JP2016124845A - Wasabia japonica extract and production method thereof, and astringent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Wasabia japonica extract and astringent that have high safety and can normalize skin condition, and to provide a production method of Wasabia japonica extract that has high safety, can normalize skin condition, and is capable of being extracted efficiently.SOLUTION: We focused on the leaf part of young Wasabia japonica after the investigation of each functionality of the root part, stem part, and leaf part, then investigated the leaf part in further detail by exchanging ethanol concentration of an extracting solvent, and enabled the effective extraction of the function of the young Wasabia japonica leaf part.SELECTED DRAWING: None

Description

  The present invention relates to a wasabi extract, a method for producing the same, and a skin conditioner. Specifically, it provides a wasabi extract and a skin conditioner that are highly safe and can condition the skin, and is highly safe, can condition the skin, and can be efficiently extracted. The present invention relates to a method for producing a wasabi extract.

  Wasabi has been prized as a spice since ancient times because of its unique spiciness and aroma. In modern times, it is an indispensable ingredient when eating sashimi and sushi.

  Wasabi stems are used for soy sauce and pickles in wasabi, and the flowers are used as decorative flowers for high-priced sashimi.

  However, the leaf portion is hard and bitter, so it is difficult to eat and is disposed of as a so-called unused resource. For this reason, an invention of a method for effectively utilizing the wasabi leaf portion is expected. For example, as shown in Patent Document 1, 1 or a root selected from roots, stems and leaves of root vegetables such as wasabi or the like Slimming effects have been found on extracts from more than one site.

JP 2012-111740 A

  However, the invention of Patent Document 1 is not a detailed comparison of each part of the root, stem, and leaf parts of wasabi, and the wasabi as a whole is merely cited as one of the root vegetables. I'm just there. For this reason, it cannot be said that the function of the wasabi leaf part has been sufficiently elucidated, and the wasabi leaf part has not been effectively utilized.

  This invention is invented in view of the above point, and relates to a wasabi extract, its manufacturing method, and a skin conditioner. Specifically, it provides a wasabi extract and a skin conditioner that are highly safe and can condition the skin, and is highly safe, can condition the skin, and can be efficiently extracted. It aims at providing the manufacturing method of a wasabi extract.

  In order to achieve the above-mentioned object, the wasabi extract having the moisturizing effect of the present invention is extracted from the wasabi leaf portion using ethanol or an ethanol-containing solvent.

  Here, by using ethanol or an ethanol-containing solvent, a fat-soluble substance contained in the wasabi leaf portion is easily extracted.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  Further, when the wasabi extract having a moisturizing effect is extracted at an ethanol concentration of a value between 20 vol% and 30 vol%, the hyaluronic acid producing ability can be enhanced.

  Moreover, also when the wasabi extract which has a moisturizing effect is extracted with the ethanol concentration of the value between 70 vol% and 80 vol%, hyaluronic acid production ability can be improved.

  Moreover, in order to achieve said objective, the wasabi extract which has the antibacterial activity of this invention is extracted from the wasabi leaf part using ethanol or an ethanol containing solvent.

  Here, by using ethanol or an ethanol-containing solvent, a fat-soluble substance contained in the wasabi leaf portion is easily extracted.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  In addition, when the wasabi extract having antibacterial activity is extracted at an ethanol concentration of 70 vol% or more, the growth of bacteria can be effectively suppressed.

  Moreover, in order to achieve said objective, the wasabi extract which has the antioxidant activity of this invention is extracted from the wasabi leaf part using ethanol or an ethanol containing solvent.

  Here, by using ethanol or an ethanol-containing solvent, a fat-soluble substance contained in the wasabi leaf portion is easily extracted.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  In addition, when the wasabi extract having antioxidant activity is extracted at an ethanol concentration between 40 vol% and 80 vol%, generation of active oxygen can be effectively suppressed.

  Moreover, in order to achieve said objective, the wasabi extract which has the lipase inhibitory activity of this invention is extracted from a wasabi leaf part using ethanol or an ethanol containing solvent.

