JP2024066503A - Antioxidant composition containing kabuchi peel extract and method for producing same - Google Patents

Abstract

[Problem] To propose effective functionality and uses of kabuchi peel. [Solution] To provide an antioxidant composition containing polymethoxyflavonoids, which are kabuchi peel extracts, as active ingredients. The kabuchi peel extract contains, as polymethoxyflavonoids, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, tangeretin, and pharma- ceutically acceptable salts thereof. The antioxidant composition can be used as an anti-aging agent, active oxygen inhibitor, radical scavenger, or elastase activity inhibitor. [Selected Figure] Figure 1

Description

The present invention relates to an antioxidant composition using ingredients contained in the pericarp of kabuchii and a method for producing the same. Such an antioxidant composition can be incorporated into, for example, cosmetics and quasi-drugs.

It has long been known that ingredients extracted from the fruit peel of the genus Citrus in the family Rutaceae or from dried orange peel have beneficial functions for the human body.

For example, Patent Document 1 discloses a lipolysis promoter consisting of the peel or leaves of a citrus plant or an extract thereof. Patent Document 1 shows that an extract of the peel of a citrus plant has a clear lipolysis promoting effect in adipose tissue and is highly effective in suppressing, preventing, and improving obesity. Furthermore, the citrus plants whose effectiveness is shown in Patent Document 1 are Citrus tangerina Tanaka, Citrus aurantium L. var daidai Makino, Evodia rutaecarpa Hook. fil et Thoms., and Citrus Junos Tanaka.

Patent Document 2 discloses a composition for inhibiting the production of fatty acid-binding protein (FABP) 5, which contains nobiletin contained in citrus peel extract as an active ingredient. FABP5 causes atherosclerosis and diabetes, and worsens skin conditions such as reduced barrier function and inflammation, and Patent Document 2 shows that citrus peel extract has the effect of inhibiting these. Patent Document 2 also shows the effectiveness of Tachibana peel extract as an extract containing nobiletin.

JP 2002-326947 A JP 2019-156743 A

By the way, one plant in the citrus genus of the Rutaceae family is the kabuchii (Citrus keraji var.kabuchii hort.ex Tanaka). Kabuchii is an endemic species native to Okinawa Prefecture, and is characterized by a thick skin but a small edible portion of the flesh. In addition, the skin of kabuchii is floating, making it relatively easy to peel. Thus, although a relatively large amount of skin can be obtained from kabuchii, most of it is discarded or used as fertilizer, and no effective method of utilizing it has been found at present.

As mentioned above, extracts from the peel of the genus Citrus in the Rutaceae family or from dried orange peel are known to have beneficial functions. However, there is no guarantee of the functionality, safety, or stability of the components derived from Kabuchi peel, and there is an issue in that their effective uses have not been clarified.

Therefore, the main objective of the present invention is to propose effective functionality and uses of kabuchi peel.

After extensive research into solutions to the above problems, the inventors of the present invention discovered that although kabuchi peel has a different component composition from other citrus fruits, it does contain a large amount of polymethoxyflavonoids. After examining the functionality, safety, and stability of kabuchi peel extract, they discovered that kabuchi peel extract primarily has antioxidant properties and is sufficiently safe and stable to be incorporated into cosmetics and quasi-drugs.

More specifically, the first aspect of the present invention relates to an antioxidant composition. The antioxidant composition of the present invention contains polymethoxyflavonoid, which is an extract from the peel of kabuchi, as an active ingredient.

In the antioxidant composition according to the present invention, the kabuchi peel extract preferably contains, as polymethoxyflavonoids, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin, or pharma- ceutically acceptable salts thereof. In particular, the kabuchi peel extract preferably contains, as polymethoxyflavonoids, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, tangeretin, and pharma-ceutically acceptable salts thereof, in a total amount of 0.01 wt% (weight %) or more and 1 wt% (weight %) or less. The remainder may be water, alcohol, or other components commonly used in cosmetics or quasi-drugs.

The antioxidant composition of the present invention can be used as an anti-aging agent.

The antioxidant composition of the present invention can be used as an active oxygen inhibitor (SOD-like agent), a radical scavenger, or an elastase activity inhibitor.

The antioxidant composition of the present invention preferably contains ethanol or butylene glycol as a solvent.

The second aspect of the present invention relates to a method for producing an antioxidant composition. The production method according to the second aspect of the present invention is basically a method for producing the antioxidant composition according to the first aspect described above, i.e., an antioxidant composition having polymethoxyflavonoid as an active ingredient, which is an extract of kabuchi peel. The production method according to the present invention includes an extraction step of extracting kabuchi peel with a solution containing ethanol or butylene glycol.

The manufacturing method according to the present invention preferably further includes a drying step and a grinding step. The drying step is a step of drying the kabuchi peel. The grinding step is a step of grinding the dried kabuchi peel. In this case, the extraction step is carried out after the grinding step.

It is preferable that the manufacturing method according to the present invention further includes a filtration step. The filtration step is a step of filtering the extracted component obtained in the extraction step at least twice using filter paper or a filtration filter having a mesh size of 0.05 μm or more and 0.22 μm or less.

The present invention provides effective functionality and uses for kabuchi peels, which have traditionally been discarded.

