for 6 hours at 1 atm (a total of 7 hours when the extraction time is included), and whether or not E6 is present in the extract is measured. However, even in the extract, the presence of E6 is 0.12 (μg / ml), which is also a numerical value below the limit of quantification as the concentration value. Even in this case, E6 is surely present. The reliability of was not obtained.

  However, the extract is heated at 98 ° C to 100 ° C for 6 hours at 1 atmosphere (7 hours in total including the extraction time), and whether or not E6 is present in the extract is evaluated. Therefore, the presence of E6 was measured using HPLC (Prominence LC-20A, Shimadzu).

Then, in the extract, the presence of E6 was found to be a concentration value that was above the limit of quantification.
That is, in this case, the reliability that E6 exists is obtained.

  In this way, when heat treatment was performed at a high temperature of around 100 ° C., it was possible to find a value above the limit of quantification as a concentration value for E6, that is, E6, even by heat treatment for 6 hours or more. is there.

  Further, the extract was subjected to heat treatment (92 ° C. to 94 ° C.) for 8 hours at 1 atmosphere (9 hours when the extraction time was included), and whether or not E6 was present in the extract was measured. .

  However, the presence of E6 in the extract could not be confirmed with certainty. That is, although a numerical value of 0.29 (μg / ml) was obtained, it was also a numerical value below the quantification limit of concentration measurement.

  Next, heat treatment (92 ° C. to 94 ° C.) is performed on the above extract for 10 hours at 1 atmosphere (11 hours when the extraction time is included), and whether or not E6 exists in the extract is measured. It was.

  Then, it was confirmed whether or not the presence of E6 having a predetermined concentration was recognized in the extract subjected to this long-time heat treatment (92 ° C. to 94 ° C.).

  Then, the numerical value exceeding the quantification limit of concentration measurement can be measured for the first time as 0.54 (μg / ml), and E6 is clearly present in the extract subjected to the heat treatment for 11 hours in total. Confirmation was obtained. This means that 215.8 μg of E6 was produced per kg of oyster.

  Furthermore, the extract was subjected to heat treatment for 12 hours at 1 atm (a total of 13 hours including the initial extraction time), and it was measured whether or not E6 having a predetermined concentration was present in the extract.

    Then, when it was confirmed whether or not E6 was present in the extract, it was possible to measure 0.86 (μg / ml) and an increased numerical value exceeding the limit of quantification.

  This means that 288.4 μg of E6 was produced per kg of oyster.

    Further, the extract was heated for 14 hours at 1 atm (a total of 15 hours when 1 hour of the extraction time was included), and whether or not E6 having a predetermined concentration was present was measured in the extract. .

    And when it was confirmed whether or not the presence of E6 was recognized in the extract, it was possible to measure a numerical value exceeding 1.42 (μg / ml) and further the limit of quantification, It was confirmed that E6 was further increased in the extract.

  And in this case, it means that 373.9 μg of E6 was produced per kg of oyster.

  Next, the extract was subjected to heat treatment at 1 atm for 16 hours (17 hours including the extraction time), and whether or not a predetermined concentration of E6 was present was measured.

  Then, when it was confirmed whether or not the presence of E6 was found in the extract, it was possible to measure the increased value again with 2.63 (μg / ml), and E6 should be heat-treated for a long time. Confirmed that it was present in an increased number.

  In this case, it means that 478.6 μg of E6 was produced per kg of oyster.

  Further, the extract was subjected to heat treatment at 1 atmosphere for 17 hours (18 hours including the extraction time), and whether or not a predetermined concentration of E6 was present was measured.

  Then, when it was confirmed whether or not the presence of E6 was recognized in the extract, it was possible to measure the numerical value of 3.42 (μg / ml) increased again, and confirmation that E6 was present was obtained. It was.

  In this case, E6 means that 519.5 μg was produced per kg of oyster.

  Next, the extract was subjected to heat treatment at 1 atm for 18 hours (19 hours including the extraction time), and it was measured whether or not E6 having a predetermined concentration was present in the extract.

