JP2017132753A - Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-derangement, and animation amplification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-derangement, and animation amplification, the oyster meat extract containing as an active ingredient 3,5-dihydroxy-4-methoxybenzyl alcohol from the oyster meat extract liquid extracted from Crassostrea gigas soft bodies.SOLUTION: The present invention is characterized by containing as an active ingredient 3,5-dihydroxy-4-methoxybenzyl alcohol that is contained in the extract extracted from oyster meat, the 3,5-dihydroxy-4-methoxybenzyl alcohol being produced from an oyster meat liquid that is obtained by heating oyster meat in raw state in which 3,5-dihydroxy-4-methoxybenzyl alcohol is not detected.SELECTED DRAWING: Figure 1

Description

The present invention is an anti-stress, anti-depressant, anti-fatigue, anti-confused, vigorous composition comprising 3,5-dihydroxy-4-methoxybenzyl alcohol extracted from oyster meat as an active ingredient. The present invention relates to an oyster meat extract for amplification.

Intra-cerebral oxidative stress has been shown to cause excessive enhancement of the HPA system, circadian rhythm abnormalities, etc. In order to prevent oxidative stress in the brain, intake of antioxidants and anti-stress agents with good brain migration It is desired.
In other words, when subjected to physical and physical stress or psychological stress, oxidative stress is generated, and the hypothalamo-pituitary-adrenal axis (HPA system) is activated and corticosteroids are activated. Is released.

  Chronic oxidative stress in the brain is over-secreted in both CRH (abbreviation for corticotropin-releasing hormone), ACTH (adrenocorticotrophic hormone), and GC (glucocorticoid). The secretory suppression mechanism due to physiological feedback is disrupted, causing excessive enhancement of the HPA system.

It is also said that the hippocampal GC receptor, which plays an important role in feedback, disappears due to oxidative stress. On the other hand, in relation to sleep, an association between cerebral oxidative stress and circadian rhythm abnormality is also being clarified.
In addition, it is said that ingestion of antioxidant substances and anti-stress acting substances having good brain migration is important for prevention of oxidative stress in the brain.

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, the oyster meat extract extracted from the oyster soft body part was added to 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient for anti-stress and anti-depression. , Succeeded in finding anti-fatigue, anti-chaos, and vitality amplification.
Furthermore, the present inventor extracted 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), which was not found at all from the original raw oyster meat, It has also been found that it can be produced by heating or pressurizing the extract of the oyster meat extract at the stage.

Therefore, as described above, the present invention uses 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient from the oyster meat extract extracted from oyster soft body as described above. An object of the present invention is to produce and provide a oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion and vitality amplification.

The oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confused, vitality amplification according to the present invention is
3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in the extract extracted from oyster meat as an active ingredient,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
It is characterized by
Or
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,
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 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. ,
It is characterized by
Or
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
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 ethyl acetate layer solution was concentrated to recover 3,5-dihydroxy-4-methoxybenzyl alcohol fraction, and the recovered 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
It is characterized by this.

According to the present invention, anti-stress, anti-depressant, anti-fatigue comprising 3,5-dihydroxy-4-methoxybenzyl alcohol extracted from oyster meat extract as an active ingredient It has an excellent effect that it can provide oyster meat extract for anti-confusing and lively amplification.

It is explanatory drawing (1) explaining the experimental result of this invention. It is explanatory drawing (2) explaining the experimental result of this invention. It is explanatory drawing (3) explaining the experimental result of this invention. It is explanatory drawing (4) explaining the experimental result of this invention. It is explanatory drawing (5) explaining the experimental result of this invention. It is explanatory drawing explaining the component content list of the standard feed to a mouse | mouth. 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 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) 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 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) extracted by this invention. It is explanatory drawing explaining the measurement principle of ORAC method. It is explanatory drawing explaining the antioxidant ability of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. It is explanatory drawing (1) explaining the structural analysis of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. It is explanatory drawing (2) explaining the structural analysis of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. It is explanatory drawing explaining a test subject's classification | category and a breakdown. It is a table | surface explaining the background of an effectiveness analysis object person. It is a table | surface explaining effectiveness. It is a table | surface explaining the effectiveness of the test subject group under 50 years old. It is explanatory drawing explaining the content of a stress load test. It is explanatory drawing explaining the breeding method. It is explanatory drawing explaining the MDA density | concentration of each organ. It is explanatory drawing explaining the 8-OHdG density | concentration in a kidney. It is explanatory drawing explaining the corticosterone density | concentration in plasma.

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

  7 and 8, 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. 7, 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, oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) to be described later is recovered using the supernatant 8 described above. Is.

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, it was confirmed that 3,5-dihydroxy-4-methoxybenzyl alcohol, which will be described later, was present in the ethyl acetate layer 11.

  Here, the amount of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) confirmed, specifically, the ethyl acetate layer 11 collected for about 2 L. It was confirmed that there was about 3 mg of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol).

  Next, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) can be separated and purified from the oyster extract by any process, Whether it was confirmed, the structure of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), and 3,5-dihydroxy The following describes how the antioxidant action of 4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) 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 extraction of 3,5-dihydroxy-4-methoxybenzyl alcohol is performed.

As can be seen from FIG. 9, the ethanol content is eliminated (step 110), and chloroform, ethyl acetate, and butanol are added to hexane from each hexane into the supernatant 8 diluted approximately 5 times to generate 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 organic solvents for 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) extraction is increased step by step, and fractions are added to each of them. Then, it is confirmed whether 3,5-dihydroxy-4-methoxybenzyl alcohol is extracted in each fraction (see FIG. 10).

  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). ) 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.

  In addition, 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, 3,5-dihydroxy-4-methoxybenzyl was observed in the ethyl acetate fraction. It is determined that alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is present (see FIG. 11, step 112 in FIG. 9).

  Next, the ethyl acetate fraction was concentrated by an evaporator, and then extracted with a so-called silica open column (step 116 in FIGS. 12 and 9), and the ethyl acetate: chloroform extraction fraction at a ratio of 3: 2 was obtained. Select (see Fig. 13), and finally, the fraction is separated and purified by HPLC (reverse phase column) to 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). (Step 116).

  Thus, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) could be separated and purified from the supernatant 8 from which the oyster meat extract was extracted.

  In addition, 3,5-dihydroxy-4-methoxybenzyl alcohol can also be separated and purified by the following operation.

  First, Trolox (Wako) dissolved in a mixed solution of 0.075mol / L phosphate buffer 2.3, 5mL, 6.3x10-7mol / LFluorescein (fluorescence probe) 0.3mL, 7% (w / v) methylated β-cyclodextrin (Wako) ) Or 0.05 mL of the test sample at 37 ° C for 10 minutes.

  Add 1.28x10-1mol / L2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH, Wako) 0.3mL, pre-warmed to 37 ° C, for example, while stirring with a stirrer, spectrofluorometer (FP-6500 , JASCO, Tokyo) Measure fluorescence intensity (excitation wavelength 493 nm, fluorescence wavelength 515 nm) every 10 seconds up to 5,000 seconds.

  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. 11) 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 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was finally obtained from the 160 mL ethanol extract of the raw material. .

  By the way, the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is caused by ultraviolet absorption spectrum (V-530, JASCO), nuclear magnetic resonance spectrum (NMR: AMX-). 500, Bruker, Karlsruhe) and mass spectrum (JMS-T100CS, JEOL, Tokyo) and structural analysis was performed (FIGS. 17 and 18). As a result, the structure of the separated and purified product was 3, 5 It is presumed to be -dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol).

conditions
UV (EtOH), λmax 270 nm; 1H-NMR (500 MHz, Acetone-d6) δH: 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); 13C-NMR (125 MHz, Acetone-d6) δC: 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.forC8H8O3, 153.05517), 171.006911 [M + H] + (calc.forC8H11O4, 171.006573).

  Here, the properties of the separated and purified 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) will be explained. Its properties are pale yellow powder, fat-soluble and water-soluble. Is shown.

  In addition, it was confirmed that the 3,5-dihydroxy-4-methoxybenzyl alcohol was a phenolic compound as shown in FIG.

