Production method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from bivalves belonging to pterioidea

Abstract

[Problem] The present invention provides a production method capable of producing a larger amount of DHMBA, which is an antioxidant, with a higher concentration from the clam meat of a bivalve of Pterioidea, which is, for example, Pinctada fucata or Isognomon ephippium, than from other bivalves, by heating or pressurizing the clam meat of Pinctada fucata or Isognomon ephippium. [Solution] The present invention is characterized by putting the clam meat of a bivalve belonging to Pterioidea in an extraction solution and heating the extraction solution, thereby making it possible to produce a larger amount of 3,5-dihydroxy-4-methoxybenzyl alcohol from the heat-treated clam meat of the bivalve belonging to Pterioidea than other bivalves.

Description

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

[0001] The present invention relates to a method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA), an antioxidant, by extracting it from the flesh of bivalve mollusks belonging to the family Oenothera.

[0002] Oysters, such as the Pacific oyster (Crassostrea gigas), are bivalve mollusks belonging to the family Ostreidae in the order Ostreida, and their habitat extends throughout East Asia, including Japan. In recent years, Pacific oysters have also been cultivated in France and Australia, and they are renowned as the most widely consumed oysters in the world. Oysters have been consumed since ancient times due to their high nutritional value, and as mentioned above, they contain large amounts of glycogen and protein, as well as minerals such as calcium, zinc, selenium, copper, and manganese. Furthermore, antioxidants reported to be derived from oysters include the enzymatic antioxidants SOD, CAT, GPx, and Prx6, and the non-enzymatic antioxidants metallothionein, uncoupling protein 5 (UCP5), α-tocopherol, and β-carotene.

[0003] Incidentally, DHMBA can also be produced from bivalves other than oysters. It was confirmed that DHMBA is generated by heating or pressurizing the shellfish meat in bivalves such as turban shell, white shell, red shell, ark shell, and scallop. Akoya oyster or Mac oyster is a kind of bivalve classified in the superfamily of cypraeidae. Akoya oyster is mainly used as the mother shell for pearl cultivation. The cultivation of akoya oyster has started since the Meiji era and is now actively carried out throughout Japan. Although the adductor muscle is used for food in some regions, most of the shellfish meat is discarded. Different from oysters, it has become one of the important industrial species for pearl production rather than for food. Incidentally, Mac oyster is not currently used for food either. In the present invention, using akoya oyster or Mac oyster, which are important industrial species together with oysters in bivalves, it was clarified whether DHMBA can be generated by heating or pressurizing, and if it can be generated, what amount and at what concentration can be generated compared to oysters.

[0004] Japanese Patent Application Laid-Open No. 2010-193756

[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 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 whether derived from oysters or not. In addition, the inventors of the present invention have succeeded in extracting and producing DHMBA from the meat of 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 the meat of bivalves of the family Oenothera, such as pearl oysters or oysters, by heating or pressurizing the meat of pearl oysters or oysters, 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 the pearl oyster (Oryzias latipes) and the common oyster (Oryzias latipes), both belonging to the same superfamily, the Buccinidae. They are also confident that other bivalves belonging to the Buccinidae superfamily, besides the pearl oyster and the common oyster, can be produced in larger quantities and at higher concentrations than other bivalves.

[0006] 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 a bivalve belonging to the superfamily Oenothera by placing the meat of the bivalve belonging to the superfamily Oenothera in an extract and heating the extract, compared to other bivalves; or, 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 a pearl oyster or a mackerel by placing the meat of the pearl oyster or a mackerel in an extract and heating the extract, compared to other bivalves; or, 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 a pearl oyster or a mackerel by placing the meat of the bivalve belonging to the superfamily Oenothera in an extract and pressurizing the extract, compared to other bivalves; The present invention is characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity than other bivalves from the pressurized bivalve meat of a bivalve belonging to the superfamily Oenothera, or by placing the meat of a pearl oyster or a mackerel in an extract and pressurizing the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a larger quantity than other bivalves from the pressurized bivalve meat of a pearl oyster or a mackerel, or by placing the meat of a bivalve belonging to the superfamily Oenothera in an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced in a higher concentration than other bivalves from the heat-treated bivalve meat of a bivalve belonging to the superfamily Oenothera, or By placing the meat of the pearl oyster or mako oyster into an extract and heating the extract, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzylThe present invention is characterized in that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration than other bivalves from the heat-treated flesh of pearl oysters or oysters, or that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration than other bivalves from the pressure-treated flesh of bivalves belonging to the superfamily Oenoidea by placing the flesh of bivalves belonging to the superfamily Oenoidea into an extract and pressurizing the extract, or that 3,5-dihydroxy-4-methoxybenzyl alcohol can be produced at a higher concentration than other bivalves from the pressure-treated flesh of pearl oysters or oysters by placing the flesh of pearl oysters or oysters into an extract and pressurizing the extract.