Then, it was possible to measure a numerical value of 4.86 (μg / ml), which was a further increased numerical value of concentration.
This means that E9.3 was produced at 609.3 μg per kg of oyster.

  Further, the extract was subjected to a long-time heat treatment for 19 hours at 1 atm (20 hours including 1 hour of extraction time), and whether or not E6 was present in the extract was measured. . Then, when it was confirmed whether or not the presence of a predetermined concentration of E6 was found in the extract, it was possible to measure a numerical value of 6.49 (μg / ml), and that E6 was further increased. Confirmation was obtained.

  In this case, E6 means that 581.3 μg was produced per kg of oyster.

  Thus, at first, E6 was not present at all in raw oysters, and was not present at all in the extract obtained by heating and extracting oyster meat extract at 1 atm for 1 hour. It was confirmed that the extract was generated as the extract was heated for an extremely long time and the heating temperature was increased to around 100 ° C.

  As a result, when the extract of oyster meat extract was heated at 1 atm for 19 hours (20 hours when 1 hour of extraction time was added) and the specific gravity of the extract was 1, 581 μg (6 (49 μg × 89.57 g) of E6 was produced.

  Furthermore, as described above, when the extract was heat-treated at 1 atm for 2 to 8 hours, E6 of a predetermined concentration was not detected at all, but for the extract (extracted liquid from which raw oysters were removed) When heat treatment is performed at 3 atm for 1 hour, a value of E6 concentration of 1.7 (μg / ml) is obtained, and from this value, a liquid volume of 21.2 kg is obtained from 20 kg of raw oysters, Assuming that the specific gravity of the extract is 1, 1820 μg of E6 is produced from 1 kg of fresh oysters by heat treatment at 3 atm for 1 hour.

  Further, when the extract was heated at 3 atm for 2 hours, a value of E6 concentration of 3.5 (μg / ml) was obtained. If 19.1 kg is obtained and the specific gravity of the extract is 1, then 3342.5 μg of E6 is generated from 1 kg of fresh oysters by heat treatment at 3 atm for 2 hours.

  In addition, when a heat treatment is performed for a predetermined time, for example, 50 minutes or more in a pressurized state of 1 atm or higher, a numerical value exceeding the limit of quantification can be found as a concentration value of E6 in combination with an increase in the heating temperature due to pressure. It will be a thing.

  As described above, it was found that E6, which is not originally found in raw oyster meat, is clearly produced by heating and / or pressurizing the extract of the oyster meat extract that has been subjected to the heat extraction. It is. Moreover, it has been found that the longer the heating time is, the more E6 is generated.

  Furthermore, it was confirmed that E6 was generated even in a short processing time when a pressure treatment of 3 atm was performed.

  In addition, this inventor crushed raw oyster meat from which E6 was not detected in a raw state, and was made into the minced state. And the crushed material made into the mince was produced | generated from the heat-processed oyster meat liquid by heating at 80 degreeC or more for 3 hours or more in the ratio of (raw oyster mince) 3: (water) 1.

  E6 could be produced in a relatively short period of time (7000 ng / mL) by crushing raw minced meat in which E6 was not detected in the raw state to a minced state.

  Here, this inventor examined the sleep improvement effect by the drink intake of the E6 containing oyster meat extract for male and female workers.

(Human test experiment 1)
In this experiment, in order to examine the effect on the subjective evaluation of sleep quality when consuming oyster meat extract beverage containing E6 for 4 consecutive weeks, the Athens Insomnia Scale (AIS) is 6 points or more, and the POMS shortened version (POMS-S) Targeting 84 adult males and females aged 30 to 60 who have a sleep problem, stress and fatigue with a "fatigue" score of 60 or higher and a "liveness" score of 40 or lower A placebo-controlled, randomized, double-blind, parallel group comparison study was conducted.

  Of the 84 participants included in the experiment, no subjects dropped out during the experiment, and all 84 completed the prescribed schedule. Among them, 78 persons (40 persons in group A) excluding 6 persons (test food group (group A) 2 persons, placebo group (group P) 4 persons) who met the efficacy analysis exclusion criteria set in advance in the experiment plan. , P group 38).