  Here, by using ethanol or an ethanol-containing solvent, a fat-soluble substance contained in the wasabi leaf portion is easily extracted.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  In addition, when the wasabi extract having lipase inhibitory activity is extracted at an ethanol concentration of 90 vol% to 100 vol%, fat decomposition and absorption can be suppressed.

  Moreover, when it has two or more functions among antibacterial activity, antioxidant activity, lipase inhibitory activity, and a moisturizing effect, a multifunctional wasabi extract and skin conditioner can be manufactured.

  In order to achieve the above object, the skin conditioner of the present invention is an antibacterial activity, an antioxidant activity, a lipase inhibitory activity and a moisturizing effect extracted from a wasabi leaf part using ethanol or an ethanol-containing solvent. Among them, it has a wasabi extract containing at least one function.

  Here, by using ethanol or an ethanol-containing solvent, a fat-soluble substance contained in the wasabi leaf portion is easily extracted.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  Moreover, the skin conditioner specialized in a specific function can be manufactured by including at least 1 function among antibacterial activity, antioxidant activity, lipase inhibitory activity, and a moisturizing effect.

  Moreover, in order to achieve said objective, the manufacturing method of the wasabi extract of this invention is equipped with the process extracted using ethanol or an ethanol containing solvent from what dried and powdered the wasabi leaf part.

  Here, the leaf portion of wasabi can be efficiently extracted by drying and pulverizing.

  Moreover, the use of ethanol or an ethanol-containing solvent facilitates extraction of a fat-soluble substance contained in the wasabi leaf portion.

  Moreover, by extracting from the leaf part of wasabi, natural components derived from nature are extracted, and it becomes easy to ensure safety.

  The wasabi extract and skin conditioner of the present invention are highly safe and can condition the skin, and the method of producing the wasabi extract of the present invention is highly safe and can condition the skin. In addition, the method enables efficient extraction.

It is the figure which represented the antioxidant activity of leaf wasabi according to part. It is the figure which represented the antibacterial activity of leaf wasabi according to the part. It is a contrast diagram of the cell survival rate of a melanoma cell, and the amount of melanin biosynthesis of a leaf wasabi. It is the figure which represented the lipase activity of the leaf wasabi according to the site | part. It is a figure showing the amount of β-hexosaminidase released by adding A23187 to leaf wasabi extract. It is a figure showing the amount of β-hexosaminidase released by adding DNP as an antigen to a leaf wasabi extract. It is a figure showing the result of having measured the cytotoxicity of the leaf wasabi extract by the MTT test. It is a contrast diagram of the hyaluronic acid production amount of the leaf portion of leaf wasabi and the cell viability of nerve cells. It is a figure showing the antibacterial activity of the leaf part of a leaf wasabi. It is a figure showing the lipase activity of the leaf part of a leaf wasabi. It is a figure showing the antioxidant activity of the leaf part of a leaf wasabi.

  Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 5 for understanding of the present invention.

Functional test of each part of leaf wasabi About the wasabi extract of the present invention, the inventor firstly has antioxidant, antibacterial activity, melanin biosynthesis inhibitory activity, lipase inhibitory activity, antiallergic, anti-inflammatory functionalities, etc. In order to examine the presence or absence of the function of adjusting the skin condition, leaf wasabi leaves, stems, and roots were compared and evaluated.

[Extraction method]
Samples commonly used in the following tests were obtained as follows.
The leaf wasabi was divided into leaves, stems and roots, frozen at minus 20 degrees, and then freeze-dried until freeze-dried by a freeze dryer.

  Each lyophilized portion was weighed 2 to 3 g, and 100 ml of 50 vol% ethanol aqueous solution was added. Thereafter, shaking extraction was performed at a speed of 180 rpm for 48 hours.