Here, various methods for measuring antioxidants in foods have been reported so far, but all have their merits and demerits, and the standardized or official method (confirmation of the validity of analytical values) There wasn't. However, in the United States, supplements and beverages that express ORAC values are already on the market and are becoming the global standard.
Therefore, 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 an antioxidant substance can be confirmed by this principle (see FIG. 15).

  Then, when the antioxidant ability of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) in the present invention was observed by the ORAC method, the so-called standard substance (Trolox) and Similarly, there was a postponement period and a strong antioxidant activity could be observed (see FIG. 16).

  In this example, in order to search from the supernatant 8 described above, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4) having high antioxidant power in the ethyl acetate fraction was used using the ORAC method. -methoxybenzyl alcohol) was discovered.

  Next, Example 2 will be described.

  Conventionally, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) has not been found in raw oyster meat. It has not been confirmed whether the active ingredient 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced.

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, 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 (3,5-dihydroxy-4-methoxybenzyl alcohol) was not detected when the extract after oyster extract extraction 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 amount of liquid 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, after adding 20 L of pure water to a 20 kg oyster and heating at standard pressure for 2 hours, the solid content (boiled oyster) was 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 liquid amount 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 per 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 by heating at 3 atmospheres for 1 hour) compared to heating experiments without pressure (oyster extract extracted at standard pressure for 18 hours). .

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

When referring to the MRM chromatogram of E6 in the sample heated for 2 hours at 3 atm for 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 amount of liquid 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.

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

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 is about 5.5 times higher than that without pressure when compared with 1 kg of oysters. It is.
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 3,5-dihydroxy-4-methoxybenzyl alcohol was present in the liquefied raw oyster.

  However, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was not detected at all in the liquid that was pressed, crushed and liquefied. . That is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) 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 3,5-dihydroxy-4-methoxybenzyl alcohol was not observed even in the extract subjected to the heat treatment for 3 hours.

  Further, the extract is subjected to heat treatment (92 ° C. to 94 ° C.) for 4 hours at 1 atm (total time including the extraction time), and 3,5-dihydroxy-4-methoxybenzyl is added to the extract. It was measured whether or not alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was present.

  In the heat treatment (92 ° C. to 94 ° C.) for 5 hours, the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol in the extract is 0.09. (Μg / ml) and concentration measurement values are below the limit of quantification, and confidence that 3,5-dihydroxy-4-methoxybenzyl alcohol exists reliably Sex was not obtained.

  Subsequently, the extract is subjected to heat treatment (92 ° C. to 94 ° C.) for 6 hours at 1 atm (total time including extraction time), and 3,5-dihydroxy-4-methoxy is added to the extract. The presence or absence of benzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was measured, but 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5- The presence of dihydroxy-4-methoxybenzyl alcohol) is 0.12 (μg / ml), and the concentration is still below the limit of quantification. In this case, 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3, the reliability that 5-dihydroxy-4-methoxybenzyl alcohol) is surely present was not obtained.

  However, the extract is heated at a high temperature of 98 ° C. to 100 ° C. for 6 hours at 1 atmosphere (7 hours in total including the extraction time), and 3,5-dihydroxy-4-methoxy is added to the extract. In order to evaluate whether benzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is present, the presence of E6 was measured using HPLC (Prominence LC-20A, Shimadzu).

And in the extract, we were able to find a value above the limit of quantification as the concentration value of the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). is there.
That is, in this case, the reliability that 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) exists was obtained.

  In this way, when heat treatment is performed at a high temperature around 100 ° C., 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) can be obtained by heat treatment for 6 hours or more. ) That is, for E6, a numerical value exceeding the limit of quantification could be found as a numerical value of concentration.

  Further, the extract is heated (92 ° C. to 94 ° C.) for 8 hours at 1 atm (9 hours when the extraction time is included), and 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3, it was measured whether 5-dihydroxy-4-methoxybenzyl alcohol) was present.

  However, the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol 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.

  Subsequently, the above extract is subjected to heat treatment (92 ° C. to 94 ° C.) for 10 hours at 1 atm (11 hours with extraction time), and 3,5-dihydroxy-4-methoxybenzyl alcohol is added to the extract. Whether or not (3,5-dihydroxy-4-methoxybenzyl alcohol) was present was measured.

  And the presence of 3, 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) at a predetermined concentration is present in the extract subjected to this long-time heat treatment (92 ° C to 94 ° C). It was confirmed whether it was recognized.

  Then, the numerical value exceeding the quantification limit of concentration measurement can be measured for the first time as 0.54 (μg / ml), and 3,5-dihydroxy-4 is contained in the extract subjected to the heat treatment for 11 hours in total. It was confirmed that -methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was clearly present. This means that 215.8 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced per kg of oyster.

  Further, the extract is subjected to heat treatment for 12 hours at 1 atm (a total of 13 hours when the initial extraction time is included), and a predetermined concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol (3 , 5-dihydroxy-4-methoxybenzyl alcohol) was measured.

  Then, when it was confirmed whether or not 3,5-dihydroxy-4-methoxybenzyl alcohol was found in the extract, 0.86 (μg / ml) And it was possible to measure the increased numerical value further exceeding the limit of quantification.

  This means that 288.4 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced per kg of oyster.

  Further, the extract is heated for 14 hours at 1 atm (a total of 15 hours when 1 hour of the extraction time is included), and the extract is subjected to a predetermined concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3, it was measured whether 5-dihydroxy-4-methoxybenzyl alcohol) was present.

  Then, when it was confirmed whether or not 3,5-dihydroxy-4-methoxybenzyl alcohol was found in the extract, 1.42 (μg / ml) And the numerical value exceeding the limit of quantification can be measured, and 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is found in the extract after prolonged heat treatment. It was confirmed that it was present even more.

  In this case, it means that 373.9 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced per kg of oyster.

  Subsequently, the extract is heated at 1 atmosphere for 16 hours (17 hours including the extraction time), and 3,5-dihydroxy-4-methoxybenzyl alcohol (3, 5) having a predetermined concentration is subjected to heat treatment. Whether or not -dihydroxy-4-methoxybenzyl alcohol) was present was measured.

  When it was confirmed whether or not 3,5-dihydroxy-4-methoxybenzyl alcohol was found in the extract, 2.63 (μg / ml) Again, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is increased by prolonged or heat treatment Confirmation was obtained.

  In this case, it means that 478.6 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was produced per kg of oyster.

  Further, the extract is heated for 17 hours at 1 atmosphere (18 hours including the extraction time), and 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5 Whether or not -dihydroxy-4-methoxybenzyl alcohol) was present was measured.

  Then, when it was confirmed whether or not the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was found in the extract, it increased again to 3.42 (μg / Ml), and it was confirmed that 3,5-dihydroxy-4-methoxybenzyl alcohol was present.

  In this case, 3,5-dihydroxy-4-methoxybenzyl alcohol (51,5-dihydroxy-4-methoxybenzyl alcohol) means that 519.5 μg was produced per kg of oyster.

  Next, the extract is heated at 1 atm for 18 hours (19 hours including the extraction time), and a predetermined concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5- Whether dihydroxy-4-methoxybenzyl alcohol) was present was measured.

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 609.3 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced per kg of oyster.

  Further, for 19 hours at 1 atmosphere (20 hours including 1 hour of extraction time), the extract was subjected to heat treatment for a long time, and 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3, it was measured whether 5-dihydroxy-4-methoxybenzyl alcohol) was present. Then, when it was confirmed whether or not the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) at a predetermined concentration was observed in the extract, 6.49 (μg / Ml), and it was confirmed that 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was further increased.

  In this case, it means that 58,3 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced per 1 kg of oyster.

  Thus, initially, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is not present at all in raw oysters, and oyster meat extract at 1 atm for 1 hour. It was not detected at all in the extract obtained by heating extraction, but it was detected but increased as the extract was heated for a very long time, and the heating temperature was increased to around 100 ° C. It was confirmed that

  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 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was produced.