  Regarding AIS, which is a subjective evaluation item of sleep, which is the main evaluation item, there was no significant difference between groups in the amount of change from before intake. For the Pittsburgh Sleep Questionnaire (PSQI-j), the amount of change in sleep efficiency from before ingestion was significantly lower in Group A compared to Group P (Group A: 0.4 ± 1.2, Group P: 1.1) ± 1.4), group A suppressed the deterioration of sleep efficiency (FIG. 4).

  An additional exploratory analysis of PSQI-j was conducted for 61 men and women (A group: 31 people, P group: 30 people) under 50 years of age among the subjects of effectiveness analysis. Significant improvement was seen in the difficult items (FIG. 5).

  From the above results, it is considered that E6 has a function of relieving mental stress and fatigue and maintaining sleep efficiency by ingesting an E6-containing oyster meat extract beverage. E6 was particularly effective in improving stress, fatigue, vitality and sleep difficulty for working men and women under 50 years of age. Moreover, there was no problem in the safety of the E6-containing oyster meat extract drink under the experimental conditions.

(Purpose and background of the experiment)
This experimental food is a oyster meat extract obtained by adding ethanol to a oyster meat extract extracted and concentrated from raw oysters, and then concentrating the obtained supernatant. In addition to essential amino acids, natural minerals such as zinc, copper, iron, selenium, chromium, cobalt, and nine types of vitamins are also included in a well-balanced manner. In addition, E6 contained in oyster meat extract is being studied as a novel antioxidant substance. In animal tests using oyster meat extract containing E6, DNA antioxidant action, lipid antioxidant action, stress mitigating action, non- Alcoholic steatohepatitis has been improved.
Therefore, in this experiment, sleep quality, stress, and fatigue were subjectively observed when men and women between the ages of 30 and 60 who felt sleep problems, stress, and fatigue were allowed to ingest E6-containing oyster meat extract for four weeks in a row. The impact on evaluation was compared with placebo as a control.

Experimental Method Experimental Design Placebo Control Randomized Double-blind Parallel Intergroup Comparative Study

Number of subjects and number of assigned subjects The number of subjects included in this experiment was 42 for the test food group and 42 for the placebo group, for a total of 84 people.

Assignment method The person in charge of assignment created an assignment table using random numbers and assigned assignment numbers to the experimental foods. The assignment table was sealed by the person in charge of assignment and kept sealed until the assignment table was opened.

Experiment period It was carried out from January 2016 to April 2016.

Selection of subjects Subjects who met the following selection criteria were selected.

Selection criteria (1) Adult male and female workers aged between 30 and 60 years (2) Those with an Athens insomnia scale score of 6 or more at the time of pre-examination (3) "Fatigue" of the shortened version of POMS at the pre-examination Those with a score of 60 or higher and a “lively” score of 40 or lower

Test target food (hereinafter referred to as experimental food)
Test food name Test food: E6-containing oyster meat extract drink Placebo: E6-containing oyster meat extract-free drink

Eating Experience of Experimental Foods Watanabe Oyster Laboratory Co., Ltd. has been experimenting and developing oyster meat processed food for the purpose of using oysters throughout the year. “Watanabe active oyster drink” has been in use for 10 years since its release, and 160,000 bottles are consumed annually.

  In addition, E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in oyster meat extract is being tested as a novel antioxidant substance, and DNA antioxidants have been tested by animal tests using oyster meat extract containing E6. Action, lipid antioxidant action, stress relieving action, etc. are recognized.

Involved ingredients
E6 (3,5-dihydroxy-4-methoxybenzyl alcohol)

Experimental food intake, intake method, intake period (1) Experimental food intake, intake method, and intake period Experimental food is twice a day, once in the morning, once at least one hour before going to bed at night, once. One bottle (50 mL) was ingested. A diary was recorded daily during the study period. The intake period was 4 weeks.

