JP7702098B2 - Antioxidant active agent, method for producing antioxidant active agent, α-glucosidase inhibitor active agent, and method for producing α-glucosidase inhibitor active agent - Google Patents

Description

The present invention relates to an antioxidant active agent, a method for producing an antioxidant active agent, an α-glucosidase inhibitor active agent, and a method for producing an α-glucosidase inhibitor active agent. More specifically, the present invention relates to an antioxidant active agent using olive branches and leaves, a method for producing an antioxidant active agent, an α-glucosidase inhibitor active agent, and a method for producing an α-glucosidase inhibitor active agent.

In recent years, the demand for health foods (including medicines) aimed at maintaining and improving health has been increasing, especially among the middle-aged and elderly. Olives contain a variety of polyphenols, including oleuropein, and are therefore used in a wide variety of functional foods.
For example, Patent Document 1 discloses an α-glucosidase inhibitor that uses components extracted from the aqueous portion of pressed olive fruits, and Patent Document 2 discloses an amylase inhibitor that uses olive leaves or extracts thereof.

JP 2005-082546 A JP 2002-010753 A

However, the technologies in Patent Documents 1 and 2 only use the components contained in olives as they are, without chemically modifying them. For this reason, with the recent increase in health consciousness, there are hopes for the development of health foods that allow people to more efficiently ingest the functions of olives that improve human health.

In view of the above circumstances, the present invention aims to provide an antioxidant active agent with improved functionality of an extract obtained from olives, a method for producing the same, and an α-glucosidase inhibitor active agent and a method for producing the same.

As a result of extensive research aimed at solving the above problems, the inventors discovered that the functionality of an extract obtained from olives can be improved by treating the extract with mycelium, leading to the completion of the present invention.

The antioxidant active agent of the present invention is characterized by containing a treated mycelium obtained by treating an extract obtained from olive branches and/or leaves with mycelium.
The method for producing the antioxidant active agent of the present invention is characterized by sequentially carrying out the following steps: a first step in which olive branches and/or leaves are irradiated with microwaves while being separated into a distillate and a solid by reduced pressure distillation; a second step in which the obtained solid is extracted with water or hot water to obtain an extract; and a third step in which the obtained extract is treated with mycelium.
The α-glucosidase inhibitor of the present invention is characterized in that it contains a treated mycelium obtained by treating an extract obtained from olive branches and/or leaves with mycelium.
The method for producing the α-glucosidase inhibitor of the present invention is characterized by sequentially carrying out a first step in which olive branches and/or leaves are irradiated with microwaves while being separated into a distillate and a solid by reduced pressure distillation, a second step in which the obtained solid is extracted with water or hot water to obtain an extract, and a third step in which the obtained extract is treated with mycelium.

According to the present invention, by treating an extract obtained from olives with mycelium, it is possible to provide an antioxidant active agent and an α-glucosidase inhibitor active agent that are expected to effectively prevent cancer, lifestyle-related diseases, infectious diseases, dementia, etc., as well as a method for producing these.

FIG. 1 is a schematic diagram of a microwave vacuum distillation apparatus 1. FIG. 1 shows the results of the antioxidant activity experiment. FIG. 1 shows experimental results of α-glucosidase inhibitory activity. FIG. 2 is a diagram showing an example of an HPLC chromatogram of a treated mycelium solution of Enoki mushroom.

An embodiment of the present invention will be described with reference to the drawings.
The antioxidant active agent of the present embodiment contains a mycelium-treated product obtained by treating an extract obtained from olive branches and leaves with mycelium, and is expected to provide efficient prevention of cancer, infectious diseases, dementia, and the like.
The α-glucosidase inhibitor active agent of this embodiment contains a mycelium processed product obtained by treating an extract obtained from olive branches and leaves with mycelium, and is expected to be effective in preventing lifestyle-related diseases and controlling blood sugar levels.

In the present specification, olive refers to a plant belonging to the genus Olea in the family Oleaceae, and the type is not particularly limited. The target parts of olives are branches and leaves, which can be used alone or in mixtures.
In the following description, when simply referring to olives, this refers to the above-mentioned target site.

In addition, there are no particular limitations on the cultivation state of the olives to be treated. For example, by using olive branches and leaves that would otherwise be discarded during the olive harvesting process, it is possible to obtain the advantage of making effective use of unused biomass resources.

The mycelium-treated product is obtained by treating an extract obtained from olives with mushroom mycelium. There is no particular limitation on the mushrooms used to prepare the mycelium-treated product. Examples include shiitake mushrooms, enokitake mushrooms, nameko mushrooms, oyster mushrooms, bunashimeji mushrooms, king oyster mushrooms, and yamabushitake mushrooms.

