A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from bivalves belonging to the family Oenothera.

Abstract

The present invention provides a method for producing DHMBA, an antioxidant, from the flesh of bivalves of the superfamily Oenothera, such as pearl oysters or oysters, by heating or pressurizing the flesh of the pearl oysters or oysters, in a larger quantity and at a higher concentration than other bivalves. [Solution] The present invention is characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the heat-treated bivalve meat of the Bivalviidae superfamily than from other bivalves by placing the meat of a bivalve belonging to the Bivalviidae superfamily into an extract and heating the extract.

Description

Technical Field

[0001] The present invention relates to a production method for extracting 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA), an antioxidant, from the meat of bivalves belonging to the superfamily Unionoida.

Background Art

[0002] Oysters, such as the Pacific oyster (Crassostrea gigas), are bivalves belonging to the family Ostreidae of the order Ostreoida, and their habitats extend throughout East Asia including Japan. In recent years, Pacific oysters are also farmed in France and Australia, and are well-known as the most commonly consumed oysters in the world. Oysters have been consumed since ancient times because of their high nutritional value. As described above, in addition to glycogen and protein, they contain large amounts of minerals such as calcium, zinc, selenium, copper, and manganese. In addition, the antioxidant substances reported from oysters include SOD, CAT, GPx, and Prx6 as enzymatic antioxidants, and metallothionein, uncoupling protein 5 (UCP5), α-tocopherol, and β-carotene as non-enzymatic antioxidants.

[0003] By the way, DHMBA can also be produced from bivalves other than oysters. It has been confirmed that DHMBA is produced by heating or pressurizing the meat of bivalves such as the ark shell, white clam, blood clam, hard clam, and scallop. The Akoya oyster, also known as the mako oyster, is a type of bivalve mollusk belonging to the family Oenothera. The Akoya oyster is primarily used as the mother-of-pearl for pearl cultivation. Akoya oyster farming began in the Meiji era and is now actively practiced throughout Japan. While the adductor muscle is consumed in some regions, much of the oyster meat is discarded. Unlike oysters, it is an important industrial species for pearl production, not for food. The mako oyster is also not currently used for food. This invention aims to clarify whether DHMBA can be produced by heating or pressurizing pearl oysters or mako oysters, which are important industrial species of bivalves along with oysters, and if so, how much DHMBA can be produced and at what concentration compared to oysters. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2010-193756 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] In recent years, the inventors of the present invention have succeeded in discovering DHMBA, an excellent novel antioxidant derived from oysters, determining its chemical structure, and also succeeding in chemically synthesizing the said antioxidant. Furthermore, they have succeeded in providing novel antioxidants and antioxidant compositions containing DHMBA as an active ingredient, which are excellent in both cases: those derived from oysters and those not derived from oysters. Furthermore, the inventors of this invention have succeeded in extracting and producing DHMBA from shellfish other than oysters, such as purple mussels, pen shells, white mussels, ark shells, and scallops. However, the present invention aims to provide a method for producing DHMBA, an antioxidant, from bivalves of the family Oenothera, such as the pearl oyster or the oyster meat, by heating or pressurizing the meat of the pearl oyster or oyster meat, in a larger quantity and at a higher concentration than other bivalves. Furthermore, the inventors of this patent have conducted DNA analysis on the bivalves used in this patent and confirmed that the species of bivalves are *Ophiopogon pulcherrimus* and *Ophiopogon pulcherrimus*, both belonging to the same superfamily, *Ophiopogon*. They are also confident that other bivalves belonging to the superfamily *Ophiopogon*, besides *Ophiopogon pulcherrimus*, can be produced in larger quantities and at higher concentrations than other bivalves. [Means for solving the problem]

[0006] The present invention By placing the flesh of a bivalve belonging to the family Oenothera in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the heat-treated flesh of the bivalve belonging to the family Oenothera than from other bivalves. It is characterized by the following: or By placing the flesh of the pearl oyster or the oyster in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the heat-treated pearl oyster or oyster flesh than from other bivalves. It is characterized by the following: or The present invention is characterized in that, by placing the flesh of a bivalve belonging to the superfamily Oenothera in an extract and pressurizing the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the pressurized flesh of the bivalve belonging to the superfamily Oenothera than from other bivalves, or by placing the flesh of a pearl oyster or a mussel in an extract and pressurizing the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the pressurized flesh of a pearl oyster or a mussel than from other bivalves, or By placing the flesh of bivalves belonging to the family Oenothera in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration from the heat-treated flesh of bivalves belonging to the family Oenothera than from other bivalves. It is characterized by the following: or By placing the flesh of the pearl oyster or the oyster in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced from the heat-treated pearl oyster or oyster flesh at a higher concentration than from other bivalves. It is characterized by the following: or The present invention is characterized by the ability to produce 3,5-dihydroxy-4-methoxybenzyl alcohol at a higher concentration than other bivalves by placing the flesh of a bivalve belonging to the superfamily Oenothera in an extract and pressurizing the extract, or, By placing the flesh of the pearl oyster or the oyster into an extract and pressurizing the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced from the pressurized pearl oyster or oyster flesh at a higher concentration than that of other bivalves. It is characterized by the following: [Effects of the Invention]

