JP2016079135A - Coprinus comatus extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a Coprinus comatus extract which dissolves into water in a given amount or more and contains an ethanol soluble component in a given amount or more, and has a more excellent anti-oxidant action and whitening action than conventional Coprinus comatus extract.SOLUTION: The Coprinus comatus extract having a more excellent anti-oxidant action and whitening action than conventional Coprinus comatus extracts can be provided by obtaining Coprinus comatus extract which has a solubility in water at room temperature of 5.0 g/100 ml or more and has a content of a soluble component to ethanol at room temperature of 1.8 mass% or more.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an extract of Sasakuhi Toyotake, which is made from Sasakuhi Toyotake. In particular, the present invention relates to a Sasahihito Toyotake extract having a solubility in water of a predetermined amount or more and an ethanol-soluble component content of a predetermined amount or more.

  Sasahihi Toyotake (scientific name: Coprinus comatus) is a kind of mushroom belonging to the genus Sasahihizoe, which is a cylindrical shape with a rounded tip, with a grayish but slightly creamy surface on its surface. Forms a white umbrella that is scattered. Inexpensive larvae have been traditionally eaten as delicious edible mushrooms, and are treated as luxury foods in Europe, especially in Italy.

  Sasa Kurehi Toyotake and its extract are used as health foods, cosmetic materials, pharmaceuticals and the like because they have various functions such as antioxidant, whitening and anti-inflammatory effects. In particular, ergothionein, which is a water-soluble component contained in a large amount of Sasahihi Toyotake in comparison with other mushrooms and plants, is known as an active ingredient exhibiting an antioxidant action and a whitening action.

  Conventionally known methods for obtaining Sasakurehi Toyotake extract are hot water extraction and solvent extraction. For example, Patent Document 1 describes an extract extracted with hot water from a dried powder of mushrooms belonging to the family Hitotake. Patent Document 2 describes water, methanol, ethanol, and the like as a solvent for obtaining Sasahihi Toyotaga extract. Non-Patent Document 1 describes that the ethanol extract of Sasahihi Toyotake has a high antioxidant effect.

JP 2010-275235 A JP 2012-092085 A

Bo Li et al., Molecules, 15,1473-1486, 2010

  However, the Sasahihito Toyotake extract obtained by conventional hot water extraction contains almost no ethanol extract having a high antioxidant action, and is a component exhibiting a high antioxidant action described in Non-Patent Document 1. Solvent-soluble components such as are not included. On the other hand, the Sasahihitake extract obtained by solvent extraction contains only a solvent-soluble component and has a problem that it hardly contains water-soluble ergothioneine. Moreover, when both extraction methods are combined, extraction operation etc. become complicated and there exists a problem that it is inferior to productivity.

  The present invention has been made in view of these problems, and the amount of water-soluble and ethanol-soluble components is not less than a predetermined amount, and is superior in antioxidative action and whitening action than conventional Sasahihitake extract. The purpose is to obtain Sasa Kurehi Toyotake extract.

  The present invention relates to a Sasahihitake extract having a solubility in water at room temperature of 5.0 g / 100 ml or more and a content of a component soluble in ethanol at room temperature of 1.8% by mass or more.

  It is preferable that the extract is a water extract of Sasa Kurehi Toyotake.

  It is preferable that the extract is a subcritical water extract of Sasa Kurehi Toyotake.

  It is preferable that the extract contains a degradation product of ergothioneine.

  According to the extract of Sasahihito Toyotake having a soluble amount in water and a content of an ethanol-soluble component of the present invention, the superoxide dismutase (SOD) -like activity and the tyrosinase inhibitory activity are higher than those of a conventional Sasahihitoyotake extract. It is possible to provide an extract of Sasahihitake, which can exhibit an antioxidant effect and a whitening effect when applied or taken.

It is a graph which shows the SOD like activity of an Example, a comparative example, and a reference example. It is a graph which shows the tyrosinase inhibitory activity of an Example, a comparative example, and a reference example. It is a graph which shows the melanin production evaluation of an Example, a comparative example, and a reference example. 2 is an HPLC chromatogram of Reference Example 1, Example 3 and Comparative Example 1.