The antioxidant active agent of this embodiment (hereinafter, simply referred to as the antioxidant active agent) contains a mycelium-treated product obtained by treating an extract obtained from olives with mycelium. By containing this mycelium-treated product, the antioxidant active agent can exhibit excellent antioxidant activity.
Therefore, it is expected that consuming this antioxidant will suppress the action of active oxygen generated in the body. Specifically, consuming this antioxidant is expected to be effective in preventing aging, cancer, infectious diseases, dementia, and other conditions caused by active oxygen.

The α-glucosidase inhibitor active agent of this embodiment (hereinafter simply referred to as the α-glucosidase inhibitor active agent) contains a mycelium-processed product obtained by treating an extract obtained from olives with mycelium, similar to the antioxidant active agent. By containing this mycelium-processed product, the α-glucosidase inhibitor active agent is expected to suppress and regulate the digestion and absorption of carbohydrates by exerting α-glucosidase inhibitory activity in the body. Specifically, it is expected that ingesting the α-glucosidase inhibitor active agent will be effective in improving a constitution that is prone to absorbing carbohydrates, preventing lifestyle-related diseases caused by eating excess carbohydrates, and controlling postprandial hyperglycemia.

Next, a method for producing the antioxidant active agent and the α-glucosidase inhibitor active agent will be described.
Since the methods for producing antioxidant active agents and α-glucosidase inhibitor active agents are the same, the method for producing an antioxidant active agent of this embodiment (hereinafter simply referred to as the method for producing an antioxidant active agent), which is a method for producing an antioxidant active agent, will be described as a representative example.

The method for producing an antioxidant active agent is to treat an extract obtained from olives with mycelium, and then add the resulting mycelium to produce an antioxidant active agent.

First, the extract to be treated with mycelium in the production method of the antioxidant active agent will be described.
The extract to be treated with the mycelium is obtained from olives. Specifically, the extract contains components resulting from solids obtained after separating at least a part of the essential oil and/or aqueous liquid from olives. For example, the extract may be a solid containing the residue (solids) itself, which is the residue obtained by performing processes such as squeezing and distillation, a liquid in which the solids are dispersed, or a liquid obtained by further extracting the solids.

The method for preparing the above-mentioned solids is not particularly limited as long as it is possible to separate at least a portion of the essential oil and/or aqueous liquid from the olives. For example, the solids can be prepared using the above-mentioned press or distillation apparatus.

When preparing the solid content using a press or distillation device, the olives can be processed to a size that can be stored in the storage section of the device before being supplied. Olive leaves, etc. may be supplied as is, but it is preferable to supply them in a small size from the standpoint of processing time and processing efficiency. Known methods can be used to reduce the size of the olives when supplying them to the device. For example, if processing such as cutting or crushing is performed, the olives can be supplied to the device in the form of chips or powder.

When a press is used, the essential oil and/or aqueous liquid is obtained as the squeezed juice, and a solid content can be obtained as the residue.
In addition, when a distillation apparatus is used, the essential oil and/or aqueous liquid can be obtained as a distillate, and the solid content can be obtained as a residue, which is a residue. In other words, when a distillation apparatus is used, the solid content is a residue remaining after at least a part of the essential oil and/or aqueous liquid is separated as a distillate from the olives fed to the distillation apparatus by distilling the olives.

Alternatively, olives may be irradiated with microwaves as described below and then pressed into a press to obtain solids. In this case, the cell walls of the olives are easily destroyed by microwaves, making it easier to obtain the desired solids.
By irradiating olives with microwaves, a microwave-irradiated product can be obtained in which the essential oils and aqueous liquids contained in the olives have partly escaped. This microwave-irradiated product is in a state in which the cell walls that make up the olives are easily destroyed by microwaves, so it can be used as a solid content as is.

In the treatment using a distillation apparatus, a solid content can be obtained by treatment such as atmospheric distillation, reduced pressure distillation, and steam distillation.
Furthermore, the heating method when using a distillation apparatus is not particularly limited, and a heater, hot water, steam, or microwave irradiation can be used.

For example, the method of irradiating microwaves is preferable because it can make the components contained in the olives in a state that is easy for the mycelium to process. Specifically, when microwaves are irradiated to olives, the moisture contained in the olives is heated and released as water vapor. At this time, the essential oils and water-soluble compounds (including volatile components) contained in the olives are obtained as distillates. On the other hand, the cell walls that make up the olives are easily destroyed by microwaves. Therefore, components that are difficult to extract by general distillation methods can be contained in the residue (i.e., solids) in a state that makes them easy to extract.
Therefore, by using an extract containing solids prepared by employing a microwave irradiation method, it becomes easier to treat the components contained in the olives with mycelium.
In particular, when the extract is prepared by subjecting the solid fraction obtained by the treatment using the distillation apparatus to the extraction treatment described below, there is an advantage in that the components contained in the solid fraction can be more easily extracted.