  Furthermore, as described above, when the extract is heated at 1 atmosphere for 2 to 8 hours, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) having a predetermined concentration is used. alcohol) was not detected at all, but when the extract (extracted liquid from which raw oysters had been removed) was heated at 3 atm for 1 hour, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5 -dihydroxy-4-methoxybenzyl alcohol) concentration of 1.7 (μg / ml) is obtained. From this value, 21.2 kg of liquid volume is obtained from 20 kg of raw oysters, and the specific gravity of the extract is 1 Then, 1820 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was produced from 1 kg of fresh oysters by heat treatment at 3 atmospheres for 1 hour.

  Furthermore, when the extract was heat-treated at 3 atm for 2 hours, the concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol (3.5) was 3.5 ( μg / ml) is obtained, and from this value, a liquid volume of 19.1 kg is obtained from 20 kg of raw oysters, and if the specific gravity of the extract is 1, 1 kg of raw oysters by heat treatment at 3 atm for 2 hours From this, 3342.5 μg of 3,5-dihydroxy-4-methoxybenzyl alcohol was produced.

  In addition, when the heat treatment is performed at a pressure of 1 atm or more for a predetermined time, for example, 50 minutes or more, the heating temperature is increased due to pressurization, and 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5 As a concentration value of -dihydroxy-4-methoxybenzyl alcohol), a value above the limit of quantification can be found.

  In this way, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-, 4-methoxybenzyl alcohol), which is not originally found in raw oyster meat, It was found that the extract was clearly produced by heating and / or pressure treatment. Moreover, it was found that the longer the heating time, the more 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-, 4-methoxybenzyl alcohol) is produced.

  In addition, when 3-atmospheric pressure treatment is performed, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-, 4-methoxybenzyl alcohol) is produced even in a short treatment time. It was confirmed that it was done.

  In addition, this inventor crushed raw oyster meat from which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was not detected in a raw state, and made it a mince state. Then, the minced crushed material is heated at 80 ° C. or more for 3 hours or more at a ratio of (raw oyster minced) 3: (water) 1, whereby 3,5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) was produced from the heat-treated oyster meat liquid.

  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 crushed and put into a minced state. Could be produced (7000 ng / mL).

  By the way, it has been reported that oxidative stress occurs when mice are reared individually.

  It is known that excessive enhancement of the HPA system due to oxidative stress causes hypersecretion of corticosterone due to a decrease in hippocampal glucocorticoid receptors and poor transmission of the HPA system.

Therefore, the anti-fractionation of fractions containing 3,5-dihydroxy-4-methoxybenzyl alcohol, a oyster soft body extract, was subsequently applied to mice that were oxidatively stressed by individual breeding. The oxidative and anti-stress effects were examined.
That is, the present inventors examined whether or not 3,5-dihydroxy-4-methoxybenzyl alcohol is a component involved in antioxidant and antistress.

  Therefore, the inventor of the present invention has found that a oyster soft body extract 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is contained in mice that have been oxidatively stressed by individual breeding. Whether anti-stress substances are present in the painting, and anti-stress substances with 3,5-dihydroxy-4-methoxybenzyl alcohol are truly involved in anti-stress Whether or not it is a component was clarified by the following experimental example.

(Preliminary experiment)
As a preliminary examination experiment, after ICR male mice were kept for 6 days, group breeding group (n = 3) and individual breeding group (n = 3) were reared for 42 days with free intake of CRF-1, Brain MDA levels were measured and examined by Student's t-test.

  As a result, the MDA value of 31.86 ± 0.77 (μmol / g of brain) in the individual breeding group was significantly higher than the group breeding group MDA value of 23.22 ± 1.71 (μmol / g of brain) at 1% risk. From this, it was confirmed that long-term individual breeding induces oxidative stress in the brain.

(Experiment 1)
18 ICR male mice 4 weeks old were group-bred for 6 days. The ICR mouse is an albino mouse originating from a Swiss mouse and is a strain selected by Dr. Hauschka for the purpose of prolific production.

  Since it was sent to various places by the Institute of Cancer Research in the United States, it was named ICR after taking the initials. This system was introduced from Charles River (USA) in 1963 and production and supply started. Characteristic features are relatively large, good growth, and temperamental nature. Therefore, it is used as a general purpose strain for studies in a wide range of fields including toxicity, pharmacology, medicinal properties, immunity, and others.

  During the group breeding, the solid feed CRF-1 was freely ingested and acclimatized. Here, the solid feed CRF-1 is a standard feed, and its component contents are as understood from the list of standard feed component contents (in 100 g) of FIG.

  After 18 days of group rearing, the 18 ICR male mice were divided into groups.

  That is, oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) and 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5- Dihydroxy-4-methoxybenzyl alcohol) does not contain oyster meat extract, for example, a control group (only group A) given only drinking water, and 3,5-dihydroxy-4-methoxybenzyl alcohol ( 3,5-dihydroxy-4-methoxybenzyl alcohol) -Free group, ie, oyster meat extract that does not contain 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) 6 groups (referred to as Group B) and 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, ie 3,5-dihydroxy-4-methoxybenzyl Contains alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) And (referred to as C group) oyster meat extract a given the group was fractionated into six three groups.

Thereafter, the mice were individually raised for 43 days with free intake of the CRF-1.
“Individual breeding” means that the aforementioned mice are individually isolated and reared individually. Mice are animals that feel stressed in groups, that is, if they are not housed in groups. Therefore, oxidative stress was caused by individual breeding.

In addition, for 14 days prior to dissection, each test object, ie, Group A, Group B, and Group C, was each 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). Does not give oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), for example, only drinking water, In addition, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Free group, that is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4- Oyster meat extract not containing methoxybenzyl alcohol) or 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, ie 3,5-dihydroxy- 4-methoxybenzyl alcohol (3,5-dihydroxy-4-m Oyster meat extract containing ethoxybenzyl alcohol) (content: 2.43μg / 0.1ml) was orally administered through a stomach tube.
In addition, oral administration with a gastric sonde means forced administration directly into the stomach from the mouth of a mouse using a device such as a syringe.
The dose of the test subject was 0.1 mL per 10 g of mouse body weight.

  In addition, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Free group, that is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4- Group B given oyster extract without methoxybenzyl alcohol) and 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, ie 3, 5 The amount of zinc and other contents in the oyster meat extract to group C given the oyster meat extract containing 3-dihydroxy-4-methoxybenzyl alcohol was the same amount .

  Thereafter, one hour after the test subject on day 14 was orally administered, blood was collected and organs were removed, and the plasma corticosterone concentration was measured.

  Here, corticosterone is a type of glucocorticoid secreted from the adrenal cortex, a substance synthesized in the body through a series of enzymatic reactions from cholesterol and controlled for secretion by the anterior pituitary hormone ACTH. is there. Corticosterone is known to increase the amount of secretion when stress is applied, and is used as an effective biomarker for measuring the degree of stress.

  8-OHdG is a substance in which a guanine base is changed to 8-hydroxy-deoxyguanosine (8-OHdG) by oxidation. DNA is composed of four types of bases: adenine, guanine, cytosine, and thymine. DNA is known to be oxidatively damaged by active oxygen, and guanine base is converted to 8-OHdG by oxidation. This 8-OHdG is excised from the gene body in the process of repairing the gene DNA, and excreted in the urine via blood. Furthermore, 8-OHdG is a relatively stable substance that is rapidly excreted in the urine without being metabolized or decomposed in the living body. Therefore, 8-OHdG is used as an excellent biomarker that reflects living body damage caused by active oxygen. ing.

  MDA is malondialdehyde (MDA). MDA is one of lipid peroxidation degradation products and is used as a main marker of lipid peroxidation.

  By the way, for statistical processing when calculating the risk factors, etc., one-way analysis of variance (ANOVA) was performed, and items with significant differences in analysis of variance were examined by multiple comparison using the Tukey method. . For both analysis of variance and multiple comparisons, the statistical significance level was 1% and 5%.