[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.

[0008] This is an explanatory diagram illustrating the production method of the present invention. This is an explanatory diagram showing the detection results of DHMBA detected by heating from bivalves belonging to the superfamily Schizoidea. This is an explanatory diagram showing the detection results of DHMBA detected by pressurization from bivalves belonging to the superfamily Schizoidea. This is an explanatory diagram showing the comparative detection results of DHMBA detected by heating from other bivalves and bivalves belonging to the superfamily Schizoidea. This is an explanatory diagram showing the comparative detection results of DHMBA detected by pressurization from other bivalves and bivalves belonging to the superfamily Schizoidea.

[0009] The following describes in detail the results of an experiment conducted to produce DHMBA from bivalves belonging to the family Oenothera. (Materials) The bivalves used as materials were the pearl oyster and the common oyster, both belonging to the family Oenothera, collected from the following two regions: Amami Oshima, Kagoshima Prefecture (common oyster), and Kamigoto, Nagasaki Prefecture (pearl oyster). (Production method) Extraction of DHMBA by heating (Figure 1) Three individuals each of the pearl oyster and common oyster, belonging to the family Oenothera, were randomly selected from the above regions, and the shell meat was extracted and weighed. The extracted shell 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 liquid shell meat was placed in a 50 ml or 15 ml Corning tube (AS ONE Corporation), and the weight was measured again. Subsequently, to facilitate the extraction of DHMBA by disrupting the cells of the bivalve oyster (Pyrus ocellatus) or oyster (Pyrus platypus), which belong to the superfamily Pyrusoidea, the 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 a 1.5 mL Eppendorf tube. Next, a DHMBA water bath extraction experiment was conducted by placing each Corning tube in water heated to 100°C using an induction heater (KZ-PH3, manufactured by National Corporation). Heating was carried out for a total of 5 hours, and sampling was performed 5 times at 1-hour intervals. During sampling, 1 mL of sample was taken from each Corning tube and stored in a 1.5 mL Eppendorf tube. Afterwards, each Eppendorf tube used for sampling was subjected to centrifugation or filtration to remove impurities and obtain an extract.