Next, this extract was concentrated to obtain a dried product.
Specifically, after the extraction with shaking, the extract was collected by suction filtration. Then, the extract was concentrated with a rotary evaporator to remove the solvent. Thereafter, the concentrated solution after removing the solvent was frozen in a deep freezer at −80 ° C. and then dried with a freeze dryer to obtain a sample.

[Antioxidant activity test]
It is already known that some of the oxygen taken into the body becomes active oxygen. When this active oxygen is generated excessively, it becomes free radicals, attacks cellular functions, and causes chronic diseases and epidemics.

  On the other hand, if it is possible to suppress the generation of excessive active oxygen using an antioxidant or the like, it is possible to suppress the generation of free radicals and maintain the homeostasis.

<Test method>
Antioxidant activity was measured by measuring fluorescence intensity using the ORAC method.
The ORAC method is a measurement method that takes advantage of the property that fluorescein emits light having a wavelength of around 520 nm when excited by being irradiated with a wavelength of 485 nm.

  Specifically, first, a peroxy radical is generated using AAPH (2,2′-azobis (2-aminopropane) dihydrochloride) which is a radical generator. When fluorescein, which is a labeling substance, is oxidized by the peroxy radical, the light emitted from the fluorescein is quenched.

  Here, when an antioxidant is present, the peroxy radical is lost, and the quenching speed of fluorescein oxidized by the peroxy radical is decreased. This is a method of measuring the degree of delay of the quenching speed and evaluating it as antioxidant activity.

<Preparation of sample solution>
The sample was weighed and dissolved in a phosphate buffer so as to have a concentration of (1 mg / 1 mL), and this was diluted as a stock solution.

<Test results>
The results of the antioxidant activity test at each site are shown in FIG. FIG. 1 represents the relative ORAC value (mean ± SD) (n = 4) in 1 mg of extract.
In FIG. 1, the highest relative ORAC value was “0.041 ± 0.0022 (mgTE / mg)” in the leaf portion.

  From this, it was found that a component having high antioxidant activity was contained from the leaf portion of leaf wasabi.

[Antimicrobial activity test]
Antibacterial activity has the property of preventing the growth of bacteria that cause allergies, which are resident bacteria on the skin surface. In this test, the antibacterial activity of each site was evaluated from the effect on the growth of E. coli and S. aureus.

<Test method>
The extract at each site was dissolved in DMSO (dimethylsulfoxide dimethyl sulfoxide) and adjusted to a concentration of 80 mg / mL. In addition, DMSO containing no extract was used as a negative control, and sorbic acid was used as a positive control.

  Staphylococcus aureus and Escherichia coli were immersed in 5 mL of NB (Nutrient broth normal broth) medium to prepare a bacterial solution, which was osmotically cultured at 120 rpm and 37 ° C. for about 18 hours to obtain stationary cells.

The obtained stationary-phase culture solution was diluted with NB medium to prepare OD ₆₆₀ = 0.4 (E. coli: 10 ⁹ CFU / mL, S. aureus: 10 ⁸ CFU / mL). Further, this was diluted with sterilized water and used as a bacterial solution having a bacterial concentration of 10 ⁴ CFU / mL for the antibacterial test. The bacterial concentration was measured by applying light having a wavelength of 660 nm and measuring the turbidity of the bacterial solution from the turbidity of the bacterial solution. In this case, the higher the turbidity, the greater the number of bacteria.

  Further, 256 μ? Of NB medium and 4 μ? Of each sample of leaf wasabi were added to 40 μ? Of this bacterial solution and stirred for about 5 seconds. In addition, the density | concentration of the solution which mixed the above is 800 microgram / ml.

  Thereafter, while keeping the concentration of the bacteria uniform, the cells were cultured with shaking at 37 ° C. for 18 hours at a rotation speed of 1150 rpm, irradiated with light having a wavelength of 630 nm, and the turbidity of the bacteria solution was measured.