Experiment schedule (1) The candidate candidates were sent to a pre-examination, and each examination of lifestyle questionnaire, POMS shortened version (POMS-S), life event questionnaire, and entry to the Athens style insomnia scale (AIS) was conducted.
(2) Subjects were selected from the results of the pre-inspection and were brought to the pre-ingestion inspection, and physical examination and measurement, general clinical examination, and filling in the Pittsburgh Sleep Questionnaire (PSQI-j) were conducted.
(3) 84 subjects were selected from the test results before ingestion. The test food and the diary during the intake period were distributed to the subjects, and the recording of the diary during the intake and the intake period of the experimental food was started.
(4) Bring the subject to the 2nd week (15th day) and 4th week (29th day) after ingestion, and conduct physical examination and measurement, POMS-S, and filling in the life event questionnaire . In the fourth week, AIS, PSQI-j, and general clinical tests were also conducted.
(5) After the start of intake, POMS-S and a life event survey form were entered at home in the first week (8th day) and the third week (22nd day).
(6) On the day before each visit to the examination and the day before the questionnaire was completed at home, he was banned from drinking, finished eating and drinking by 10:00, and went to bed before 12:00 and instructed him to sleep well.

Evaluation of effectiveness Evaluation index of effectiveness Primary endpoint: AIS, PSQI-j

Evaluation method
PSQI-j and AIS compared the test food intake group with the placebo intake group using the Mann-Whitney U test for the amount of change from the pre-intake value at each time point after intake. For reference, the amount of change from each time point after ingestion in each group was evaluated using Wilcoxon's signed rank sum test.

Numerical display and significance level Numerical values are shown as mean ± standard deviation, and the significance level of the test was 5% on both sides.

Experimental Results In the following, when the average value is xxx and the standard deviation is yyy, it is indicated as xxx ± yyy.

Selection of test subjects A total of 84 test subjects were selected from 37 males and 47 females, and the experiment was started.

Breakdown of analysis subjects Of the 84 people who started the experiment, no subjects dropped out of the experiment, and 84 subjects completed the prescribed schedule and examination contents.
Of the 84 subjects, a total of 6 subjects who met the efficacy analysis exclusion criteria decided to exclude the subjects from the efficacy analysis before opening the assignment table. Therefore, 78 subjects were analyzed for effectiveness (Fig. 1 explained in Fig. 1).

Background factors of effectiveness analysis subjects Table 2-1 described in Fig. 2 shows the background factors of efficacy analysis subjects (gender, age, height, weight, BMI, systolic blood pressure, diastolic blood pressure, pulse, POMS- S, AIS). There was no significant difference in the AIS used as selection criteria.

Efficacy assessment Key endpoints (AIS, PSQI-j)
Table 2-2-2 explained in Fig. 3 and Table 2-2-3 explained in Fig. 4 show the transition of AIS and PSQI-j as the main evaluation items.

AIS
There was no significant difference between groups in the amount of change from before ingestion (Table 2-2-2 explained in Fig. 3). In the group comparison, both groups fluctuated (improved).

PSQI-j
FIG. 4 shows pre-intake values, PSQI-j scores at 4 weeks after ingestion, and the amount of change. In the test food intake group, the change in the "sleep efficiency" score was 0.4 ± 1.2, and in the placebo food intake group 1.1 ± 1.4, which was significantly lower in the test food intake group, and the deterioration of sleep efficiency was suppressed It was shown that.

  In the intra-group comparison of each group, the total score (Global Score), sleep quality, sleep time, sleep time, sleep difficulty, and daytime wakefulness significantly changed (improved) in the A group compared to before intake. . In the P group, the total score, sleep quality, sleep time, sleep time, and difficulty in waking up during the day significantly fluctuated (improved), and sleep efficiency fluctuated significantly (worsened) before ingestion. )did.

Exploratory effectiveness analysis (PSQI-j)
Since it is considered that younger people have higher stress adaptability, 61 men and women (group A: 31 people, group P: 30 people) younger than 50 years among the efficacy analysis subjects were analyzed as exploratory efficacy analyses. Additional analysis was performed on PSQI-j as a target (Table 2-2-5 described in FIG. 5).
PSQI-j
The amount of change from the ingestion of sleep difficulty was significantly lower in the A group than in the P group (A group: -0.3 ± 0.4, P group: 0.0 ± 0.4).