<Sasakurehi Toyotake Extract>
The Sasahihito Toyotake extract of the present invention is an extract made from Sasahihitoyotake as a raw material, characterized in that the amount dissolved in water and the content of ethanol-soluble components are not less than a predetermined value, and the functionality of the Sasahihi Toyotake extract has It is a Sasahihitake extract that can efficiently exhibit (SOD-like activity and tyrosinase inhibitory activity).

  The components extracted from Sasa Kurehi Toyotake Extract are roughly divided into three components: those that are soluble only in water, those that are insoluble in water and only soluble in solvents such as ethanol, and those that are soluble in both water and solvents such as ethanol. can do.

  100% by mass of the Sasahihi Toyotake extract of the present invention is a component that is substantially soluble in water. That is, the Sasahi Toyota extract of the present invention is an extract component that does not contain a water-insoluble component, and is a component that is substantially soluble only in water (also referred to as an ethanol-insoluble component) and a component that is soluble in water and ethanol (ethanol) It is also called a soluble component.

  Since components exhibiting high SOD-like activity and tyrosinase inhibitory activity are generally water-soluble, it is desirable that the amount of the extract of the salamander Toyotake of the present invention dissolved in water at room temperature (25 ° C.) is large, and sufficient activity can be obtained. Therefore, it is 5.0 g / 100 ml or more. The upper limit of the amount dissolved in water is not particularly limited.

  The above-mentioned components soluble only in water (ethanol-insoluble components) include ergothionein, which is an active ingredient that exhibits antioxidant and whitening effects, and some of the degradation products of ergothioneine described below, and has high SOD-like activity and tyrosinase inhibition Shows activity.

  The water-soluble and ethanol-soluble component (ethanol-soluble component) includes a degradation product of ergothioneine, which will be described later, and exhibits high SOD-like activity and tyrosinase inhibitory activity.

  The content of the ethanol-soluble component in the extract of the salamander of the present invention is 1.8% by mass or more, more preferably 1.9% by mass or more, and further preferably 2.0% by mass or more. When the content of the ethanol-soluble component is less than 1.8% by mass, the effects of SOD-like activity and tyrosinase inhibitory activity tend not to be confirmed. Further, the content of the ethanol-soluble component is preferably 10% by mass or less, and more preferably 5% by mass or less. When it exceeds 10 mass%, there exists a possibility that content of a water-soluble component may become inadequate.

  It is preferable that the extract of Sasahihi Toyotake of the present invention contains a degradation product of ergothioneine because it is more excellent in SOD-like activity and tyrosinase inhibitory activity. The degradation product of ergothioneine is a degradation product produced by degradation of ergothioneine by subjecting ergothioneine to subcritical treatment, etc., and exhibits an SOD-like activity and tyrosinase inhibitory activity higher than ergothioneine before degradation. Found them.

  The degradation product of ergothioneine will be described with reference to the attached FIG. (I) of FIG. 4 is an HPLC chromatogram of Sasahihito Toyotake extract (the whole subcritical water extract) obtained in Example 3 described later. (II) is an HPLC chromatogram of a subcritical processed product of commercially available ergothioneine (manufactured by Sigma-Aldrich). Further, the same (III) is an HPLC chromatogram of the Sasahihitake extract (water extract) obtained in Comparative Example 1 described later. A single peak indicated as “ERG” in each chromatogram is an ergothioneine peak. Here, although the commercially available ergothionein used in (II) is almost 100% ergothioneine, since it is a subcritical processed product, a plurality of peaks indicated by “▽” can be confirmed in addition to the peak of ergothioneine. These “▽” peaks are the degradation products of ergothioneine. Since (I) is a subcritical water extract of Sasahihi Toyotake, which contains a large amount of ergothioneine, in addition to the peak of ergothioneine, the peak of ergothioneine degradation products indicated by “▽” and the peak of other components can also be confirmed. . On the other hand, since (III) is a hot water extract of Sasa Kurehi Toyotake, the peak of water-soluble ergothioneine can be confirmed, but the peak of the degradation product of ergothioneine cannot be confirmed.