<Test results>
As a result of measuring the turbidity due to Staphylococcus aureus using a sample for each part, the result was that the sample of the leaf portion had the lowest turbidity. 2 represents the final concentration (μg / mL), and the significant difference between the control and each extract was determined by t-test. (: * P &lt; 0.05 ** P &lt; 0.01)

  Thereby, it turned out that a leaf part has a strong growth inhibitory action (antibacterial activity) with respect to Staphylococcus aureus and Escherichia coli compared with a root part and a stem part.

[Melanin biosynthesis inhibition test]
When the skin is exposed to ultraviolet rays, melanin is made from the melanocytes in the basal layer of the skin, causing stains. Since melanin is produced by melanoma cells, if melanin biosynthesis of melanoma cells can be inhibited, the occurrence of spots can be prevented.

<Test method>
A 50% ethanol extract of each part of leaf wasabi was dissolved in DMSO, and three types of samples having a concentration of 80 mg / mL, a concentration of 40 mg / mL, and a concentration of 20 mg / mL were prepared for each part of leaf, stem and root. I made 9 kinds in total.

1 mL of B16 melanoma cells at a concentration of 1.0 × 10 ⁵ cells / mL were inoculated into each well of the well plate and statically cultured for 24 hours in a 5% CO ₂ incubator set at 37 ° C.

  Here, a part of the cultured melanoma cells was used for a test for measuring melanin production, and the rest was used for a test for measuring cell viability. Each will be described below.

[Measurement of melanin biosynthesis]
EMEM medium (Eagle'S minimal essential medium Eagle's minimum essential medium) is removed from the well plate, and an aqueous NaOH solution is added. This is because melanin is eluted by utilizing the property that the aqueous NaOH solution is strongly alkaline.

<Cell reductivity measurement test>
Remove EMEM medium from the well plate and add MTT-PBS solution. When MTT-PBS solution is taken up into living cells, it is reduced and colours and precipitates. Using this property, the reducing power of living cells is measured.

  Moreover, since the reducing power of a cell is proportional to the number of cells, the survival rate of the cell can be measured by measuring the reducing power of the cell. In addition, since hydrochloric acid can be added to enhance the color development, it is easier to measure.

<Test results>
The sample subjected to the above test was measured with a plate reader. The result is shown in FIG.
In addition, the white bar graph of FIG. 3 shows cell viability, and a black bar graph shows melanin content.

  From FIG. 3, it was found that a sample having a concentration of 160 μg / ml in the root part contained a component that suppressed melanin biosynthesis to about 50% while maintaining a cell viability of about 80%.

  Next, melanin biosynthesis is suppressed to about 65% while maintaining the cell viability (about 80%) in the root part of the sample with a concentration of 40 μg / ml and the root part of the sample with a concentration of 160 μg / ml. It was found that the ingredients were included.

  In the sample of the leaf portion having a concentration of 160 μg / ml and a concentration of 80 μg / ml, the cell viability is as extremely low as about 30%, so that it is presumed that a cytotoxic component is contained.

  In addition, the stem portion has a high cell survival rate of about 90% at all concentrations, but the melanin content is also high at 85 to 110%, so that it does not contain a component that inhibits melanin biosynthesis of melanoma cells. Conceivable.

  In addition, in the samples with root concentration of 80 μg / ml and 40 μg / ml, the cell viability is as high as 90 to 95%, but the melanin content is as high as 80 to 90%. It is thought that the component which inhibits is not contained.

  As described above, it was confirmed that the root portion having a concentration of 160 μg / ml and the leaf portion having a concentration of 40 μg / ml contained a component that effectively inhibits melanin biosynthesis without damaging the cells.

<Lipase inhibitory activity test>
It is well known that lipase is an enzyme that breaks down fat (triglyceride). When the lipase present in human skin breaks down the fat content of the skin, fatty acids are produced, causing pore clogging and acne.

  Once acne occurs, acne bacteria release lipase, decompose triglycerides and release fatty acids. For this reason, the environment where acne bacteria are easy to propagate is made, and the skin becomes rough.

  Therefore, if the lipase can inhibit the function of decomposing fat, the growth of acne bacteria can be suppressed, and the skin condition is adjusted.