(Results and discussion of Experiment 1)
In addition, group A in FIG. 1 includes oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) and 3,5-dihydroxy-4- For example, the control group to which oyster meat extract containing no methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was not given, for example, only drinking water, was given, and group B was 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Free group, that is, oysters that do not contain 3,5-dihydroxy-4-methoxybenzyl alcohol The group to which the meat extract was given, and the group C was 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, that is, 3,5-dihydroxy-4-methoxy Contains benzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) A group to which oyster meat extract was given.

  FIG. 1 shows the plasma corticosterone concentration. According to the SMIRNOFF rejection test method, an abnormality was found in one data of group B, which was rejected. As can be seen from FIG. 1, the plasma corticosterone concentration value of group C is 119.8 ± 5.7 (ng / mL), whereas the plasma corticosterone concentration value of group A is 149.9 ± 7.5 (ng / mL).

  From the results, it was confirmed that the plasma corticosterone concentration value of group C was lower than the plasma corticosterone concentration value of group A. Therefore, the risk rate was 1%, which was significant.

  As a result of the test, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Containing group, that is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy Group (Group C) given oyster meat extract containing -4-methoxybenzyl alcohol) contains 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) A control group (A, for example, which was given only drinking water without giving oyster meat extract or oyster meat extract without 3,5-dihydroxy-4-methoxybenzyl alcohol) It can be understood that the 1% risk rate was significantly lower than the group).

  Therefore, from the measurement result of plasma corticosterone concentration value, 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, that is, 3,5-dihydroxy-4- It can be judged that the stress relieving action of group C to which the oyster meat extract containing methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was given was observed.

  However, as a result of comparing the plasma corticosterone concentration values of the B group and the A group, there was no significant difference.

  That is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Free group, that is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4- This is because no anti-stress effect was observed in group B to which oyster meat extract containing no methoxybenzyl alcohol) was given.

  The difference between the components of Group B and Group C is that they do not contain or only contain 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). Yes, and other components such as zinc were the same.

  Therefore, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Containing group, ie, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4 The stress relieving effect observed only in group C given oyster meat extract containing (-methoxybenzyl alcohol) was 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). It became clear that it originated.

  That is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was shown to act as a functional component of the anti-stress action of oyster meat extract.

(Experiment 2)
In Experiment 2, in order to accurately grasp the stress response status of each group due to individual rearing stress, the dose of each test diet was doubled and the number of animals increased, the 8-OHdG concentration in the brain, Changes in MDA concentration, plasma corticosterone concentration and hippocampal corticosterone receptor number were observed.

In Experiment 2, after the same preliminary breeding as described above, the group was divided into three groups, A group (n = 10), B group (n = 10), and C group (n = 10), and then ICR males. Mice were individually raised for 50 days with free intake of CRF-1 and distilled water.
From the 43rd day after the start of individual breeding, each sample was forcibly administered orally with a stomach tube every day at 9:00 AM for 8 days. The dose was 0.2 mL per 10 g body weight.

Group A is pure water, Group B is 3,5-dihydroxy-4-methoxybenzyl alcohol-Free group, ie 3,5-dihydroxy-4- The oyster soft body extract fraction not containing methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is classified into group C as 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4 -methoxybenzyl alcohol) -Containing group, ie, oyster soft body extract fraction containing 3,5-dihydroxy-4-methoxybenzyl alcohol was forcibly orally administered. Dissection was performed from 1 hour after administration (AM10: 00) on the 8th day. For dissection, laparotomy was performed under anesthesia with somnopentyl (50 mg / kg), and heparinized blood was collected from the inferior vena cava. Thereafter, the whole brain was removed and the hippocampus was isolated.

(Results and discussion of Experiment 2)
Next, FIG. 2 shows the 8-OHdG concentration in the brain. As can be understood from FIG. 2, the 8-OHdG concentration value in the brain of group C is 7.51 ± 0.71 (ng / g of brain), whereas the 8-OHdG concentration value in the brain of group A is 11.32 ± 0.86 (ng / g of brain).

  From the results, it was confirmed that the 8-OHdG concentration value in the brain of group C was lower than the 8-OHdG concentration value in the brain of group A. Therefore, the risk rate was 1%, which was significant.

  Next, FIG. 3 shows the MDA concentration in the brain. As can be seen from FIG. 3, the MDA concentration value in the brain of group C is 22.13 ± 0.58 (μmol / g of brain), whereas the MDA concentration value in the brain of group A is 28.11 ± 1.33. (μmol / g of brain).

  From the results, it was confirmed that the MDA concentration value in the brain of group C was lower than the MDA concentration value in the brain of group A. Therefore, the risk rate was 1%, which was significant.

Therefore, from the measurement results of the 8-OHdG concentration value in the brain and the MDA concentration value in the brain, 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, that is, , 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) group was given a significant antioxidant effect on brain DNA and lipid It was.

FIG. 4 shows the plasma corticosterone concentration.
Plasma corticosterone level 141.4 ± 9.8 (ng / ml) in group C is significantly lower at 1% risk than group A 193.9 ± 9.1 (ng / ml), and group B 177.3 ± 6.7 (ng / ml) It was significantly lower at a 5% risk (Fig. 4).

  Similar to the results of Experiment 1, it was observed that the C group had a significantly higher effect of suppressing homeostasis against stress than the A group and the B group, and suppressed reproducibility.

Next, FIG. 5 shows the hippocampal glucocorticoid receptor Alpha concentration.
As can be seen from FIG. 5, the GR Alpha value 0.812 ± 0.038 (ng / mg of hippocampus) in the hippocampus of group C is significantly higher at 1% risk than group A 0.590 ± 0.036 (ng / mg of hippocampus). In addition, it was significantly higher at a 5% risk rate than Group B 0.638 ± 0.049 (ng / mg of hippocampus) (FIG. 5).

  As a biological response to stress, corticosterone is released into the blood through activation of the HPA system, and when corticosterone binds to hippocampal GR Alpha, a rest signal of the stress response is transmitted to the hypothalamus. The homeostasis is maintained by terminating the stress response.

However, oxidative stress due to individual rearing stress decreases the hippocampal GR Alpha and fails to transmit a resting signal to the hypothalamus, thereby continuing corticosterone release, increasing plasma corticosterone concentration, The situation seems to have been prolonged.
In addition, it is said that when the stress state is prolonged, the neurites of the hippocampus itself atrophy and disappear, thereby making the brain vulnerable to stress.

  In this experiment, the plasma corticosterone concentration was high even under individual rearing stress environment, but 3,5-dihydroxy-4-methoxybenzyl alcohol-Containing group, In other words, ingestion of oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol alleviates oxidative stress in the brain, thereby increasing hippocampal GR Alpha, GR Since the expression was increased, it was suggested that quiescent signal transmission to the hypothalamus was smooth, corticosterone release was reduced, and homeostasis of the living body was maintained.

  That is, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) -Containing group, ie, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4 of the hippocampal GR Alpha by antioxidant activity in the brain derived from 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) It appears that the plasma corticosterone concentration decreased due to the resting signal transmission to the hypothalamus, and the homeostasis of the living body against stress was maintained, and 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5- Dihydroxy-4-methoxybenzyl alcohol) was observed to relieve stress.

  Next, we will explain experimental examples of anti-stress, anti-depression, anti-fatigue, anti-confused and vitality amplification effects by taking E6 (3, 5-dihydroxy-4-methoxybenzyl alcohol) -containing oyster meat extract for men and women workers .

  Here, in order to examine the impact on the subjective evaluation of stress and fatigue when consuming oyster meat extract containing E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) for 4 weeks in a row, POMS shortened version (POMS -S) Targeting 84 adult males and females aged 30 to 60 who feel stress and fatigue in daily life such as work 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 enrolled in the study, no subjects dropped out during the study period, and all 84 completed the prescribed schedule. Among them, 78 people (40 people in A group) excluding 6 people (test food group (Group A) 2 people, placebo group (Group P) 4 people) who met the efficacy analysis exclusion criteria defined in the research plan in advance. Name, P group 38).