[0010] Extraction of DHMBA by pressurization (Figure 1) Three individuals each of the bivalves *Ophiopogon pulcherrimus* and *Platypleura punctata*, belonging to the superfamily *Platypleura*, were randomly selected from the above region, and 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 *Ophiopogon pulcherrimus* or *Platypleura punctata* bivalves belonging to the superfamily *Platypleura*, and facilitate the extraction of DHMBA, the Corning tubes were subjected to ultrasonic treatment for 1 hour using an ultrasonic cleaner (ASU-10, AS ONE Corporation). After 1 hour, 1 ml of sample was taken from each Corning tube and stored in a 1.5 ml Eppendorf tube. A Kajiwara pressure cooker was used for pressurization. Each sample was placed in a pressure vessel 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 vessel 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, and the pressure was released. Then, 0.5 mL of the sample was sampled 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 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 after 100-fold dilution. The separation column used was an ODS (Optical Spectroscopy) Shim-Pack VP-ODS (length 150 mm × inner diameter 2.0 mm, particle size 5 μm). A gradient analysis was performed using mobile phase A: 0.05% aqueous acetic acid solution and mobile phase B: acetonitrile (mobile phase B: 0 min 5% → 5 min 100% → 7.5 min 5% → 12 min 5%). 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, and analysis was performed at 10V, 20V, and 30V. For quantitative analysis, electrospray ionization (ESI) was used as the ionization method, 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 were DL temperature: 250°C, nebulizer gas flow rate: 3 L / min, heat block temperature: 400°C, dry-in gas flow rate: 15 L / min. (Results) Figure 2 shows the results of the heat extraction. For the bivalve mollusk *Macrobrachium* belonging to the superfamily *Macrobrachium* from Amami Oshima, DHMBA was not detected before heating or 1 hour after heating. After 2 hours after heating, 0.84 μg / g of DHMBA was obtained, and after 3 hours after heating, 1.68 μg / g of DHMBA was obtained. Furthermore, it was confirmed that 1.96 μg / g of DHMBA was obtained 4 hours after heating, and 2.95 μg / g of DHMBA was obtained 5 hours after heating. In addition, for the pearl oyster, a bivalve belonging to the superfamily Oenothera, from Kamigoto, no DHMBA was detected before heating, and 0.89 μg / g of DHMBA was obtained 1 hour after heating. 1.70 μg / g of DHMBA was obtained 2 hours after heating, and 2.23 μg / g of DHMBA was obtained 3 hours after heating. Furthermore, 3.00 μg / g of DHMBA was obtained 4 hours after heating, and 3.36 μg / g of DHMBA was obtained 5 hours after heating. Neither the *Macrobrachium nipponense* bivalve from Amami Oshima nor the *Platypleura kobus* bivalve from Kamigoto Islands, both belonging to the same superfamily, contained DHMBA in their raw, unheated state. This revealed that DHMBA is extracted and produced from the flesh of *Platypleura kobus* or *Macrobrachium nipponense* bivalves, both belonging to the same superfamily, through heat treatment.

[0013] The results of pressurized extraction are shown in Figure 3. For *Makgai*, a bivalve belonging to the superfamily *Machinoidea* from Amami Oshima, 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 obtained. In addition, for *Akoya-gai*, a bivalve belonging to the superfamily *Machinoidea* from Kamigoto, 27.12 μg / g of DHMBA was obtained after 1 hour of pressurization. Furthermore, after 2 hours of pressurization, 48.46 μg / g of DHMBA was obtained, and after 3 hours of pressurization, 66.70 μg / g of DHMBA was obtained. 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, it was confirmed that DHMBA, an antioxidant, can be extracted and produced from the flesh of the bivalve mollusks belonging to the family Odontocephala, specifically the pearl oyster (Orychophragmus flumineus) and the Pacific oyster (Orychophragmus flumineus), by heating or pressurizing the flesh of these mollusks. To compare the amount of DHMBA produced by the pearl oyster (Orychophragmus flumineus) and the Pacific oyster (Orychophragmus flumineus), the extraction results of other bivalves belonging to the same family are shown in Figures 4 and 5. Heat extraction and pressurized extraction were carried out under the same conditions as the extraction of the pearl oyster (Orychophragmus flumineus) and the Pacific oyster (Orychophragmus flumineus). When the flesh of other bivalves belonging to the same family as the pearl oyster (Orychophragmus flumineus) was heated or pressurized, the Pacific oyster (Orychophragmus flumineus) 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 bivalve *Cypraea tigris*, belonging to the family Cypraeidea, was able to produce over 20 μg / g of DHMBA in just one hour of pressurized treatment, more than twice the amount produced by pressurizing *Cypraea japonica* for three hours. Furthermore, pressurized treatment of the flesh of *Cypraea tigris* and *Platycodon oyster*, both belonging to the family Cypraeidea, for three hours produced approximately 70 μg / g of DHMBA. This is about seven times the amount of DHMBA produced by pressurizing *Cypraea japonica* for the same three hours. This demonstrates that *Cypraea tigris* and *Platycodon oyster*, both belonging to the family Cypraeidea, can efficiently produce large amounts of DHMBA in a short time compared to other bivalves, including *Cypraea japonica*. 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. 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 heat-treated 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 heating the extract.

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

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 oysters, 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 oysters than from other bivalves, by placing the meat of pearl oysters or mackerel oysters in an extract and pressurizing the extract.

5. 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 heat-treated 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 heating the extract.

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

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. 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 from the pressurized meat of pearl oysters or mackerel at a higher concentration than other bivalves, by placing the meat of pearl oysters or mackerel in an extract and pressurizing the extract.

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