<Test method>
When triglyceride reacts with pancreatic lipase solution and separates at the ester bond, a fluorescent substance is generated. This fluorescent material is excited when irradiated with light having a wavelength of 355 nm, and emits light having a fluorescent color of 460 nm. By measuring the fluorescence intensity of this light, the influence on the lipase activity is evaluated.

To 25 μL of an extract solution of leaf wasabi leaves, 50 μL of 0.1 mM 4-methylbelliferyl oleate (4-methylumbelliferyl oleate; 4-MUO 13 mM Tris-HCl, 150 mM NaCl, 1.3 mM CaCl ₂ buffer) 150 μg / mL pancreatic lipase 25 μL.

  Then, it left still for about 30 minutes in the incubator set to 25 degreeC.

<Test results>
FIG. 4 shows the results of measurement for three types of concentrations of 0.6 mg / ml, 1.2 mg / ml and 2.4 mg / ml for each part of the leaf, stem and root. The unit of orlistat concentration used as a positive control is μg / ml.

  From FIG. 4, the lipase activity showed the lowest value (about 10%) in the sample with a leaf portion concentration of 2.4 mg / ml. In addition, the lipase activity was as low as about 35% even in the sample having a leaf concentration of 1.2 mg / ml.

  In particular, even the 0.6 mg / ml sample that showed the highest lipase activity among the leaf samples showed comparable or slightly stronger lipase inhibitory activity than the root and stem samples. Compared with the root part and stem part, very remarkable results were obtained.

  From the above, it can be considered that the leaf part contains many components that inhibit lipase activity.

<Anti-allergy and anti-inflammatory test>
It is already known that allergic reactions are caused when calcium enters cells and the concentration of calcium in the body increases.

  A23187 (calcium ionophore) is a reagent for increasing the permeability of calcium. When A23187 is added to RBL-2H3 cells, calcium contained in the medium enters RBL-2H3 cells, an enzyme called β-hexosaminidase is released (allergic reaction), and so-called degranulation occurs.

  If there is a substance that can suppress the release of β-hexosaminidase, it can be used as a countermeasure against hay fever and allergy because it has an antiallergic action.

<Test method>
Various types of allergic reactions are currently identified. Therefore, in this study, we tested the main mechanism of allergic reaction, the release rate of β-hexosaminidase by A23187, and the release rate of β-hexosaminidase by DNP-BSA and anti-DNP IgE. went.

  Β-hexosaminidase activity in granules contained in RBL-2H3 cells released by IgE stimulation from RBL-2H3 cells (rat mast cell-like cell line) was measured, and the inhibitory action was examined.

  RBL-2H3 cells were dispensed into a well plate and immersed in a leaf wasabi sample for 30 minutes, then A23187 was added, and further immersed for 30 minutes. Then, a substrate (p-nitrophenyl-n-acetyl-β-D-glucosamine p-nitrophenyl-N-acetyl-β-D-glucosaminide) was added in an amount of 50 μ ?, incubated at room temperature for 1 hour, and then cultured at 405 nm. The wavelength was applied and the absorbance was measured.

  Anti-DNP IgE was dispensed into the well plate and immersed in a leaf wasabi sample for 30 minutes, and then DNP-BSA was added and immersed for 30 minutes. Then, 50 μL of a substrate (p-nitrophenyl-n-acetyl-β-D-glucosamine) was added and incubated at room temperature for 1 hour, after which the wavelength of 405 nm was applied and the absorbance was measured.

<Test results>
The results of the above test are shown in FIGS. 5A to 5C. FIG. 5A is a view showing the amount of β-hexosaminidase released by adding A23187 to leaf wasabi extract. FIG. 5B is a diagram showing the amount of β-hexosaminidase released by adding DNP as an antigen to a leaf wasabi extract. FIG. 5C is a diagram showing the results of measuring cytotoxicity by the MTT test.

  The negative control is a result using a sample to which A23187 is not added, and the positive control is a result using a sample to which A23187 is added.