As the distillation apparatus for irradiating microwaves, a known apparatus can be used, for example, a microwave reduced pressure distillation apparatus 1 as shown in FIG.
This microwave vacuum distillation apparatus 1 comprises a distillation tank 2 corresponding to a storage section for storing olives as a raw material, a microwave heating device 3 for irradiating microwaves, an air inlet pipe 4 for supplying air or an inert gas such as nitrogen gas to the distillation tank 2, a distillate outlet pipe 5 for introducing the distilled distillate, a cooling device 6 for cooling the distillate outlet pipe 5, a heating control device 7 for controlling the temperature of the distillation tank 2, and a pressure control device 10 for controlling the internal pressure of the distillation tank 2. The pressure control device 10 comprises a vacuum pump 8 and a pressure control valve 9.
With the above-mentioned configuration, the microwave reduced pressure distillation apparatus 1 is configured so that the pressure inside the distillation tank 2 and the like are adjusted via the heating control device 7 and the pressure control device 10, respectively.

When using a distillation apparatus, known distillation conditions can be used. The conditions vary depending on the capacity, water content, microwave strength, etc., but for example, the pressure inside the storage section of the apparatus can be adjusted to a reduced pressure of 10 to 90 kPa and the treatment time to 0.2 to 8 hours.

The mixture of essential oil and aqueous liquid obtained as a distillate can be separated into an oily fraction and a water-soluble fraction by processes such as standing, redistillation, and liquid-liquid separation. Of the separated fractions, the oily fraction corresponds to the above-mentioned essential oil, and the water-soluble fraction corresponds to the above-mentioned aqueous liquid. By separating these fractions, they can be used for the desired purposes.

As the extract, as described above, a solid extract containing the solid matter itself prepared by the above-mentioned method, a liquid extract in which the prepared solid matter is dispersed in a solution, an extract containing a liquid obtained by further extracting the prepared solid matter, etc. In other words, any of the extracts is prepared so as to contain the components contained in the solid matter.
The first extract can be easily prepared by taking a predetermined amount of the prepared solid matter. The second extract is prepared by immersing the prepared solid matter in a liquid such as water that does not affect the mushroom mycelium, so that the components contained in the solid matter can be easily extracted. And the last extract is prepared to contain a liquid that contains only the components contained in the prepared solid matter.

The method for preparing the final extract is not particularly limited as long as it is a method capable of extracting the components contained in the solid matter.
For example, a general extraction method using water, hot water, organic solvent, etc. can be adopted. When performing extraction treatment with water or hot water, the extraction treatment is performed with each liquid for a predetermined time, and then the supernatant obtained using a centrifuge can be used as the extract. When performing extraction treatment with an organic solvent, it is necessary to perform treatment that does not affect the mycelium. For example, the organic solvent is removed from the liquid after extraction, and then the liquid is replaced with a solvent that does not affect the mycelium, such as water, to obtain an extract.

Next, the extract prepared as described above is inoculated with a seed mycelium of a desired mushroom. After inoculation, the mycelium is cultured for a predetermined period of time to prepare a treated mycelium product.
This treated mycelium is obtained by treating an extract obtained from olives, which contains an antioxidant active agent and an α-glucosidase inhibitor active agent, with mycelium.

In summary, the method for producing an antioxidant active agent is a method for producing the antioxidant active agent by sequentially carrying out a step of preparing an extract obtained from olives and a step of treating the prepared extract with mycelium.
The former process for preparing an extract obtained from olives includes a process for preparing a solid content obtained from olives. This process includes a process for adjusting the solid content by microwave irradiation treatment, a process for preparing the solid content by a pressing method, or a process for preparing the solid content by a distillation method. The process for preparing the solid content by a distillation method includes a method of vacuum distillation while irradiating microwaves.
In addition, when the solid content is further subjected to an extraction treatment, the method may include a step of further extracting the prepared solid content using water, hot water, or the like.

The above-mentioned method of preparing solid matter from olives by vacuum distillation while irradiating them with microwaves corresponds to the first step in the claims. The step of further extracting the prepared solid matter using water or hot water corresponds to the second step in the claims.

By using the above manufacturing method, it is possible to produce an antioxidant active agent that exhibits excellent antioxidant activity. In addition, by using a similar manufacturing method, it is possible to produce an α-glucosidase inhibitor active agent that exhibits excellent α-glucosidase inhibitory activity.