  The degradation product of ergothioneine exhibits higher SOD-like activity and tyrosinase inhibitory activity than ergothioneine before degradation (from comparison with Reference Examples 1 and 2 described later). In addition, from the chromatograms of (I) and (II) in FIG. 4, ergothioneine is a component that is soluble only in water and difficult to dissolve in ethanol, but ergothioneine degradation products can be dissolved in water and ethanol. It turns out that a soluble component is contained. In other words, the content of the ethanol-soluble component in the extract of Sasahihiru Toyotake is a value that is an indicator of the content of the degradation product of ergothioneine, and the Sasahihitoyotake extract exhibits high SOD-like activity and tyrosinase inhibitory activity, and is an antioxidant. It becomes an index indicating whether or not it is a Sasakehi Toyotake extract that can exert its action and whitening effect.

<Manufacturing method>
As an example of the method for producing Sasahihi Toyotake of the present invention, an extraction process for extracting Sasahihi Toyotake as a raw material and a solid-liquid separation process for separating an extract from a raw material residue will be described. According to this production method, the extract of Sasahihi toyoke consists of a component that is soluble only in water and a component that is soluble in water and ethanol, and the amount of water-soluble and ethanol-soluble component is a predetermined amount or more. It is possible to produce a Hitoyotake extract.

Raw material The raw material for the extraction of the salamander Toyotake of the present invention is the Sacrahi Toyotake (scientific name: Coprinus comatus), which belongs to the genus Sasahihikarie. The form and state are not particularly limited, and those in a raw state, dried or powdered, etc. are preferably used. When using the raw material, it is preferable to wash dirt such as soil before the extraction step.

Extraction step The extraction step is a step of obtaining an extraction-processed product by subjecting the Sasakuhi Toyotake raw material to extraction processing. Examples of the extraction solvent used for extraction include water, ethylene, ethane, propane, carbon dioxide, methanol, ethanol and mixtures thereof, and water is most preferably used from the viewpoint of extraction efficiency and safety of water-soluble components. . Examples of the extraction treatment method include continuous extraction, immersion extraction, supercritical extraction, subcritical water extraction, and the like, but Sasahihi Toyotake extract having a water-soluble amount and an ethanol-soluble component content of a predetermined amount or more is used. Subcritical water extraction is preferred because it can be obtained efficiently.

  The subcritical water extraction is a method in which a subcritical fluid as an extracting agent brought into a subcritical state under a predetermined temperature and pressure is brought into contact with a raw material to be extracted (in the present invention, Sasakurehi Toyotake) from the extracted raw material. The component is extracted. For example, when water is raised to a pressure of 22.12 MPa and a temperature of 374.15 ° C., it shows a state where it is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure lower than the critical point is called subcritical water. This subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product.

  When water is used as an extractant used for subcritical water extraction, it can be used in a liquid state or a gas state as long as it is a high-temperature water treatment. That is, steam may be supplied to the treatment tank for subcritical water extraction, water may be supplied, or both of them may be supplied. The temperature of water or water vapor is desirably 100 ° C. or higher, and the desired reaction field is more likely to proceed in a liquid state than in a gas. The use of water in a critical state is preferred. More specifically, put the water that is the extractor and the extractant into a pressure vessel such as metal or ceramics to make it close to the sealed state, and the subcritical state of water (temperature: 100 ° C or higher, pressure: saturated vapor pressure) As described above, the extract obtained by performing the contact between the two for a predetermined time or more can be used as the subcritical water extract.

  Examples of the extractant used for subcritical water extraction include, in addition to water, ethylene, ethane, propane, carbon dioxide, methanol, ethanol, and mixtures thereof. Among these, it is most preferable to use water from the viewpoint of safety. Next, processing conditions when the extractant is water will be described.

  The subcritical water extraction temperature of Sasahihi Toyotake is preferably between 140 and 200 ° C. for the reason that it is easy to efficiently obtain a Sasahihitake extract with a water-soluble amount and a content of ethanol-soluble components of a predetermined amount or more. 160-195 ° C is more preferable, and 180-195 ° C is more preferable. When the temperature of subcritical water extraction is less than 140 ° C., the obtained extract tends to have insufficient SOD-like activity and tyrosinase inhibitory activity. Moreover, when the temperature of subcritical water extraction exceeds 200 degreeC, it will decompose excessively and there exists a tendency for the effect of the SOD like activity and tyrosinase inhibitory activity of the obtained extract to fall.