  In addition, for comparison of test results, data on Gallic acid (gallic acid) and Quercetin (quercetin), which are pharmaceuticals having anti-inflammatory action, were posted.

From FIG. 5A and FIG. 5B, when the leaf part sample was used, the strongest suppression with respect to (beta) hexosaminidase activity was seen rather than the root part and the stem part.
In addition, as shown to FIG. 5C, it adds that the cytotoxicity of the extract of a leaf wasabi was not confirmed.

  In the above-mentioned antioxidant test, antibacterial test, melanin biosynthesis inhibitory activity test, lipase inhibitory activity test, antiallergy and anti-inflammatory test, the extract of the leaf part should show the most remarkable value. I understood.

  Accordingly, the applicant focused on the leaf portion of the leaf wasabi and conducted the following test using samples extracted by changing the ethanol concentration in order to examine the function of the leaf portion in more detail.

[Extract Extraction Method and Sample]
About 2 g of lyophilized leaf wasabi was weighed, ethanol 0 vol% (water 100 vol%), ethanol 25 vol% (water 75 vol%), ethanol 50 vol% (water 50 vol%), ethanol 75 vol% (water 25 vol%), ethanol 100 vol. Extraction was carried out for each of the five types of extraction solvents of% (water 0 vol%).

A specific extraction method is described below.
After performing osmotic extraction at a rotational speed of 180 rpm for 48 hours, the extract was collected by suction filtration. Furthermore, after concentrating the extract with a rotary evaporator, ethanol was completely removed in a desiccator to obtain a dried product. In the case of extraction with 0 vol% ethanol (100 vol% water), the extraction was followed by centrifugation, and the supernatant was freeze-dried to obtain a water extract.

[Hyaluronic acid production test]
Fibroblasts are known to produce dermal parts such as hyaluronic acid. When the function of neuroblasts is reduced, hyaluronic acid is lost, the amount of water held by the skin is reduced, and wrinkles are caused.

  If there is a substance that activates the function of neuroblasts and enhances the ability to produce hyaluronic acid, it can keep the skin moisturized and firm.

<Test method>
Each of the extracts obtained at the above five ethanol concentrations was further adjusted to three concentrations to obtain a total of 15 samples.

After inoculating neuroblasts into a well plate and incubating under conditions of 5% CO ₂ and 37 ° C. for 24 hours, after adding the above sample and further incubating for 72 hours, the amount of hyaluronic acid was determined by a method called ELISA. Measured with Cell viability was determined by the MTT method.

<Test results>
The results of the above test are shown in FIG. Black bar graphs indicate hyaluronic acid production, and white bar graphs indicate cell viability. The line graph shows the amount of hyaluronic acid per living cell.

  From FIG. 6, the sample with the highest amount of hyaluronic acid per cell viability was the ethanol 75% extract (concentration 12.5 μg / ml), which was about 135% when the control (DMSO) was 100%. .

  The sample with the second highest amount of hyaluronic acid per cell viability was a sample of ethanol 25% extract (concentration 25 μg / ml), which was about 128% when the control (DMSO) was 100%.

  According to this test, it was found that the leaf portion of leaf wasabi was extracted with an ethanol concentration of 75% and adjusted to a concentration of 12.5 μg / ml to exhibit the highest hyaluronic acid producing ability.

[Antimicrobial activity test]
As was done in the functional test of each part of the leaf wasabi, the antibacterial activity of the sample of each extract concentration was also determined in this test because of its influence on the growth of E. coli and S. aureus. Activity was evaluated.

<Test method>
Each ethanol concentration extract was dissolved in DMSO (dimethylsulfoxide). Further, DMSO containing no extract was used as a negative control, and sorbic acid as a preservative was used as a positive control.

  Staphylococcus aureus and Escherichia coli were immersed in 5 mL of NB (Nutrient broth) medium to prepare a bacterial solution, which was subjected to osmotic culture at 160 rpm and 37 ° C. for about 18 hours to obtain stationary cells.