Next, the present invention will be described in more detail with reference to examples. Note that these examples are merely examples of the present embodiment, and the present invention is not limited in any way by the following examples.

Pruned olive branches and leaves were crushed in a grinder, and the crushed material was placed in the distillation tank of the microwave reduced pressure distillation apparatus shown in Figure 1, and distillation treatment was carried out with the conditions inside the distillation tank set as follows.
Pressure in distillation tank: reduced pressure of 0.25 KPa Distillation time: 30 minutes

The residue (solids) and distillate were obtained by distillation using the microwave vacuum distillation apparatus shown in Figure 1.

The resulting residue was removed from the distillation tank, and a specified amount was taken and extracted with water to prepare a water extract. A specified amount was also taken from the residue and extracted with hot water to prepare a hot water extract.

The prepared water extract and hot water extract correspond to the extract obtained from olives in this embodiment.

(Preparation of Water Extract)
The aqueous extract was prepared as follows.
1. 50 ml of ultrapure water was added to 1 g of olive branches and leaves and left to stand at 4°C for 24 hours.
2. The mixture was centrifuged (8000 rpm, 4°C, 30 minutes) to collect the supernatant.
3. The mixture was centrifuged again (8000 rpm, 4°C, 15 minutes) and the collected supernatant was used as the water extract.

(Preparation of hot water extract)
1. 1 g of olive branch and leaves was added to 50 ml of boiled ultrapure water and left to stand at 100°C for 20 minutes.
2. After cooling to room temperature, the mixture was centrifuged (8000 rpm, 4°C, 30 minutes) to recover the supernatant.
3. The mixture was centrifuged again (8000 rpm, 4°C, 15 minutes) and the collected supernatant was used as the hot water extract.

(Mycelium Treatment)
The prepared water extract and hot water extract were subjected to mycelium treatment as follows.
Enokitake mushroom was used as the type of mycelium mushroom.
Mushroom seed cultures were added to each extract solution, and the solution was left to stand for 24 hours at 25° C. in a dark place to prepare mycelium treatment solutions.

This mycelium treatment liquid corresponds to the mycelium treatment product obtained by treating the extract obtained from olives with mycelium in this embodiment.

Solids were filtered out from the prepared mycelium treatment liquid to prepare measurement samples for measuring antioxidant activity and α-glucosidase inhibitory activity.

(Antioxidant Activity Analysis)
The measurement samples were used to analyze the antioxidant activity.
Antioxidant activity was evaluated in two ways: superoxide radical scavenging activity and DPPH radical scavenging activity.

(Superoxide radical scavenging activity)
Measurement was performed by luminescence method using a commercially available kit (ATTO, AB-2970 CLETA-S kit).
The amount of luminescence was measured using an ATTO AB-2270 Luminescence Sensor.

(DPPH radical scavenging activity)
To 180 μL of DPPH solution (DPPH 1.0 mg, 100% ethanol 5.0 ml, 0.1 M MES buffer (pH 6.0) 5.0 ml), 20 μL of the sample or extraction solvent (negative control) was added and reacted at room temperature in the dark for 30 minutes.
After the reaction, the absorbance at 517 nm was measured using a microplate reader (CORONA ELECTRONICS CO., LTD., SH-1200 Lab).

(Analysis of α-glucosidase inhibitory activity)
The measurement samples were used to analyze α-glucosidase inhibitory activity.
The α-glucosidase inhibitory activity was evaluated using α-glucosidase (Sigma-Aldrich, model number: I1630).
A given amount of the measurement sample was taken, mixed with α-glucosidase, and left to stand at room temperature for 10 minutes. Then, an aqueous solution of p-nitrophenyl α-D-glucopyranoside was added to the mixture and reacted with stirring at 37°C for 30 minutes. After that, an aqueous Tris solution was added, and the absorbance at 400 nm was measured using a microplate reader (CORONA ELECTRONICS SH-1200 Lab).

The α-glucosidase inhibitory activity was expressed as the α-glucosidase inhibitory activity rate (%), which was simply indicated as "inhibition rate (%)" in the figure.
The α-glucosidase inhibitory activity rate (%) was calculated by subtracting the blank value from each analytical value and dividing the result by the blank value.