Safety assessment Adverse events Regarding adverse events, some subjective complaints and objective findings were observed, but all symptoms were mild and no serious adverse events were observed. . In addition, the investigator's chief investigator determined that there was no causal relationship with the research food, and there was no side effect.

Discussion I feel sleep problems, stress, and fatigue with an Athens style insomnia scale of 6 points or more, a POMS shortened version (POMS-S) with a “fatigue” score of 60 points or more and a “liveness” score of 40 points or less. A comparative study of the effects of the intake of E6-containing oyster meat extract on subjective quality of sleep quality, stress and fatigue for 84 weeks of male and female adults aged 30 to 60 years Went.

  Regarding AIS, which is a subjective evaluation item of sleep, there was no significant difference between groups in the amount of change from before ingestion (Table 2-2-2 described as FIG. 3). Regarding PSQI-j, the amount of change in sleep efficiency from before ingestion was significantly lower in Group A compared to Group P (Group A: 0.4 ± 1.2, Group P: 1.1 ± 1.4) (Fig. Table 2-2-3) described as 4), both groups are deteriorating, so it can be said that Group A suppressed the deterioration of sleep efficiency.

  One factor in improving sleep efficiency is thought to be a reduction in mid-wakeness. It has been reported that there is a positive correlation between evening plasma cortisol levels and the number of subsequent awakenings in sleep in healthy and insomnia patients. By oral administration of oyster meat extract containing E6, the effect of decreasing the corticosterone level in plasma for the time corresponding to the time of sleep of mice was confirmed. In addition, an E6-containing oyster meat extract ingestion of one beverage confirmed that the sleep awakening increase suppression effect was observed in the test food intake group compared to the placebo intake group. Based on these findings, the effect of suppressing the deterioration of sleep efficiency in the test food group observed in this experiment may be due to the inhibitory effect on plasma corticosterone secretion due to the antioxidant effect of E6 and the inhibitory effect on mid-wakefulness induced thereby. Conceivable.

  Based on the results of stress biomarkers, it was considered that younger people had higher ability to adapt to stress. Therefore, 61 men and women under the age of 50 (group A: 31 people, group P: 30 people) were analyzed. An additional exploratory analysis was performed on PSQI-j.

  In PSQI-j, regarding the sleep difficulty, the A group showed a significant improvement compared to the P group. From these results, E6-containing oyster meat extract drinks are particularly effective in improving stress, fatigue, vitality, confusion, and sleep difficulty for working men and women under 50, who are considered to have a relatively high ability to adapt to stress. It can be said that it demonstrated.

Conclusion As a result of a placebo-controlled, randomized, double-blind, parallel group comparison study of working men and women aged 30 to 60 who are experiencing problems with sleep, PSQI-j Suppression of sleep efficiency score was suppressed. Therefore, E6 was considered to have a function of maintaining sleep efficiency. E6 was particularly effective in improving sleep difficulty for working men and women under the age of 50.
Moreover, there was no problem in the safety of the E6-containing oyster meat extract drink under the experimental conditions.

(Human test experiment 2)
In this experiment, a placebo that ingests E6-containing oyster meat extract beverage (test food) for 4 weeks for working men and women who feel stress and fatigue from the age of 30 to 60 years of age and have low sleep satisfaction A controlled randomized, double-blind, parallel group comparison study was conducted. As the placebo food, a beverage containing no E6-containing oyster meat extract was used.

Of the 32 subjects who were assigned so that their gender, age, and the proportion of those who were awake were not balanced among the groups, 31 subjects completed the prescribed schedule of the experiment. In addition, the effectiveness evaluation example of the questionnaire excluding the OSA sleep questionnaire MA version (hereinafter referred to as “OSA-MA”) was: Test food intake group (Group A): 13 people, Placebo food intake group (P group): 15 A total of 20 people were evaluated, and EEG and OSA-MA efficacy evaluation examples were group A: 9 people and group P: 11 people for a total of 20 people.