  It is preferable that the subcritical water extraction pressure of Sasa Kurehi Toyotake is at or above the saturated vapor pressure at each temperature (for example, 0.60 MPa or more at 150 ° C., 1.3 MPa or more at 190 ° C.). By using this pressure, it becomes easy to efficiently obtain a Sasahihi Toyotatake extract in which the amount dissolved in water and the content of ethanol-soluble components is a predetermined amount or more. In addition, although the upper limit of the pressure of subcritical water extraction is not specifically defined, it is preferable to suppress around 20-30 MPa on the specification of a high voltage | pressure apparatus.

  The subcritical water extraction time for Sasaclehi toyoke is preferably 5 to 60 minutes, more preferably 10 to 30 minutes. By setting the treatment time within the range, it becomes easy to efficiently obtain a Sasakuhi Toyotake extract in which the amount dissolved in water and the content of the ethanol-soluble component is a predetermined amount or more. When the subcritical water extraction time is less than 5 minutes, the effect of the SOD-like activity and tyrosinase inhibitory activity of the obtained extract tends to be insufficient. Moreover, when subcritical water extraction time exceeds 60 minutes, it will decompose excessively and there exists a tendency for the effect of the SOD-like activity and tyrosinase inhibitory activity of the obtained extract to fall.

  That is, as extraction conditions by subcritical water extraction of Sasakuhi Toyotake when the extractant is water, the processing temperature is 140 to 200 ° C., the processing pressure is equal to or higher than the saturated vapor pressure of each temperature, and the processing time is 5 to 60 minutes. Preferably it is done. By carrying out under these conditions, it is possible to obtain a Sasahihitake extract, which has a water-soluble amount and an ethanol-soluble component content of a predetermined amount or more, and has an excellent antioxidant effect and whitening effect. Furthermore, the treatment temperature is 180 to 195 ° C., the treatment pressure is equal to or higher than the saturated vapor pressure at each temperature, and the treatment time is 10 to 30 minutes. It is preferable because it is possible to obtain an extract of Sasahihito Toyotake, which is excellent in water.

Solid-Liquid Separation Step and Drying Step The sub-critical water extraction product obtained from the extraction step is a sub-critical water-extracted liquid (hereinafter referred to as an extract) and unreacted mainly plant fiber. It is a mixture with the solid component (henceforth a raw material residue) which consists of. Since this treated product contains Sasahihi Toyotake extract, which is excellent in antioxidant and whitening effects, it can be used as it is or in a dried state, but it is more excellent in antioxidant and whitening effects. In order to obtain an active component, it is desirable to go through the following solid-liquid separation process.

  The solid-liquid separation step is a step of separating an extraction treatment product such as a subcritical water extract into an extract and a raw material residue (solid material). Specific solid-liquid separation processes include filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressure dehydration, Belt press, centrifugal concentration dehydration, multiple disk dehydration and the like can be mentioned. Among these, filtration is preferable because the operation is simple and the separation efficiency is excellent.

  The extract obtained by the solid-liquid separation process can be used as it is or in a concentrated state as a Sasahihitake extract with excellent antioxidant and whitening effects. It is desirable to dry to enhance. By drying the extract, a solid Sasahihitake extract is obtained. As a drying method, a general drying method can be used. Naturally, it is allowed to stand naturally, heat transfer drying such as box drying or spray drying which is a heating system, internal heat drying such as microwave drying, non-heating system Freeze drying, vacuum drying, suction drying, pressure drying, ultrasonic drying, and the like are possible. Of course, it is acceptable to dry using a general and simple oven and thermostat. Moreover, you may perform the decoloring using adsorption agents, such as activated carbon and an ion exchange resin, before a drying process suitably. The salamander extract obtained in this way can be used for the intended purpose as it is, or other ingredients may be formulated, blended and processed into powder, capsule, tablet form, water, alcohol, etc. Of course, it may be used in the form of a liquid after being dissolved in a soluble solvent.

  Use organic solvents (alcohols, ketones, acetones, etc.) for extracts that have undergone a solid-liquid separation process and a drying process to identify active ingredients and to selectively obtain more functional ingredients. Extraction may be performed. Of these, ethanol is preferred because it has little effect on the human body and is easy to use in foods and cosmetics.