The obtained stationary-phase culture solution was diluted with NB medium to prepare OD ₆₆₀ = 0.4 (E. coli: 10 ⁹ CFU / mL, S. aureus: 10 ⁸ CFU / mL). Further, this was diluted with sterilized water and used as a bacterial solution having a bacterial concentration of 10 ⁵ CFU / mL for the antibacterial test.

  Further, 445 μ４ of NB medium and 5 μ? Of leaf wasabi sample were added to 50 μ? Of this bacterial solution and stirred for about 5 seconds. In this test, the sample concentration is 800 μg / ml.

  Thereafter, 150 μ? Each was dispensed to the well plate and cultured with shaking at 37 ° C. for 18 hours at a rotation speed of 1150 rpm. Thereafter, light having a wavelength of 630 nm was applied to measure the turbidity of the bacterial solution.

<Test results>
FIG. 7 shows the results of measuring the turbidity due to E. coli and Staphylococcus aureus using samples for each extraction concentration. The black bar graph is the turbidity of Escherichia coli, and the white bar graph is the turbidity of Staphylococcus aureus.

  As shown in FIG. 7, it was found that a sample of ethanol 75 vol% (water 25 vol%) had the lowest turbidity and strong antibacterial activity against E. coli and Staphylococcus aureus. In particular, the antibacterial activity against Staphylococcus aureus is stronger than sorbic acid.

  In addition, the antibacterial activity against E. coli was also about one-third to about one-half that of a sample having an ethanol concentration of 50 vol% or less, indicating that a remarkable antibacterial action was exhibited.

  It was also found that the sample extracted with 100 vol% ethanol (water 0 vol%) also has low turbidity and exhibits strong antibacterial activity. In particular, the antibacterial activity against Escherichia coli is stronger than that of ethanol 75 vol% (water 25 vol%).

  From the above test results, when the leaf wasabi leaf part was extracted at a concentration between 75% ethanol (25% water) and 100 vol% ethanol (0 vol% water), strong antibacterial activity against S. aureus and E. coli. It was found to have activity.

[Lipase inhibitory activity test]
In this test, in order to efficiently use the leaf portion of leaf wasabi, each of the extracts obtained at the above five ethanol concentrations was further adjusted to two concentrations (400 mg / ml and 800 mg / ml). A total of 10 samples were obtained.
<Test method>
25 μL of the extract solution was mixed with 50 μL of 0.1 mM 4-methylbelliferate oleate (4-methylumbelliferyl oleate; 4-MUO 13 mM Tris-HCl, 150 mM NaCl, 1.3 mM CaCl ₂ buffer solution) and 150 μg / mL pancreas 25 μL of the derived lipase was mixed and dissolved in a buffer solution.

  Then, it incubated for about 30 minutes on 25 degreeC conditions. Then, 100 μ て of 0.1 M citrate buffer (pH 4.2) was added to stop the reaction, and light at 355 nm was applied to measure fluorescence intensity at 460 nm.

<Test results>
The test results are shown in FIG. In addition, the result of having performed the test without including the leaf wasabi leaf extract was displayed as control (BL) 100%, and the value of Orleystat (Orl) used as an anti-obesity drug was also displayed.

  From FIG. 8, it was found that the higher the ethanol concentration of the extraction solvent, the stronger the lipase inhibitory activity. In particular, a sample with a concentration of 800 mg / ml of ethanol 100 vol% (water 0 vol%) has the highest lipase inhibitory activity, which is about 1/5 of the control value.

  From the above test results, it was found that higher lipase inhibitory activity can be obtained by extracting leaf wasabi leaves with higher ethanol concentration.

[Antioxidant activity test]
<Test method>
A peroxy radical is generated using AAPH (2,2′-azobis (2-aminopropane) dihydrochloride) which is a radical generator. When fluorescein, a kind of fluorescent dye, is oxidized by peroxy radicals, the light emitted from fluorescein is quenched.