[0007] According to the present invention, the antioxidant DHMBA can be produced in a larger quantity and at a higher concentration than other bivalves by heating or pressurizing the meat of bivalves belonging to the superfamily Oenoidea, such as pearl oysters or oysters. [Brief explanation of the drawing]

[0008] [Figure 1] This is an explanatory diagram illustrating the production method of the present invention. [Figure 2] This is an explanatory diagram showing the detection results of DHMBA detected by heating in bivalves belonging to the superfamily Oenothera. [Figure 3] This is an explanatory diagram showing the detection results of DHMBA detected under pressure from bivalves belonging to the superfamily Oenothera. [Figure 4] This is an explanatory diagram showing the comparative detection results of DHMBA detected by heating in other bivalves and bivalves belonging to the superfamily Oenanthes. [Figure 5] This is an explanatory diagram showing the comparative detection results of DHMBA detected under pressure from other bivalves and bivalves belonging to the superfamily Buccinidae. [Modes for carrying out the invention]

[0009] The following describes in detail the results of an experiment conducted to produce DHMBA from bivalves belonging to the superfamily Oenothera. (material) Bivalves belonging to the superfamily Ctenogastropoda collected from the following two regions, pearl oysters and Maco oysters, were used as materials. Amami Oshima, Kagoshima Prefecture (Maco oyster), Kamigoto, Nagasaki Prefecture (Pearl oyster) (Production method) Extraction of DHMBA by heating (Figure 1) Three individuals each of pearl oysters or Maco oysters, which are bivalves belonging to the superfamily Ctenogastropoda in the above regions, were randomly selected, and the shellfish meats were removed and weighed. The removed shellfish meats were placed in separate beakers, 90 ml of ultrapure water was added, and then homogenization treatment was performed. For homogenization, a POLYTRON PT10-35 manufactured by KINEMATICA was used. An appropriate amount of the liquefied shellfish meat was placed in a 50 mL or 15 ml Corning tube (AS ONE Corporation), and the weight was measured again. Subsequently, in order to break the cells of the shellfish meat of pearl oysters or Maco oysters belonging to the superfamily Ctenogastropoda and make it easier to extract DHMBA, the above-mentioned Corning tube was subjected to ultrasonic treatment for 1 hour using an ultrasonic cleaner (ASU-10 manufactured by AS ONE Corporation). After 1 hour, 1 mL of the sample was sampled from each Corning tube and stored in a 1.5 mL Eppendorf tube. Next, each Corning tube was placed in hot water heated to 100 °C using an IH heater (KZ-PH3 manufactured by National) to conduct an extraction experiment of DHMBA by decoction. Heating was performed for a total of 5 hours, and sampling was performed 5 times every hour. At the time of sampling, 1 mL of the sample was sampled from each Corning tube and stored in a 1.5 mL Eppendorf tube. Thereafter, for each Eppendorf tube that had been sampled, centrifugation or filtration was performed in order not to cause clogging in the analyzer, and an extract from which impurities had been removed was obtained.

[0010] Extraction of DHMBA by pressurization (Figure 1) Three individuals each of the Akoya oyster (Oryzias latipes) and the Maccaenidae bivalve (Oryzias latipes), belonging to the superfamily Oryzias superfamily, were randomly selected from the above-mentioned region. The oyster meat was extracted and weighed. The extracted oyster meat was placed in separate beakers, 90 ml of ultrapure water was added, and then homogenization was performed. A KINEMATICA Polytron PT10-35 was used for homogenization. An appropriate amount of the liquefied oyster meat was placed into 50 ml or 15 ml Corning tubes (AS ONE Corporation), and the weight was measured again. Subsequently, in order to break down the cells of the bivalve oyster (Pyrus ocellatus) or oyster (Pyrus nigricans), which belong to the superfamily Pyrusoidea, and facilitate the extraction of DHMBA, the aforementioned Corning tubes were subjected to ultrasonic treatment for 1 hour using an ultrasonic cleaner (ASU-10, manufactured by AS ONE Corporation). After 1 hour, 1 mL of sample was taken from each Corning tube and stored in 1.5 mL Eppendorf tubes. A Kajiwara pressure cooker was used for pressurization. Each sample was placed in a pressure cooker filled with water, the lid was closed, and the pressurization temperature was set to 135°C. Heating (pressurization) was then started. It took approximately 90 minutes for the internal temperature of the cooker to reach 135°C, and the pressure gauge showed approximately 0.2 MPa (2 atmospheres). One hour after the internal temperature reached 135°C, pressurization was stopped, the pressure was released, and 0.5 mL of the sample was taken from each Corning tube and stored in a 1.5 mL Eppendorf tube. The above process was repeated until the total pressurization time at 135°C reached 2 hours, then 3 hours. After that, each Eppendorf tube from which the samples were taken was subjected to centrifugation or filtration to remove impurities and obtain an extract.