Sacherhi toyoke extract obtained by subcritical water extraction Sasakuhi toyoke extract obtained by subcritical water extraction has an amount of water soluble in a predetermined amount or more and an ethanol-soluble component content of a predetermined amount or more. And a decomposition product of ergothioneine, which is superior in antioxidative action and whitening action than the conventional extract of Sasahihito Toyotake.

  The present invention will be described based on examples, but the present invention is not limited to the examples.

<Preparation of Sasakuhi Toyotake extract for testing>
The preparation process of the test Sasahihitake extract in each Example, Comparative Example and Reference Example will be described. Table 1 shows the main extraction conditions for the examples and comparative examples.

Example 1 (subcritical water extraction)
100 g of dried Sasakuhi Toyotake (purchased from Healthcare Systems Co., Ltd.) was placed in a subcritical water treatment can, and subcritical water extraction was performed at a treatment temperature of 150 ° C., a treatment pressure of 0.5 MPa, and a treatment time of 30 minutes. After the subcritical water extraction is completed, the processed product in the processing can is collected, suction filtered with a cellulose filter paper (5C manufactured by Advantec), and the filtrate is freeze-dried to obtain 55% of the test Sasakuhi Toyotake extract A1. 9 g was obtained.
Furthermore, 10 g of the extract A1 was added to 200 ml of ethanol at 25 ° C. (purity 99.5%, manufactured by Kanto Chemical Co., Ltd.), and extracted at 25 ° C. for 2 hours. Then, it filtered with the filter paper made from cellulose (5C made from Advantec), and the ethanol insoluble component A2 in extract A1 was obtained by drying the residue on a filter paper. Moreover, the ethanol soluble component A3 in the extract A1 was obtained by freeze-drying the filtrate. The weight of the ethanol-soluble component A3 was 0.18 g, and 10 g of the extract A1 was used, so that the ethanol-insoluble component A2 was found to be 9.82 g.

Example 2 (Subcritical water extraction)
Under the same conditions as in Example 1 except that subcritical water extraction was performed at a processing temperature of 170 ° C., a processing pressure of 0.8 MPa, and a processing time of 20 minutes, 69.6 g of a test Sasakuhi Toyotake extract B1 was obtained.
Furthermore, ethanol extraction was carried out in the same manner as in Example 1, and 9.81 g of ethanol-insoluble component B2 and 0.19 g of ethanol-soluble component B3 were obtained from 10 g of extract B1.

Example 3 (Subcritical water extraction)
Under the same conditions as in Example 1 except that subcritical water extraction was performed at a processing temperature of 190 ° C., a processing pressure of 1.3 MPa, and a processing time of 20 minutes, 67.6 g of a test Sasakuhi Toyotake extract C1 was obtained.
Furthermore, ethanol extraction was performed in the same manner as in Example 1, and 9.75 g of ethanol-insoluble component C2 and 0.25 g of ethanol-soluble component C3 were obtained from 10 g of extract C1.

Comparative Example 1 (water extraction)
50 g of dried Sasakuhi Toyotake (purchased from Healthcare Systems Co., Ltd.) was subjected to hot water extraction (85 ° C., 2 hours) with 200 ml of 85 ° C. water. The treated product was filtered through a cellulose filter paper (5C manufactured by Advantec), and the filtrate was freeze-dried to obtain 25.7 g of a test Sasahihitake extract D1.
Furthermore, ethanol extraction was performed in the same manner as in Example 1, and 9.83 g of ethanol-insoluble component D2 and 0.17 g of ethanol-soluble component D3 were obtained from 10 g of extract D1.

Comparative Example 2 (ethanol extraction)
Solvent extraction (25 ° C., 2 hours) of 200 g of dried Sasakuhi Toyotake (purchased from Healthcare Systems Co., Ltd.) with 200 ml of ethanol at room temperature (25 ° C.) was performed. The treated product was filtered through a cellulose filter paper (5C manufactured by Advantec), and the filtrate was freeze-dried to obtain 0.41 g of a test Sasahihitake extract E1.

Reference Examples 1 and 2
A commercially available ergothioneine reagent (Sigma Aldrich purity 98% or more) was used as Sample F1 (Reference Example 1). Further, an aqueous solution in which 20 mg of ergothioneine was dissolved in 10 ml of water was placed in a subcritical water treatment can, subjected to subcritical treatment at a treatment temperature of 190 ° C., a treatment pressure of 1.3 MPa, a treatment time of 20 minutes, and freeze-dried. This was designated as Sample F2 (Reference Example 2).