  Regarding the evaluation item POMS-S, the amount of change from the previous “depression” score to the third and fourth weeks was significantly lower in the A group than in the P group (the third week: the A group: -17.5 ± 13.4, P group: −11.1 ± 12.2, 4th week: Group A: −19.0 ± 11.8, P group: −13.6 ± 11.0). In addition, the amount of change in the “fatigue” score from the previous week to the second week is significantly lower in the A group than in the P group (A group: -15.1 ± 11.1, P group: -9.9 ± 10.0). It was. In addition, the “liveness” score did not show a statistically significant difference in the comparison between the groups, but the change amount from the prior of the A group was larger (improvement) than the P group at each time point.

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

(The purpose and background of this experiment)
The oyster meat extract used in this experiment is an oyster meat extract obtained by concentrating the obtained supernatant after adding ethanol to an extract extracted and concentrated from raw oysters.
E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in oyster meat extract is being researched as a novel antioxidant substance, and as described above, it has been confirmed by animal tests using oyster meat extract containing E6. Antioxidant and stress relieving action is recognized.
Therefore, in this experiment, the effects of placebo on the subjective evaluation of stress and fatigue when men and women between the ages of 30 and 60 who are experiencing stress and fatigue have taken E6-containing oyster meat extract for four consecutive weeks. Was compared as a control.

Study Methods Study 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 study was 42 for the test food group and 42 for the placebo group, for a total of 84 people.

Allocation method The allocation staff created an allocation table using random numbers and assigned an allocation number to the research food. The assignment table was sealed by the person in charge of assignment and kept sealed until the assignment table was opened.

Research period: Conducted from January 2016 to April 2016.

Subject selection and exclusion criteria Subjects who met the following selection criteria and did not violate the exclusion 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

Research target food (hereinafter referred to as research food)
Name of research food Test food: Beverage of oyster meat extract containing E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) Placebo: Beverage without oyster meat extract containing E6 (3,5-dihydroxy-4-methoxybenzyl alcohol)

Eating experience of research food E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in oyster meat extract is being studied as a new antioxidant, and E6 (3,5-dihydroxy-4-methoxybenzyl alcohol) Antioxidant stress mitigating action and the like have been recognized by animal tests using oyster meat extract containing

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

Research food intake, intake method, intake period, and related ingredient intake settings (1) Research food intake, intake method, and intake period Research food is twice a day, once in the morning, at least one hour at night. One dose (50 mL) was taken once before. A diary was recorded daily during the study period. The intake period was 4 weeks.

Study schedule and examination content Study schedule (1) The candidate candidates visited the pre-examination, and each examination of the lifestyle questionnaire and POMS shortened version (POMS-S) was completed.
(2) Subjects were selected from the pre-examination results, and they visited the pre-ingestion examination, and physical condition confirmation / measurement and general clinical examinations were conducted.
(3) 84 subjects were selected from the test results before ingestion. Subjects were distributed research food and diary during the intake period, and they began recording the diary of the intake and the intake period of the research food.
(4) The subject will come to the hospital on the 2nd week (15th day) and 4th week (29th day) after ingestion, and perform physical condition confirmation / measurement and POMS-S entry tests. A general clinical test was also conducted at the 4th week.
(5) After starting intake, POMS-S was entered at home in the first week (8th day) and in 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 Evaluation item: POMS-S

Evaluation method
POMS-S compared the test food intake group and the placebo intake group using a 2-sample t-test for the change from the pre-intake value of the test value at each time point after intake. As a reference, the amount of change from before ingestion at each time point after ingestion in each group was evaluated using a one-sample t-test.

Numerical display and significance level Numerical values are expressed as mean ± standard deviation, and significance levels of both p <0.05 and p <0.01 were used for the test.

Results of the study 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 men and 47 women, and the study was started.

Breakdown of analysis subjects Among the 84 subjects who started the study, no subjects dropped out of the study, and 84 subjects completed the prescribed schedule and examination contents.
Six subjects decided to exclude the subject from the efficacy analysis before opening the assignment table.
Therefore, 78 subjects were analyzed for effectiveness (Fig. 1 explained in Fig. 19).

Background factors of effectiveness analysis subjects Table 2-1 described in FIG. 20 shows the background factors of the effectiveness analysis subjects (sex, age, height, weight, BMI, systolic blood pressure, diastolic blood pressure, pulse, POMS- S, AIS). There was no significant difference in POMS-S and AIS used as selection criteria.

Efficacy Evaluation Key Evaluation Items (POMS-S)
Table 2-2-1 described in FIG. 21 and Table 2-2-4 described in FIG. 22 show the transition of POMS-S as an evaluation item.

POMS-S
The amount of change from pre-depression (D) to the 3rd and 4th weeks was significantly lower in the A group than in the P group (3rd week: A group: -17.5 ± 13.4, P group:- 11.1 ± 12.2, 4th week: Group A: −19.0 ± 11.8, Group P: −13.6 ± 11.0). The amount of change in fatigue (F) from the previous week to the second week was significantly lower in the A group than in the P group (A group: -15.1 ± 11.1, P group: -9.9 ± 10.0) ( Table 2-2-1).
In each group, in group A, tension / anxiety (TA) (at each time point), depression (D) (at each time point), anger / hostility (AH) (week 1, week 3, week 4) ), Vitality (V) (1st, 3rd, 4th week), fatigue (F) (at each time point), confusion (C) (1st, 3rd, 4th week) compared with previous Significantly changed (improved). In group P, tension / anxiety (TA) (at each time point), depression (D) (at each time point), anger / hostility (AH) (1st week, 3rd week, 4th week), vitality (V) (1 (Week, Week 3, Week 4), Fatigue (F) (Week 1, Week 3, Week 4), Confusion (C) (Week 1, Week 3, Week 4) Compared with, it changed significantly (improved).

Exploratory effectiveness analysis (POMS-S)
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 POMS-S.

POMS-S (subject group under 50)
The amount of change at each time point after ingestion of depression (D) was significantly lower in group A than in group P (1 week: group A: -14.0 ± 12.0, group P: -7.4 ± 10.1, 2nd week: Group A: -16.9 ± 11.6, P group: -9.8 ± 9.2, 3rd week: Group A: -18.8 ± 2.6, P group: -9.7 ± 11.1, 4th week: Group A: -19.9 ± 11.9, P group: -12.3 ± 10.6). The amount of change at each time point after ingestion of vitality (V) was significantly higher in group A than in group P (1 week: Group A: 9.2 ± 10.3, Group P: 3.9 ± 5.1, 2 weeks) : A group: 9.3 ± 9.0, P group: 4.1 ± 5.3, 3rd week: A group: 11.1 ± 11.1, P group: 4.1 ± 7.1, 4th week: A group: 10.0 ± 10.4, P group: 3.7 ± 4.9 )showed that. The amount of change at each time point after the intake of fatigue (F) was significantly lower in group A than in group P (1 week: group A: -12.7 ± 10.6, group P: -6.5 ± 8.4, Week 2: Group A: -15.6 ± 11.3, Group P: -7.6 ± 8.2, Week 3: Group A: -16.4 ± 11.5, Group P: -10.1 ± 9.7, Week 4: Group A: -17.7 ± 10.8, P group: -11.6 ± 11.1). The amount of change at each time point after the ingestion of confusion (C) was significantly lower in the A group than in the P group (1 week: A group: -13.5 ± 12.1, P group: -7.7 ± 9.4, 2nd week: Group A: -14.5 ± 12.3, P group: -8.9 ± 7.4, 3rd week: Group A: -18.0 ± 11.8, P group: -10.5 ± 11.3, 4th week: Group A: -18.0 ± 12.4, P group: -10.6 ± 10.7) (Table 2-2-4).

  In each group, in group A, tension / anxiety (TA) (at each time point), depression (D) (at each time point), anger / hostility (AH) (1st week, 3rd week, 4th week) ), Vitality (V) (1st, 3rd, 4th week), fatigue (F) (at each time point), confusion (C) (1st, 3rd, 4th week) compared with previous Significantly changed (improved). In group P, tension / anxiety (TA) (weeks 1, 3, 4), depression (D) (weeks 1, 3, 4), anger / hostility (AH) (3 Week (4th week), Vigor (V) (1st week, 3rd week, 4th week), Fatigue (F) (1st week, 3rd week, 4th week), Confusion (C) (1 (Week, Week 3, Week 4) significantly changed (improved) compared to the previous time.