[0011] Quantitative analysis of DHMBA The DHMBA concentration in each collected sample was quantified using a liquid chromatogram / tandem mass spectrometer (LC-MS / MS: Prominence high-pressure gradient HPLC, triple quadrupole mass spectrometer LCMS-8040 system, Shimadzu Corporation). DHMBA standard (Ushio Chemix Co., Ltd.) was dissolved in pure water to prepare calibration curve solutions at concentrations of 1, 5, 10, 50, and 100 μg / mL. Each sample was subjected to quantitative analysis by LC-MS / MS at a 100-fold dilution. A Shim-Pack VP-ODS (150 mm length × 2.0 mm inner diameter, 5 μm particle size) ODS column was used for separation. A gradient analysis (mobile phase B: 0 min 5% → 5 min 100% → 7.5 min 5% → 12 min 5%) was performed using mobile phase A: 0.05% aqueous acetic acid solution and mobile phase B: acetonitrile. The flow rate was set to 0.25 mL / min and the column oven temperature to 40°C. The sample injection volume was 1 μL, and DHMBA was detected in negative ion mode. Product ion scanning was used for quantitative analysis. Product ion scanning was performed using the deprotonated ion [MH]- of DHMBA with a m / z of 169.1 as the precursor ion, at 10V, 20V, and 30V. For quantitative analysis, electrospray ionization (ESI) was used, and multiple reaction monitoring (MRM) was employed. MRM transitions were determined by automated optimization.

[0012] Q1 / Q3 = 169.1 / 154.1 (quantitative transition), 169.1 / 136.9, 169.1 / 125.1 (qualitative transition) were used. The collision energies were set to 15V, 28V, and 13V respectively. Other MS parameters included DL temperature: 250°C, nebulizer gas flow rate: 3 L / min, heat block temperature: 400°C, and dry-in gas flow rate: 15 L / min. (result) Heating extraction results Figure 2 shows the results of the heat extraction. Regarding *Makakui*, a bivalve mollusk belonging to the superfamily *Makakui* found on Amami Oshima Island, DHMBA was not detected before heating or one hour after heating. Two hours after heating, 0.84 μg / g of DHMBA was obtained, and three hours after heating, 1.68 μg / g of DHMBA was obtained. Furthermore, it was confirmed that 1.96 μg / g of DHMBA was obtained four hours after heating, and 2.95 μg / g of DHMBA was obtained five hours after heating. Furthermore, for the pearl oyster (Orychophragmus violaceus), a bivalve belonging to the superfamily Orychophragmus, found in Kamigoto, no DHMBA was detected before heating. After 1 hour, 0.89 μg / g of DHMBA was obtained. After 2 hours, 1.70 μg / g of DHMBA was obtained, and after 3 hours, 2.23 μg / g of DHMBA was obtained. After 4 hours, 3.00 μg / g of DHMBA was obtained, and after 5 hours, 3.36 μg / g of DHMBA was obtained. Neither the *Makakugai* bivalve, belonging to the superfamily *Makakugai*, from Amami Oshima, nor the *Akoya-gai* bivalve, belonging to the superfamily *Makakugai*, from Kamigoto, contained DHMBA in their raw, unheated state. This revealed that DHMBA is extracted and produced from the flesh of *Akoya-gai* or *Makakugai* bivalves, both belonging to the superfamily *Makakugai*, through heat treatment.

[0013] Pressurized extraction results Figure 3 shows the results of pressurized extraction. For *Macrobrachium nipponense*, a bivalve belonging to the superfamily *Macrobrachium nipponense* found on Amami Oshima Island, no DHMBA was detected before pressurization. After 1 hour of pressurization, 23.15 μg / g of DHMBA was obtained. Furthermore, after 2 hours of pressurization, 53.08 μg / g of DHMBA was obtained, and after 3 hours of pressurization, 69.66 μg / g of DHMBA was confirmed to be obtained. Furthermore, for the pearl oyster (Orychophragmus violaceus), a bivalve belonging to the superfamily Orychophragmus, from Kamigoto, DHMBA was obtained at a concentration of 27.12 μg / g after 1 hour of pressurization. After 2 hours of pressurization, DHMBA was obtained at a concentration of 48.46 μg / g, and after 3 hours of pressurization, DHMBA was obtained at a concentration of 66.70 μg / g. Neither the *Macrobrachium nipponense* (a bivalve belonging to the family *Macrobrachium* superfamily from Amami Oshima) nor the *Ophiopogon scoparius* (a bivalve belonging to the family *Macrobrachium* superfamily from Kamigoto) contained DHMBA in their raw, unpressurized state. This revealed that DHMBA is extracted and produced from the flesh of *Ophiopogon scoparius* or *Macrobrachium nipponense*, both bivalves belonging to the family *Macrobrachium* superfamily, through pressurization. Furthermore, it was confirmed that both *Macrobrachium nipponense* from Amami Oshima and *Ophiopogon scoparius* from Kamigoto could produce a larger amount of DHMBA by pressurizing their flesh rather than heating it.