<Evaluation>
The following evaluation was performed with respect to the obtained Sasakuhi Toyotake extract for test and the sample obtained. The results are shown in Tables 1-3.

Ethanol dissolution test The content (mass%) of ethanol-insoluble components and ethanol-soluble components in the obtained extract was calculated. The results are shown in Table 1.

Water dissolution test 5.0 g of the extract shown in Table 1 was added to 100 ml of water at 25 ° C. and stirred, followed by filtration with cellulose filter paper (5C manufactured by Advantec), and drying of the filtrate lyophilizate and the residue on the filter paper. The mass of the object was measured. Water-soluble component content (mass%) was computed from the obtained mass. The results are shown in Table 1. Table 1 also shows whether the amount dissolved in water is 5 g / 100 ml or more.

SOD-like activity A sample solution was prepared by dissolving in a solvent so that the concentration of each sample was 100 mg / ml. The ethanol-soluble component was dimethyl sulfoxide (manufactured by Wako Pure Chemical Industries, Ltd.), and the others were distilled water. The SOD-like activity of the obtained sample solution was measured using an SOD measurement kit (Dojindo Laboratories Co., Ltd.), the SOD inhibition rate was determined, and the IC50 value (final concentration) was calculated. The average value of the values measured at n = 3 is the result, and is shown in Table 2 and FIG. In addition, the error bar in a figure shows the standard deviation of 3 times of measurement.

Tyrosinase inhibitory activity The following reagents were prepared and used. In addition, all the phosphate buffers used the same thing of pH = 6.7.
Enzyme solution: Reference Examples F1 and F2 are phosphate buffer containing 300 units / ml tyrosinase, others are phosphate buffer containing 30 units / ml tyrosinase Substrate solution: phosphate containing 0.2 mg / ml L-DOPA Buffer sample solution: Solution in which the concentration of each sample is 100 mg / ml, dissolved in a solvent (ethanol soluble component is dimethyl sulfoxide, and others are distilled water), and filtered through a 0.45 μm membrane filter [Sample (S)]
25 μl of each sample solution and 100 μl of the enzyme solution were sequentially added to each well of the microplate and allowed to stand at 37 ° C. for 10 minutes. Thereafter, 125 μl of the substrate solution was added to each well to make a total volume of 250 μl, and further allowed to stand at 37 ° C. for 10 minutes. Thereafter, the absorbance (S) at 492 nm was measured using an absorbance plate reader Multiscan JX (Thermo Fisher Scientific Co., Ltd.).
[Control (C)]
Absorbance (C) was measured by reacting under the same conditions as in (S) except that 25 μl of each solvent was used instead of 25 μl of the sample solution.
[Sample blank (SB)]
Absorbance (SB) was measured by reacting under the same conditions as (S) except that 100 μl of phosphate buffer was used instead of 100 μl of enzyme solution.
[Control blank (CB)]
Other than using 25 μl of solvent (ethanol-soluble component is dimethyl sulfoxide, others are distilled water) instead of 25 μl sample solution, and using 100 μl phosphate buffer instead of 100 μl enzyme solution ( The reaction was carried out under the same conditions as in S), and the absorbance (CB) was measured.
[Calculation of tyrosinase inhibition rate and IC50 value]
Using each absorbance measurement result, the tyrosinase inhibition rate (%) of each test Sasahihitake extract was calculated according to the following formula, and the IC50 value (final concentration) was determined. The average value of the values measured at n = 3 is the result and is shown in Table 2 and FIG. In addition, the error bar in a figure shows the standard deviation of 3 times of measurement.
Formula: ((C-CB)-(S-SB)) / (C-CB) × 100