Discussion I feel sleep problems, stress, and fatigue with an Athens 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. Compared with placebo as a control for 84 adult males and females aged between 30 and 60 years of age, the effects of drinking E6-containing oyster meat extract on subjective quality of sleep quality, stress, and fatigue after four consecutive weeks of intake Study was carried out.

  Regarding POMS-S, which is the primary endpoint, the amount of change from pre-depression (D) to weeks 3 and 4 is significantly lower in group A than in group P (week 3: group A) : -17.5 ± 13.4, P group: -11.1 ± 12.2, 4th week: A group: -19.0 ± 11.8, P group: -13.6 ± 11.0). In addition, the change in fatigue (F) from the previous week to the second week is significantly lower in Group A than in Group P (Group A: -15.1 ± 11.1, Group P: -9.9 ± 10.0). (Table 2-2-1). The vitality (V) used for the selection criteria was not significantly different in the comparison between groups, but at each time point, the amount of change from Group A in comparison with Group P was larger (improvement). there were. Depression means “depression with a feeling of loss of confidence”, fatigue means “decreased motivation and vitality / feeling of fatigue”, and vitality means “energy, vigor or vitality”. Therefore, it is considered that E6-containing oyster meat extract drinks can help relieve mental stress and fatigue and improve vitality.

  When subjected to physical / physical stress or psychological stress, it produces oxidative stress in the brain, and the hypothalamic-pituitary-adrenal axis (HPA system) is enhanced. The release of certain glucocorticoids is increased. When the glucocorticoid binds to the glucocorticoid receptor in the hippocampus, the negative feedback of the HPA system functions and the stress response is terminated. In chronic brain oxidative stress, the amount of glucocorticoid receptor in the hippocampus decreases, leading to the enhancement of the HPA system, but the glucocorticoid receptor decreased by vitamin E, an antioxidant, increases, causing anxiety in rats. It is known that behavior decreases.

  E6 translocation into the brain has been confirmed from brain samples of the blood-brain barrier in vitro remodeling system model test and mouse oral administration test, but the safety of E6 to the central nervous system has been confirmed by observation of general conditions in rats Has been. In addition, a reduction in oxidative stress markers (8-OHdG, MDA) in the brain has been confirmed by oral administration of E6-containing oyster meat extract to mice stressed by long-term individual breeding. Furthermore, a significant increase in hippocampal glucocorticoid receptors and a significant decrease in plasma corticosterone levels have been confirmed with a decrease in brain oxidative stress. Therefore, it is considered that in humans, mental stress and fatigue were alleviated by reducing the oxidative stress state in the brain by the antioxidant ability of E6.

The stress biomarker study results suggest that younger people have higher stress adaptability, and therefore, 61 males and females (A group: 31 people, P group: 30 people) who are under 50 years old among the efficacy analysis subjects. An additional exploratory analysis of POMS-S was performed on the subjects. In POMS-S, in addition to depression (D) and fatigue (F), group A showed a significant improvement compared to group P in vigor (V) and confusion (C).
These results show that E6-containing oyster meat extract is particularly effective in improving stress, fatigue, confusion, and vitality 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.

  With regard to safety, although there were some fluctuations in the test values, the responsible physician judged that there was no problem with the safety of the E6-containing oyster meat extract beverage. In addition, no side effect was observed with the E6-containing oyster meat extract beverage. Therefore, the safety of E6-containing oyster meat extract beverages was demonstrated under the present study conditions.

Conclusions: A placebo-controlled, randomized, double-blind, parallel group comparison study of working men and women aged 30 to 60 who feels stress and fatigue. The evaluation score of depression and fatigue of POMS-S was improved. Therefore, E6 is thought to relieve mental stress and fatigue.

E6 was particularly effective in improving stress, fatigue, confusion, and vitality for working men and women under 50 years of age.
In this experiment, there was no problem with the safety of the E6-containing oyster meat extract beverage.

  In addition, we will investigate the antioxidant and stress mitigating actions of 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as E6), a novel antioxidant identified from oyster soft body extract, on oxidative stress mice.

Two types of extracts, E6-containing extract (24.3 μg / mL) and E6-free extract, were prepared from the oyster soft body.
30 SLc: ICR male mice were individually reared for 42 days to create oxidative stress mice. These mice were divided into 3 groups, and water, E6 non-extracted solution, and E6-containing extract were orally administered for 8 days, respectively. After that, blood sample, liver, kidney including adrenal gland (hereinafter referred to as kidney), pancreas and spleen are removed, malondialdehyde (MDA) value in each organ, 8-hydroxy-2'-deoxyguanosine (8-OHdG) value in kidney And plasma corticosterone levels were measured.

  Results: MDA in each organ and 8-OHdG in the kidneys were significantly decreased and plasma corticosterone concentration was significantly decreased only in the group ingesting the E6-containing extract (hereinafter referred to as E6 group).

  Conclusion: E6 contained in the soft body of oysters showed a lipid antioxidant effect in each organ, a fatigue relieving effect by a DNA antioxidant effect in the kidney, and a stress relieving effect by lowering plasma corticosterone.

(Introduction)
In a human study, we took a Japanese oyster soft body extract (test drink) to a subject who had sleep problems and felt fatigue and stress. The Japanese version of the Profile of Moods States, a shortened version of the psychological index ( POMS) and the Athens Insomnia Scale (AIS) confirmed the usefulness of the test beverage for stress such as depression, fatigue, and sleep quality. Furthermore, in a placebo-controlled, randomized, double-blind, parallel-group comparison study, we observed sleep-improving effects and inhibitory effects on mid-wakefulness by AIS and human sleep electroencephalogram measurements when the test beverage was ingested. As described above, the stress relieving action, fatigue reduction, and sleep quality improving action of the oyster soft body extract have been observed, but the components involved are unknown.

  It is shown that cortisol concentration decreases as well as the depression of POMS depression occurs by massage, etc., plasma and salivary cortisol as stress markers as emotional approach, POMS as emotional approach and objective approach It is used. Increased plasma cortisol levels due to stress have been reported to cause depression-like behavior. It has been shown that in patients with depression, blood cortisol concentration, which is a stress hormone, is elevated at the time of admission, and cortisol concentration decreases as depression improves with treatment.

  Antioxidants such as ascorbic acid have been reported to prevent disorders involving the endocrine system caused by reactive oxygen species. She-Fang Ye et al. Show that when the kidneys, including the adrenal gland, are affected by oxidative stress, the blood corticosterone level is affected by the secretion function, but the value is suppressed by administration of antioxidants, and normal It was shown that

  In other words, it is suggested that oxidative stress induces an increase in plasma glucocorticoid and raises the numerical value of depression of psychological index POMS, followed by a decrease in blood glucocorticoid due to the intake of antioxidants A decline in POMS has been reported.

  We isolated and identified a novel antioxidant substance, 3,5-dihydroxy-4-methoxybenzyl alcohol (E6), which exhibits strong amphipathic properties from the oyster soft body. The antioxidant capacity of E6 was 1.24 ± 0.35 μmol of TE / μmol as evaluated by Oxygen Radical Absorbance Capacity Assay (ORAC method), which is an antioxidant capacity measurement method. The ORAC value of L-ascorbic acid, an antioxidant also called vitamin C, was 0.53 ± 0.13 μmol of TE / μmol, and the ORAC value of E6 was about 2.3 times that of L-ascorbic acid.

  In this test, the hypothesis that E6 is a component involved in reducing oxidative stress and stress mitigating action of the oyster soft body extract. Based on this, it was examined whether or not E6 in the oyster soft body extract exhibited an antioxidant action and a stress mitigating action on mice in an oxidative stress state caused by individual breeding.