In the questionnaire, the amount of change from the value before sleep intake to the 4th week of the QOL questionnaire was significantly lower in group A than in group P (improvement) (FIG. 20). In the electroencephalogram, group A maintains the total number of wakefulness in the amount of change in the middle awakening time in the 4th week and the amount of change in the total number of wakefulness in the 4th week. (FIG. 21).

  From the above results, it has been clarified that the intake of E6-containing oyster meat extract drink improves sleep-related QOL for adult working men and women who are usually tired and are not satisfied with sleep. In Group P, mid-term arousal increased during the experiment period, but it was not observed in Group A. Therefore, the intake of E6-containing oyster meat extract beverages exerted a good sleep state maintenance and improvement effect. It was suggested that

(Purpose and background of the experiment)
The Applicant previously conducted an open experiment in which this E6-containing oyster meat extract drink was ingested for 4 weeks for adult working men and women who are experiencing problems with sleep. It was suggested that the amount of δ power in the first period recovered and improved.
Therefore, this time, we examined the sleep improvement effect by continuous intake of E6-containing oyster meat extract beverages for working men and women who feel that there is a problem with sleep, using brain waves and subjective evaluation during sleep as indices, In addition, a double-blind parallel group comparison test was conducted in which the E6-containing oyster meat extract-free beverage was used as a control for the purpose of comparing the EEG and subjective evaluation indices.

Experimental method Experimental design Double-blind parallel group comparison test

Number of subjects and number of assigned subjects The number of subjects included in this experiment was 16 for the test food group and 16 for the placebo group, for a total of 32 people.

Allocation method The allocation staff created an allocation table using random numbers and assigned an allocation number to the test food. The assignment table was sealed by the person in charge of assignment and kept sealed until the assignment table was opened. In the assignment, consideration was given so that the gender, age of the subjects and the proportion of those who were awake were not balanced among the groups.

  After fixing the analysis subject and data, the person in charge of assignment opened the assignment table and disclosed the information. However, if a serious adverse event occurs and an emergency is required, the allocation table will be opened as necessary and only the minimum necessary information will be disclosed.

Experiment period
Conducted from March 2013 to June 2013.

Selection of subjects Subjects who satisfy all of the following selection criteria were enrolled.

Selection criteria (1) Males and females of adult workers aged 30 to 60 (2) Mainly having a serum Zn concentration of less than 80 μg / dL (3) Normal bedtime PM 9: 00-AM 1:00 (4) Athens insomnia scale score at the time of prior examination is 6 points or more (5) POMS shortened version "fatigue" score is 50 points or more and "liveness" score is 50 points or less (6) Awakening during sleep To be 70% or more of all subjects (1 to 3 times a week)

Test target food (hereinafter referred to as experimental food)
Name of experimental food Test food: E6-containing oyster meat extract drink Placebo: E6-containing oyster meat extract-free drink

Eating experience of experimental food E6-containing oyster meat extract beverage used in this experiment is sold by Watanabe Oyster Laboratories Co., Ltd. as “Watanabe active oyster drink” and has been drunk in a wide range of ages.

Involved ingredients
E6 (3,5-dihydroxy-4-methoxybenzyl alcohol)

Intake, intake method and intake period The test food was taken once a day (50 mL) 1 hour before bedtime. Subjects were instructed to record the time and quantity of test food intake in a diary. The intake period was 4 weeks (28 days).