Melanin Production Evaluation Sample solutions (10 mg / ml) of Example 3 (Test Sacherhi Toyotake Extract C1) and Comparative Example 1 (Test Sacherhi Toyotake Extract D1) were prepared, and these melanin production inhibition evaluation tests were conducted. . In the melanin production inhibition test, human-derived melanocyte cells (manufactured by Kurabo Industries, trade name: Normal human Epidermal melanocyte (NHEM) AD) are used to place a sample solution on the surface of the cells, and calculate the amount of melanin. The skin whitening was evaluated.
[Melanocyte culture]
Melanocyte Culture Human-derived melanocyte cells were pre-cultured for about 24 hours in a dedicated medium Derma Life MaComp kit. Thereafter, 50 μl of the test sample and 450 μl of Derma Life MaComp kit were added to the well containing the cells, and cultured at 37 ° C. and 5% carbon dioxide concentration for 72 hours. As a comparison, a test in which 500 μl of medium was added instead of the sample was also performed (control). n = 3.
[Melanin determination]
After completion of the culture, the medium in each well was removed by suction, washed twice with phosphate buffered saline, 100 μl of 0.25% trypsin-ethylenediaminetetraacetic acid solution was added, and the mixture was allowed to stand at 37 ° C. for 5 minutes. Thereafter, Dulbecco's Modified Eagle Medium medium was added to neutralize trypsin activity, the cells were detached, and centrifuged to remove the entire supernatant. 200 μl of 1N NaOH was added to the precipitate to elute intracellular melanin, the absorbance at 405 nm was measured, and the amount of melanin was calculated from a standard curve prepared in advance using synthetic melanin. In addition, the production inhibition rate of melanin shows the value which each converted, assuming the measured value of the light absorbency of control as 1. The average value of the values measured at n = 3 is the result and is shown in Table 2 and FIG. In addition, the error bar in a figure shows the standard deviation of 3 times of measurement.

  From the results of Tables 1 and 2 and FIGS. 1 to 3, the extract of Sasahihi toyoke, which has a water-soluble amount and an ethanol-soluble component content of the present invention above a predetermined value, has high SOD-like activity and tyrosinase inhibitory activity. In addition, it can be seen that this is a Sasahihitake extract that can exert an antioxidant effect and a whitening effect when applied or taken.

High Performance Liquid Chromatography (HPLC) Analysis Example 3 (Extract C1), Reference Example 2 (Sample F2) and Comparative Example 1 (Extract D1) were subjected to HPLC analysis under the following conditions. The gradient conditions for the mobile phase are shown in Table 3. The obtained HPLC chromatogram of Example 3 is shown in FIG. 4 (I), the HPLC chromatogram of Reference Example 2 is shown in FIG. 4 (II), and the HPLC chromatogram of Comparative Example 1 is shown in FIG. 4 (III).
Measuring device: LC-2010HT manufactured by Shimadzu Corporation
Column: COSMOSIL (registered trademark) MS-II Waters (φ4.6 × 250 mm) manufactured by Nacalai Tesque Co., Ltd.
Flow rate: 0.6 ml / min
Detection wavelength: 250 nm
Column temperature: 35 ° C
Injection volume: 10 μl
Mobile phase (A): water: formic acid = 100: 0.1
Mobile phase (B): Methanol

  The single peak indicated as “ERG” in each chromatogram is the peak of ergothioneine. Here, in the chromatogram (II) which is the result of Reference Example 2 (Sample F2) obtained by subjecting commercially available ergothioneine to subcritical treatment, in addition to the ergothioneine peak, a plurality of peaks indicated by “▽” can be confirmed. These “▽” peaks are considered to be peaks indicating degradation products of ergothioneine. Since (I) is an extract (Example 3) obtained by subcritical treatment and extraction of Sasahihi Toyotake containing a large amount of ergothioneine, in addition to the peak of ergothioneine, the degradation product of ergothioneine indicated by “▽” Peaks and peaks of other components can also be confirmed. On the other hand, since (III) is a hot water extract of Sasahihi Toyotake (Comparative Example 1), it can be seen that the peak of water-soluble ergothioneine can be confirmed, but the peak of the degradation product of ergothioneine cannot be confirmed.

Claims (4)

The amount dissolved in water at room temperature is 5.0 g / 100 ml or more,
Sasahihi Toyotake extract containing 1.8% by mass or more of a component soluble in ethanol at room temperature. The extract of Sasahihito Toyotake according to claim 1, wherein the extract is a water extract of Sasahihitake. The Sasahihito Toyotake extract according to claim 1, wherein the extract is a subcritical water extract of Sasahihiyotake. The extract of Sasahihi Toyotake according to any one of claims 1 to 3, wherein the extract contains a degradation product of ergothioneine.