(Materials and methods)
1. Experimental animals and breeding conditions
Four-week-old Slc: ICR male mice (Sankyo Lab Service Co., Ltd.) were given free intake of solid feed CRF-1 (Oriental Yeast Co., Ltd.) and drinking water. Habituated. As breeding conditions, set temperature and humidity: 24 ± 1 ℃, 55 ± 5%, air conditioning equipment: All Fresh method, lighting time: 12 hours automatic on / off method (lighting from 8:00 AM to 8:00 PM), breeding Equipment: Plastic cage.
This experiment is based on the animal experiment guideline of the Natural Materials Research Institute, Inc. “Standards for the breeding and storage of experimental animals and relief of pain” (Ministry of the Environment Notification No. 88, April 28, 2006, final revision 2013) This was done in compliance with Ministry of the Environment Notification No. 84).

2. Preparation of E6-free extract and E6-containing extract Water was added to a oyster soft body part, and the extract from which the oyster soft body part was removed after water extraction by heating was used as a first extract. Next, the E6 concentration in the first extract was measured with a triple quadrupole high-performance liquid chromatograph mass spectrometer (LC-MS / MS: LCMS-8040, Shimadzu Corp.). Confirmed (detection limit: 0.04 μg / mL).
Watanabe Oyster Laboratories Co., Ltd. developed an E6 generation method by heating and pressurizing extraction of soft shells and applied for a patent.
The first extract was divided into two. Since the first extract was not treated and E6 was not detected, this extract was designated as an E6-free extract. The extract intake group was designated as an E6 non-extract extract intake group (hereinafter, E6 no group).
The second extract was heated and pressurized to produce E6. This was designated as an E6-containing extract, and the extract intake group was designated as an E6-group. The E6 concentration in the E6-containing extract is 24.3 μg / mL.

3. Method for Evaluating that E6 is a Component Involved in Antioxidation State that E6 contained in the extract containing E6 in the protocol of this test is a component influencing the antioxidant activity is described.
We have already reported the main antioxidants in oyster soft body extract. The heat-treated oyster soft body extract (E6-containing extract in this test) was separated by thin layer chromatography to obtain 11 fractions, and each fraction was tested for its antioxidant ability by the ORAC method. As a result of evaluation, the sixth fraction showed the highest antioxidant ability, but almost no antioxidant ability was observed in the other fractions. The 6th fraction was purified by HPLC (LC-20A, Shimadzu Corp.), then using ¹ H NMR, ¹³ C NMR, heteronuclear multiple-bond correlation (HMBC) and electrospray ionization-mass spectrometry (ESI-MS) E6 was identified. E6 was the main antioxidant in the oyster soft body extract.

In addition, zinc, copper, selenium and the like are involved in the functional expression of antioxidant enzymes. Therefore, in order to compare the amount of trace elements between the extract containing no E6 and the extract containing E6, zinc was used as a representative marker of all trace elements, and the zinc content of both extracts was measured using an atomic absorption spectrophotometer (AA- 7000, Shimadzu Corporation). As a result, the zinc content of the extract containing no E6 and the extract containing E6 was 2.23 ppm for both extracts, and there was no difference. From this result, it was considered that there was no great difference in the content of other trace elements including zinc between the E6-free extract and the E6-containing extract.
From these results, it was shown that the difference in the components involved in the antioxidant ability between the E6-free extract and the E6-containing extract is the difference between “not containing” or “containing” E6.

4). Stress load examination
Huong et al. Observed an increase in the level of malondialdehyde (MDA), a marker of oxidative stress, after 42 days of individual breeding of Slc: ICR male mice, and reported that individual breeding induced oxidative stress. This oxidative stress situation seemed to be similar to that of modern people living in a stressed society. In this study, the long-term individual breeding method of Huong et al. Was used to produce oxidative stress mice.

In order to confirm whether or not a stress condition was caused by long-term individual breeding, the MDA of the oxidative stress marker and the plasma corticosterone value of the stress marker were compared between the group breeding and the individual breeding.
As a stress load study, Slc: ICR male mice were preliminarily reared, and as a group rearing group, water and solid feed CRF-1 were freely fed, and 50-day group rearing (n = 10) was performed. As an individual breeding group of mice, water and solid feed CRF-1 were freely ingested, individual breeding was performed for 50 days (n = 10), and pure water was forcibly administered orally with a stomach tube for the last 8 days. . On day 50, urinary 8-OHdG levels in both groups, liver and kidney MDA levels, and stress marker plasma corticosterone levels were measured, and oxidative stress and stress status were compared.

  8-OHdG, which is a DNA oxidative stress marker, was measured using a highly sensitive ELISA kit for 8-OHdG (Japan Institute for Aging Control) after urine collection. The MDA value used as the main marker, which is the final product of lipid peroxidation, was measured using the Malondiadehyde assay kit (Japan Institute for Aging Control) after excising the liver and kidney.

  From the results of the stress load test, urinary 8-OHdG values, liver and kidney MDA values, and plasma corticosterone values of the individually bred mice were significantly higher than those of the group bred mice (FIG. 23: stress load test). . From this, it was confirmed that oxidative stress was induced in the whole mouse body, liver, and kidney by long-term individual breeding.

  Furthermore, the increase in stress due to long-term individual breeding was confirmed from the fact that plasma corticosterone, which is said to be a stress-hormone or stress marker, increased significantly by long-term individual breeding.

5. Breeding method and grouping Mice in an oxidative stress state induced by long-term individual breeding were used as a control group. In order to confirm the reproducibility of the antioxidant effect and stress relieving effect under conditions similar to the existing test, the control group (pure water was orally administered) and the E6 group with the same ingredients as the existing food were established. . Moreover, in order to confirm that E6 is a component involved in the antioxidant action and the stress relieving action, an E6 group and an E6 group were provided (FIG. 24).

  After pre-breeding, the group was divided into three groups: control group (n = 10), E6 group (n = 10), and E6 group (n = 10), and then Slc: ICR male mice Individual feeding was performed for 50 days with free intake of solid feed CRF-1 and distilled water.

  From day 43, for 8 days, pure water was used for the control group, E6-free extract was used for the E6 group, E6-containing extract was used for the E6 group, and gavage was performed daily at 9:00 AM with a gastric sonde. Administered. The dose of each feed was 0.2 mL per 10 g body weight, and the body weights of the control group, E6 group, E6 group were 45.2 ± 1.1 g, 42.8 ± 1.0 g, and 43.4 ± 1.1 g.

  Dissection was performed 1 hour after administration (10:00 AM) on the 8th day of administration. For dissection, laparotomy was performed under anesthesia with somnopentyl (50 mg / kg), and heparinized blood was collected from the inferior vena cava. Thereafter, the kidney including the liver and adrenal gland (hereinafter: kidney), pancreas and spleen were removed.

  Measurement items were liver, kidney, pancreas, spleen MDA value, kidney 8-OHdG, plasma corticosterone value. MDA and 8-OHdG values were measured in the same manner as in the stress load study.

5-1 Measurement of plasma corticosterone Corticosterone in plasma was measured using Assay Max Corticosterone ELISA kit (Assaypro). Corticosterone standard solution or plasma sample was added to each 96 μl of 25 μL, and 25 μL of biotinylated corticosterone was added, followed by incubation at room temperature for 2 hours. After washing by adding 200 μL of washing buffer, 50 μL of Streptavidin-Peroxidase Conjugate was added and incubated for 30 minutes. After washing by adding 200 μL of washing buffer, 50 μL of Chromogen Substrate was added and incubated for 20 minutes. Subsequently, 50 μL of Stop Solution was added to each well to stop the reaction, and then the absorbance at 450 nm was measured with a microplate reader (ImmunoMiniNJ-2300, Biotech). The corticosterone concentration was calculated from the absorbance data.

6). Statistical processing method The statistical analysis of the stress load examination was expressed as mean ± standard error, and the test for significant difference was performed by Student's t-test. The statistical significance level was 1% and less than 5%.
Statistical analysis of this breeding test was performed by one-way analysis of variance (ANOVA), and items with significant differences in ANOVA were examined by multiple comparison using the Tukey method. In both analysis of variance and multiple comparisons, the statistical significance level was 1% and less than 5%. Excel Statistics 2015 (Social Information Service Co., Ltd.) was used for the analysis software.