  Here, when an antioxidant is present, the peroxy radical is lost, and the quenching speed of fluorescein oxidized by the peroxy radical is decreased. In this method, the degree of the quenching speed delay is correlated with the antioxidant activity of the sample.

<Preparation of sample solution>
The sample was weighed and dissolved in a phosphate buffer so as to have a concentration of (1 mg / 1 mL), and this was diluted as a stock solution.

<Test results>
The test results are shown in FIG. A sample extracted with an ethanol concentration of 50 vol% (water 50 vol%) had the highest antioxidant activity. The sample extracted with an ethanol concentration of 75 vol% (water 25 vol%) had the second highest antioxidant activity.

  From the above test results, the extract containing antioxidant activity can be extracted by extracting leaf wasabi leaves at a concentration between ethanol concentration 50 vol% (water 50 vol%) and 75 vol% (water 25 vol%). can do.

<Summary>
From all the above test results, the leaf wasabi leaf part was extracted with an ethanol concentration of about 75 vol% (water 25 vol%) of the extraction solvent, so that hyaluronic acid producing ability, antibacterial activity, lipase inhibitory activity, antioxidant activity were obtained. It became clear that functions could be extracted most efficiently.

  By extracting the leaf wasabi extract under the above conditions, an extract containing a plurality of components such as hyaluronic acid producing ability, antibacterial activity, lipase inhibitory activity, and antioxidant activity can be extracted.

  Further, if an extract containing a plurality of components such as hyaluronic acid producing ability, antibacterial activity, lipase inhibitory activity, and antioxidant activity is used as a cosmetic component, skin tone can be effectively adjusted.

  In addition, ingredients such as hyaluronic acid producing ability, antibacterial activity, lipase inhibitory activity, and antioxidant activity are components contained in plants called leaf wasabi, so they are safer than cosmetics containing synthetic chemicals. Can be used with confidence.

  In addition, with such an extraction method, there is no literature that details the function of the leaf portion of the leaf wasabi, and it is possible to efficiently use the function of the leaf portion of the leaf wasabi by changing the extraction solvent. Was not.

  Therefore, it is considered impossible to predict the use of such leaf wasabi extract as a skin conditioner for cosmetics and the like in the industry dealing with cosmetics and the like.

Claims (12)

A horseradish extract having a moisturizing effect extracted from the leaf portion of horseradish using ethanol or an ethanol-containing solvent. The wasabi extract having a moisturizing effect according to claim 1, wherein the extract was extracted at an ethanol concentration of 20 vol% to 30 vol%. The horseradish extract having a moisturizing effect according to claim 1, which is extracted at an ethanol concentration of a value between 70 vol% and 80 vol%. Wasabi extract having antibacterial activity extracted from wasabi leaves using ethanol or ethanol-containing solvent. The wasabi extract having antibacterial activity according to claim 4, which is extracted at an ethanol concentration of 70 vol% or more. Wasabi extract with antioxidant activity extracted from wasabi leaves using ethanol or ethanol-containing solvent. The horseradish extract having antioxidant activity according to claim 6, which is extracted at an ethanol concentration of a value between 40 vol% and 80 vol%. A horseradish extract having lipase inhibitory activity extracted from the leaf portion of horseradish using ethanol or an ethanol-containing solvent. The horseradish extract having a lipase inhibitory activity according to claim 8, which is extracted at an ethanol concentration of 90 vol% to 100 vol%. The wasabi extract according to claim 1, 4, 6, or 8, which has two or more functions among antibacterial activity, antioxidant activity, lipase inhibitory activity and moisturizing effect. A skin conditioner having a wasabi extract containing at least one function among antibacterial activity, antioxidant activity, lipase inhibitory activity and moisturizing effect, extracted from a leaf portion of wasabi using ethanol or an ethanol-containing solvent. A method for producing a wasabi extract comprising a step of extracting a leaf portion of wasabi from a powdered product using ethanol or an ethanol-containing solvent.

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