FIG. 2 shows the evaluation results of antioxidant activity.
2(A), the superoxide radical scavenging activity of the water extract (olive branch and leaf water extract in the figure) was 15.7% before treatment (left graph) and 44.5% after treatment (right graph). Also, the hot water extract (olive branch and leaf hot water extract in the figure) was 30.8% before treatment (left graph) and 58.8% after treatment (right graph).
2(B), the DPPH radical scavenging activity of the water extract (olive branch and leaf water extract in the figure) was 23.1% before treatment (left graph) and 35.1% after treatment (right graph). Also, the hot water extract (olive branch and leaf hot water extract in the figure) was 30.8% before treatment (left graph) and 50.8% after treatment (right graph).
As shown in Figure 2, the activity after mycelium treatment (water extract or hot water extract) was approximately two times higher than that before treatment. It was also confirmed that the hot water extract had higher activity than the water extract.

FIG. 3 shows the evaluation results of α-glucosidase inhibitory activity (α-glucosidase inhibitory activity rate (%)).
3, the α-glucosidase inhibitory activity of the water extract (olive branch and leaf water extract in the figure) was 57.1% before treatment (left graph) and 68.2% after treatment (right graph). Also, the activity of the hot water extract (olive branch and leaf hot water extract in the figure) was 48.6% before treatment (left graph) and 90.5% after treatment (right graph).
As shown in Figure 3, similar to the evaluation of antioxidant activity, the activity after mycelium treatment was improved compared to before treatment. Also, it was confirmed that the hot water extract was more effective.

(HPLC analysis of mycelium treatment solution)
The mycelium treatment solution (using hot water extract) was analyzed using HPLC (Shimadzu Corporation).
In the experiment, a treated liquid of Enoki mushroom mycelium was used.

The measurement conditions were as follows.
Column used: Shim-pack CLC-ODS 250 x 4.6 mm (Shimadzu Corporation)
Mobile phase: Solution A (2% acetic acid), Solution B (100% methanol)
Flow rate: 0.5ml/min
Measurement wavelength: 280nm

The measurement results are shown in FIG.
Oleuropein, which is known to be contained in olive leaves, was not detected in the mycelium treatment liquid after the mycelium treatment. In addition, a peak not seen before the treatment was confirmed in the mycelium treatment liquid after the mycelium treatment.
This suggests that oleuropein was denatured by the enzymes in the mycelium, producing a component different from oleuropein. It is possible that this different component activates various health benefits beyond those of oleuropein.
The color of the liquid changed before (water extract or hot water extract) and after (mycelium treatment liquid) the mycelium was treated. The liquid turned brown by treating the mycelium. The browning indicates that polyphenols have polymerized. Although it is unclear whether this is a substance that can be detected by HPLC, it is possible that this may have contributed to the improvement of functionality.

The antioxidant active agent and α-glucosidase inhibitor active agent of the present invention are suitable for preventing cancer, lifestyle-related diseases, infectious diseases, dementia, etc. Furthermore, the manufacturing method of the antioxidant active agent and the manufacturing method of the α-glucosidase inhibitor active agent of the present invention are suitable for manufacturing the antioxidant active agent and the α-glucosidase inhibitor active agent of the present invention, respectively.

Reference Signs List 1 Microwave vacuum distillation apparatus 2 Distillation tank 3 Microwave heating apparatus 4 Air flow inlet pipe 5 Distillate outlet pipe 6 Cooling apparatus 7 Heating control apparatus 8 Vacuum pump 9 Pressure control valve 10 Pressure control apparatus

Claims (4)

The present invention contains a mycelium treatment product obtained by adding mycelium of the genus Enokitake to an extract obtained from olive branches and/or leaves,
The extract is a liquid ;
This antioxidant active agent is obtained by subjecting olive branches and/or leaves to vacuum distillation while irradiating them with microwaves to separate at least a portion of the essential oil and/or aqueous liquid, and then extracting the residue with water or hot water. A method for producing an antioxidant active agent, comprising the steps of: a first step of irradiating olive branches and/or leaves with microwaves and separating the distillate and residue by reduced pressure distillation; a second step of extracting the resulting residue with water or hot water to obtain a liquid extract; and a third step of adding mycelium of the genus Enokitake to the resulting extract. The present invention contains a mycelium treatment product obtained by adding mycelium of the genus Enokitake to a liquid extract obtained from olive branches and/or leaves,
The extract,
The α-glucosidase inhibitor is characterized in that it is obtained by subjecting olive branches and/or leaves to reduced pressure distillation while irradiating them with microwaves, separating at least a portion of the essential oil and/or aqueous liquid, and extracting the residue with water or hot water. A method for producing an α-glucosidase inhibitor, comprising the steps of: a first step of separating olive branches and/or leaves into a distillate and a residue by vacuum distillation while irradiating them with microwaves; a second step of extracting the resulting residue with water or hot water to obtain a liquid extract; and a third step of adding mycelium of the genus Enokitake to the resulting extract.

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