[0014] (summary) As the results show, we confirmed that the antioxidant DHMBA can be extracted and produced from the bivalve oyster (Platypleura japonica) or the oyster (Platypleura punctata), which belong to the superfamily Platycephalaenopsis, by heating or pressurizing the meat of the oyster or oyster. To compare the amount of DHMBA produced by the bivalve oyster (Platypleura scoparius) or the Pacific corydoras (Platypleura punctata), both belonging to the family Platypleura superfamily, the extraction results of other bivalves belonging to the same family are shown in Figures 4 and 5. Heat extraction and pressure extraction were carried out under the same conditions as extraction for the Platypleura scoparius or the Pacific corydoras. When the flesh of the other bivalves belonging to the same family was heated or pressurized, the Pacific oyster (Platypleura punctata) was able to extract and produce the most DHMBA, producing 10.66 μg / g of DHMBA after 3 hours of pressurized treatment. On the other hand, the Pacific corydoras, a bivalve belonging to the family Platypleura superfamily, was able to produce more than twice the amount of DHMBA produced by pressurized Pacific oysters for 3 hours, with only 1 hour of pressurized treatment, producing more than 20 μg / g of DHMBA. Furthermore, by pressurizing the flesh of the bivalves *Macrobrachium nipponense* and *Pyrus oyster*, both belonging to the superfamily *Macrobrachium*, for 3 hours, approximately 70 μg / g of DHMBA was produced. This is about seven times the amount of DHMBA produced by pressurizing *Cornus flavipes* in the same 3 hours. This demonstrates that *Macrobrachium nipponense* and *Pyrus oyster*, both belonging to the superfamily *Macrobrachium*, are capable of efficiently producing large amounts of DHMBA in a short time compared to other bivalves, including *Cornus flavipes*. As described above, the bivalves *Macrobrachium nipponense* and *Platypleura kobus*, which belong to the superfamily *Macrobrachium nipponense*, possess excellent DHMBA production capabilities, and it was confirmed that *Macrobrachium nipponense* and *Platypleura kobus*, which belong to the superfamily *Macrobrachium nipponense*, are the most efficient bivalves in producing DHMBA.

Claims

1. By placing the flesh of a bivalve belonging to the family Oenothera in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the heat-treated flesh of the bivalve belonging to the family Oenothera than from other bivalves. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the flesh of a bivalve mollusk belonging to the superfamily Oenothera, characterized by the following:

2. By placing the flesh of the pearl oyster or the oyster in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the heat-treated pearl oyster or oyster flesh than from other bivalves. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the flesh of pearl oysters or mako oysters, characterized by the following:

3. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the meat of bivalves belonging to the superfamily Scutellaria, characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the meat of bivalves belonging to the superfamily Scutellaria than from other bivalves, by placing the meat of bivalves belonging to the superfamily Scutellaria in an extract and pressurizing the extract.

4. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the meat of pearl oysters or mackerel, characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity from the pressurized meat of pearl oysters or mackerel than from other bivalves, by placing the meat of pearl oysters or mackerel in an extract and pressurizing the extract.

5. By placing the flesh of bivalves belonging to the family Oenothera in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration from the heat-treated flesh of bivalves belonging to the family Oenothera than from other bivalves. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the flesh of a bivalve mollusk belonging to the superfamily Oenothera, characterized by the following:

6. By placing the flesh of the pearl oyster or the oyster in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced from the heat-treated pearl oyster or oyster flesh at a higher concentration than from other bivalves. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the flesh of pearl oysters or mako oysters, characterized by the following:

7. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the meat of bivalves belonging to the superfamily Scutellaria, characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration from the meat of bivalves belonging to the superfamily Scutellaria than from other bivalves, by placing the meat of bivalves belonging to the superfamily Scutellaria in an extract and pressurizing the extract.

8. By placing the flesh of the pearl oyster or the oyster into an extract and pressurizing the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced from the pressurized pearl oyster or oyster flesh at a higher concentration than that of other bivalves. A method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the flesh of pearl oysters or mako oysters, characterized by the following:

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