(result)
As shown in the table of Fig. 25 (MDA concentration of each organ), the MDA value of 180.1 ± 5.1 μmol / g of wet tissue in the E6 group is more dangerous than the MDA value of 210.7 ± 7.6 μmol / g of wet tissue in the control group The rate was significantly low at 1%. The MDA value of 106.5 ± 3.1 μmol / g of wet tissue in the E6 group was significantly lower than the control group of 120.0 ± 4.1 μmol / g of wet tissue at a risk rate of 5%. The MDA value of 87.92 ± 4.2 μmol / g of wet tissue in the pancreas of E6 group was significantly lower than that of the control group in the MDA value of 102.3 ± 3.2 μmol / g of wet tissue at a risk rate of 5%. The MDA value of 144.5 ± 4.5 μmol / g of wet tissue in the spleen of the E6 group was significantly lower than that of the control group at a risk rate of 5% than the MDA value of 159.5 ± 3.9 μmol / g of wet tissue.
The MDA values in the liver, kidney, pancreas, and spleen in the E6 group did not tend to be lower than the MDA values in each organ of the control group, but no significant difference was observed.

  The 8-OHdG value in the kidney of the E6 group was 25.9 ± 1.7 ng / g of wet tissue, and no significant difference was observed with the 8-OHdG value in the control group of 30.5 ± 2.0 ng / g of wet tissue. The EOH group kidney 8-OHdG value 18.7 ± 1.3 ng / g of wet tissue was significantly lower than the control group kidney 8-OHdG value by 1% risk. In addition, the 8-OHdG value in the kidney of the E6 group was significantly lower at a 5% risk rate than the 8-OHdG value in the kidney of the E6 group (FIG. 26).

  As shown in FIG. 27, there was no significant difference between the plasma corticosterone value of 177.3 ± 6.7 ng / mL in the E6 group and the control group 193.9 ± 9.1 ng / mL. The plasma corticosterone level of 141.4 ± 9.8 ng / mL in the E6 group was significantly lower than the control group plasma corticosterone level of 193.9 ± 9.1 ng / ml at 1% risk. In addition, the plasma corticosterone level in the E6 group was significantly lower than the plasma corticosterone level in the E6 group at a 5% risk rate.

(Discussion)
In this test, it was examined whether or not the participating component expressing the oxidative stress mitigating action, anti-fatigue action and stress mitigating action of the oyster soft body extract was E6.
From FIG. 25, the MDA values in the liver, kidney, pancreas and spleen of the E6 group were significantly lower than those in the control group, and lipid antioxidant activity in each organ by administration of the E6-containing extract was confirmed. .

  On the other hand, the MDA value in each organ without the E6 group was not significantly decreased as compared with the control group, and no lipid antioxidant effect was observed by administration of the E6-free extract. We also reported a significant decrease in liver MDA levels in an oral administration test of E6-containing extract to non-alcoholic steatohepatitis (NASH) model mice in which the liver is in an oxidative stress state.

  Note that the difference between the components of the E6 containing extract and the E6 non-containing extract is only the difference between “contains” and “does not contain” E6, and other components are similar. From this, E6 was suggested as a participating component in the lipid antioxidant action of the oyster soft body extract.

  As shown in the kidney data in FIG. 26, the 8-OHdG value in the kidney of the E6 group was significantly lower than that of the control group and significantly lower than that of the E6 group. The 8-OHdG level in the kidneys of each group increased by long-term individual breeding was significantly decreased in the E6-group only by the comparison test of the 3 groups, and only in the E6-containing extract DNA antioxidant activity was observed.

  The difference between the components of the extract containing E6 and the extract containing no E6 is only whether or not it contains E6, and the components involved in the DNA antioxidant activity in the kidney observed only when the extract containing E6 is administered Was suggested to be E6, a novel antioxidant extracted from the soft part of the oyster.

  Fatigue is said to be a state in which cells and protein parts in the cells are damaged by excessive production of active oxygen, and there are few energy and substances necessary to repair the cell parts. In humans, it has been suggested that antioxidant intake is effective in improving fatigue.

  From these, it is suggested that the significant fatigue-reducing action in the POMS test by ingestion of E6-containing food observed in the human double-blind comparative group test is derived from the antioxidant action of the E6-containing extract in each organ. It was done. In particular, E6, which was suggested as a component involved in the DNA antioxidant activity in the kidney, was also suggested as a component involved in the fatigue reduction effect in the kidney.

  As shown in FIG. 27, the plasma corticosterone concentration in the E6 group was significantly lower than that in the control group and that in the E6 group. A significant reduction in plasma corticosterone concentration, which was increased by the stress of long-term individual breeding, was observed only in the E6 group, according to the comparison of the three groups. A significant decrease in corticosterone, which is a stress hormone, was observed in the E6 group, indicating that a stress relieving action by administration of the E6-containing extract was observed.

  This phenomenon is caused by damage to the endocrine system due to reactive oxygen species, and prevention of its malfunction by ingestion of antioxidants. In addition, in vivo oxidative stress induces an increase in plasma corticosterone concentration, and antioxidants Was the same as the report that the plasma corticosterone concentration decreased due to the improvement of renal function regulation including adrenal gland. From this, the mechanism of the decrease in plasma corticosterone concentration observed only by administration of the extract containing E6 is that the functional regulation of the kidney including the adrenal gland is improved by the antioxidant action of E6, and then cortisol from the adrenal gland is improved. This was thought to be due to suppression of excessive secretion of costerone.

  The difference between the components of the extract containing E6 and the extract containing no E6 is only the difference between “contains” and “does not contain” the antioxidant E6, and the stress observed only in the administration of the extract containing E6 It was suggested that the component involved in the relaxation action is E6.

  The previously reported depression-reducing effect of POMS in the human test by administration of the E6-containing extract seemed to be due to the stress-relieving effect due to the decrease in plasma corticosterone concentration observed only in the E6-group in this study.

  The relationship between the stress relieving action and sleep shown by the decrease in plasma corticosterone concentration in the E6 group observed in this study will be considered. We observed a placebo-controlled randomized, double-blind, parallel-group comparison study in humans of AIS and human sleep electroencephalogram measurements at the time of ingestion of the test drink, and sleep-inhibition and mid-wake increase suppression.

  In both healthy and insomnia patients, there is a positive correlation between plasma cortisol levels in the evening and onset and the number of subsequent wakefulness, and increased cortisol in the evening is important to induce and maintain sleep disorders It is pointed out that this may be an element.

  The blood collection time of 10:00 AM in this study appears to be the equivalent of evening or falling asleep for nocturnal mice. The plasma corticosterone concentration in the control group showed a high value due to the stress caused by individual breeding, but the plasma corticosterone concentration significantly decreased only in the E6 group. From this fact, the administration of the extract containing E6 to humans whose plasma corticosterone concentration is increased due to stress and whose sleep quality has been lowered has the function of reducing plasma cortisol concentration and improving sleep quality. Expected to generate.

(Conclusion)
A significant antioxidant effect on lipids in the liver, kidney, pancreas and spleen was observed only in the E6 group against oxidative stress mice by long-term individual breeding. Only in the E6 group, a significant antioxidant effect on DNA in the kidney was observed.
It was suggested that E6 in the E6-containing extract had a fatigue-reducing action derived from lipid antioxidant action in each organ and DNA antioxidant action in the kidney.

A significant decrease in plasma corticosterone concentration was induced only in the E6 group, suggesting a stress relieving effect by E6.
The difference between the components of the E6 containing extract and the E6 non-containing extract is only the difference between “contains” and “does not contain” E6. From this, it was suggested that the component involved in the lipid antioxidant effect in each organ, the DNA antioxidant effect in the kidney, and the decrease in plasma corticosterone concentration observed only in the E6 group was E6.

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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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. ,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.
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 ethyl acetate layer solution was concentrated to recover 3,5-dihydroxy-4-methoxybenzyl alcohol fraction, and the recovered 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
Oyster meat extract for anti-stress, anti-depression, anti-fatigue, anti-confusion, and vitality amplification.

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