Experiment schedule (1) As a pre-test for candidate subjects who have obtained consent, physical condition confirmation, physical measurement, general clinical test, serum Zn concentration, serum Se concentration test, lifestyle questionnaire, POMS shortened version (POMS-S ), Life Event Questionnaire, and Filling in the Athens Insomnia Scale (AIS).
(2) After selecting candidate subjects, as early two tests, POMS-S, Life Event Questionnaire, AIS, Pittsburgh Sleep Questionnaire (PSQI-j), QOL Questionnaire, JUMACL, Mental health of workers Each inspection of the check (CHCW) entry was conducted.
(3) The usage of the portable electroencephalograph was explained at the briefing session for the included subjects.
(4) From the day of the briefing session, I wrote a diary every day. In addition, each week from Monday night to Friday morning one week before the start of test food intake, the EEG from bedtime to waking up was recorded with a portable electroencephalograph, and the OSA sleep survey form MA version (with limited time) OSA-MA).
(5) The diary was written every day even after the start of test food intake. In addition, the brain wave from bedtime to bedtime is recorded with a portable electroencephalograph every 4 nights from Monday night to Friday morning in the 1st, 2nd and 4th week of test food intake, and within the time limit I made OSA-MA complete.
(6) One week later (7 days after ingestion) In the inspection, POMS-S was entered on the specified date.
(7) In the two-week test (14 days after ingestion), the subjects visited the hospital on the specified day, and were checked for physical condition, physical measurement, and filling in the QOL questionnaire.
(8) After 4 weeks (28 days after ingestion), the subjects were sent to the hospital on the specified day, and physical examination, physical measurement, and filling in the QOL questionnaire were conducted.
(9) We banned alcohol the day before each examination visit and instructed them not to take medicines as much as possible. I went to bed before 12:00 and tried to get enough sleep.

Evaluation method effectiveness evaluation index Main evaluation items: QOL questionnaire score, EEG

Effectiveness evaluation method
The score of the QOL questionnaire was compared using the Mann-Whitney U test for the amount of change from before the intake at each time point after the intake of the test food intake group and that of the placebo food group. In addition, the actual measurement value at each time point after ingestion in each group was compared with that before ingestion using a one-sample Wilcoxon test.

Display and significance level Each test value is expressed as mean ± standard deviation, and the significance level of the test was 5% on both sides.

Experimental results Selection of subjects A total of 32 subjects were selected and the experiment was started.

  A total of 3 people (test food group: 2 people, placebo food group: 1 people) who met the criteria for exclusion from the analysis were excluded from those subject to efficacy analysis. As a result, the number of subjects subject to efficacy analysis was 28 (test food group: 13 people, placebo food group: 15 people).

  EEG was measured for 4 days in each week of each measurement period, but 8 subjects were excluded from the analysis of EEG, 4 in the test food group and 4 in the placebo food group for which the analyzable data was less than 2 days. A total of 20 subjects (9 test food groups, 11 placebo food groups) were subjects for EEG and OSA-MA efficacy analysis.

Background factors of effectiveness analysis subjects Table II-1-1 explained in Fig. 18 shows 28 subjects of effectiveness analysis, Table II-1-2 explained in Fig. 19 shows EEG and OSA-MA analysis subjects of 20 people As background factors, age, sex, height, weight, BMI, POMS-SF (fatigue), POMS-SV (liveness) were shown.

Efficacy evaluation main evaluation items (QOL questionnaire, EEG)
Table II-2-5 described with reference to FIG. 20 and Table II-2-10 described with reference to FIG.

QOL questionnaire At the 4th week of the amount of change from the value before sleep intake, Group A showed a significantly lower value than Group P (Group A: -1.2 ± 0.6, Group P: -0.5 ±) 0.7).

Mid-wakefulness and sleep efficiency As can be seen from Table II-2-10 explained in Fig. 21, in the fourth week of the change from the pre-intake value of the total number of awakenings, the A group was significantly different from the P group The values were low (Group A: 1.6 ± 7.5 times, Group P: 19.9 ± 24.1 times).
Regarding sleep efficiency, there was no difference between groups in the amount of change from the value before intake, but only in group P, compared with 88.2 ± 7.1% before intake of the actual measurement value, it was 85.1 ± 7.8% at 4 weeks. Significantly low. On the other hand, in group A, a decrease in sleep efficiency was not confirmed.

Discussion For 32 workers who have a total AIS score of 6 or more, a POMS-S “fatigue (F)” T score of 50 or more, and a “liveness (V)” T score of 50 or less. A double-blind parallel group comparison test was conducted in which E6-containing oyster meat extract beverage was the test food, E6-containing oyster meat extract-free beverage was the placebo food, and was ingested continuously for 4 weeks (28 days).

  A questionnaire excluding OSA-MA, with 31 subjects completing the prescribed experiment schedule out of 32 subjects assigned and incorporated, taking into account that the gender, age, and proportion of those who are awake are not balanced among the groups. In addition, 28 subjects were analyzed for the effectiveness of serum Zn and serum Se, and 20 subjects were analyzed for the effectiveness of electroencephalographs and OSA-MA.

  Regarding the questionnaire, in the fourth week of the amount of change from the pre-sleep intake value in the QOL questionnaire, the test food intake group (Group A) was significantly lower than the placebo food intake group (P group) (Improved).

  Regarding EEG, group A was significantly lower than group P in the amount of change from the pre-intake value of midweek awakening time in week 4 and the amount of change from the pre-intake value in total number of awakenings in week 4 showed that.

Conclusion It has become clear that the intake of E6-containing oyster meat extract drink improves sleep-related QOL for adult working men and women who feel tired and are not satisfied with sleep. Moreover, from the electroencephalogram analysis, since the increase in mid-term arousal during the experiment period observed in the P group was not observed in the A group, the E6-containing oyster meat extract beverage suppressed the deterioration of the sleep state and the sleep state. It was suggested that the improvement effect is exhibited.
Moreover, there was no problem in the safety of the E6-containing oyster meat extract drink under the experimental conditions.

DESCRIPTION OF SYMBOLS 1 Extraction container 2 Extraction solution 3 Raw oyster meat 4 Ethanol solution 5 Ethyl acetate 6 Concentrate 7 Precipitate 8 Supernatant 9 Supernatant concentrate 10 Diluent 10a Water layer 11 Ethyl acetate layer

Claims (13)

3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in the extract extracted from oyster meat as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
By heating raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol is not detected in the raw state, 3,5-dihydroxy-4-methoxybenzyl alcohol is heated. (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heat-treated oyster meat liquid, and the produced 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) ) As an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
Raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol is not detected in the raw state is crushed, and the crushed material is kept at 80 ° C or higher for more than 3 hours. By heating, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heat-treated oyster meat liquid, and the produced 3,5-dihydroxy- 4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
By heating raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not detected in the raw state for 6 hours or more at 98 ° C to 100 ° C, , 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heat-treated oyster meat liquid, the produced 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
By heating raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not detected in a raw state for at least 9 hours at 90 ° C. or higher, 5-Dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heat-treated oyster meat liquid, and the produced 3,5-dihydroxy-4-methoxybenzyl alcohol (3 , 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
Raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not detected in the raw state is placed in the extraction solution, and the oyster meat extract is extracted. Heat the meat extract at 1 atm for at least 10 hours at 90 ° C or higher to heat 3,5-dihydroxy-4-methoxybenzyl alcohol. Produced from the oyster meat extract, and the produced 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
In raw state, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not detected. -Dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from oyster meat fluid heated in the pressurized state, and the produced 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
Raw oyster meat in which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not detected in a raw state is at least 1 hour at a pressure of 3 atmospheres or more, 90 ° C. or more 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the oyster meat fluid heated in the pressurized state, and the produced 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
A fraction of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was recovered from oyster meat using ethyl acetate, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol was recovered. Alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
3,5-dihydroxy-4-methoxybenzyl alcohol fraction was recovered from oyster meat using ethyl acetate and ethanol, and the recovered 3,5-dihydroxy-4- Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethyl acetate is added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4-methoxybenzyl The alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) fraction is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is used as an active ingredient. ,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
The oyster meat is stored in the extraction container in which the extraction solution is stored, and the solution containing the oyster meat extract is collected. Ethyl acetate and ethanol are added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4- The fraction of methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is an active ingredient. And
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethanol is added to the collected solution containing the oyster meat extract, and the supernatant liquid and the precipitate are separated. The separated supernatant is taken out, and ethyl acetate is added to the supernatant to separate the ethyl acetate layer and the aqueous layer,
The solution of the separated ethyl acetate layer is volatilized and concentrated to recover a 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) fraction. Dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
The oyster meat extract which has the suppression effect on sleep efficiency deterioration, the sleep difficulty improvement effect, the improvement of sleep based on the sleep items of QOL, and the suppression of the increase in mid-wakefulness by maintaining the total number of awakenings.

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