FIG. 1 is a flow chart showing one embodiment of the method for producing Kabuchi Citrus Peel extract according to the present invention. FIG. 2 shows the measurement results of the extraction rate and polymethoxyflavonoid content of Kabuchi Citrus Fruit Peel Extract. FIG. 3 shows the results of measuring the dependence of the extraction rate on the solvent concentration in the extraction of Kabuchii Citrus Fruit Peel. FIG. 4 shows the measurement results of the solvent concentration dependence of the polymethoxyflavonoid content in Kabuchi Citrus unshiu peel extract. FIG. 5 shows the results of measuring the solvent concentration dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 6 shows the results of measuring the solvent concentration dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 7 shows the measurement results of the solvent concentration dependence of the SOD inhibitory activity of Kabuchi Citrus unshiu peel extract. FIG. 8 shows the measurement results of the solvent concentration dependence of the SOD inhibitory activity of Kabuchi Citrus unshiu peel extract. FIG. 9 shows the measurement results of the solvent concentration dependency of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 10 shows the measurement results of the solvent concentration dependency of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 11 shows the results of measuring the temperature dependence of the extraction rate in the extraction of Kabuchii Citrus Fruit Peel. FIG. 12 shows the results of measuring the temperature dependence of the polymethoxyflavonoid content in Kabuchi Citrus unshiu peel extract. FIG. 13 shows the results of measuring the temperature dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 14 shows the results of measuring the temperature dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 15 shows the results of measuring the temperature dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 16 shows the results of measuring the temperature dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 17 shows the results of measuring the temperature dependence of the SOD inhibitory activity of Kabuchi Citrus unshiu peel extract. FIG. 18 shows the results of measuring the temperature dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 19 shows the results of measuring the temperature dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 20 shows the results of measuring the temperature dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 21 shows the results of measuring the temperature dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 22 shows the results of measuring the temperature dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 23 shows the results of measuring the temperature dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 24 shows the results of measuring the temperature dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 25 shows the measurement results of the time dependence of the extraction rate in the extraction of Kabuchii dried citrus fruit peel. FIG. 26 shows the measurement results of the polymethoxyflavonoid content in Kabuchi Citrus Fruit Peel extract. FIG. 27 shows the results of measuring the time dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 28 shows the results of measuring the time dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 29 shows the results of measuring the time dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 30 shows the results of measuring the time dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 31 shows the results of measuring the time dependence of the ABTS radical scavenging activity of Kabuchi Citrus Fruit Peel extract. FIG. 32 shows the results of measuring the time dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 33 shows the results of measuring the time dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 34 shows the results of measuring the time dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 35 shows the results of measuring the time dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 36 shows the results of measuring the time dependence of the SOD inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 37 shows the results of measuring the time dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 38 shows the results of measuring the time dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 39 shows the results of measuring the time dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 40 shows the results of measuring the time dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. FIG. 41 shows the results of measuring the time dependence of the elastase inhibitory activity of Kabuchi Citrus Fruit Peel extract. Figure 42 shows the polymethoxyflavonoids (PMFs) content of Kabuchi Citrus Fruit Peel extract, and the advantages of the solvent concentration dependency, temperature dependency, and time dependency of various functionalities. FIG. 43 shows the results of measuring the SOD inhibitory activity, ABTS radical scavenging activity, and elastase inhibitory activity of standard polymethoxyflavonoids. FIG. 44 shows the results of a safety test on Kabuchi Citrus Fruit Peel Extract.

Below, the embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and includes appropriate modifications of the embodiments described below within the scope obvious to those skilled in the art.

In this specification, the numerical range "A to B" means "A or more and B or less."

[1. Antioxidant Composition]
The present invention relates to an antioxidant composition. The antioxidant composition is a composition containing a substance having antioxidant activity. The antioxidant composition according to the present invention contains polysaccharides that are an extract from the rind of Kabuchi. It contains methoxyflavonoids as active ingredients. Polymethoxyflavonoids extracted from the kabuchi peel mainly contain the following components:
(A) sinensetin (or a pharma- ceutically acceptable salt thereof)
(B) isosinensetin (or a pharma- ceutically acceptable salt thereof)
(C) Nobiletin (or a pharma- ceutically acceptable salt thereof)
(D) heptamethoxyflavone (or a pharma- ceutically acceptable salt thereof)
(E) natudaidine (or a pharma- ceutically acceptable salt thereof)
(F) tangeretin (or a pharma- ceutically acceptable salt thereof)
The antioxidant composition according to the present invention may contain the above-mentioned components (A) to (F) and their pharma- ceutically acceptable salts in an amount of 0.01 to 1 wt %. It is possible to add other ingredients to the product as long as the antioxidant effect is not lost. One of the characteristics of Kabuchi peel is that it contains all of these ingredients in a well-balanced manner.

The above components (A) to (F) are all types of citrus flavonoids and can be extracted or isolated from the peel of kabuchi. When kabuchi peel is extracted using ethanol or butylene glycol, which can be used in cosmetics or quasi-drugs, as an extraction solvent, the contents of each component in the extract are generally in the following order:
1. Tangeretin 2. Nobiletin 3. Heptamethoxyflavone 4. Natsudaidine 5. Sinensetin 6. Iso-sinensetin The difference in the content of Natsudaidine, Sinensetin, and Iso-sinensetin in the extract is slight, and the order may be reversed. The order of Tangeretin, Nobiletin, and Heptamethoxyflavone is basically as above.

More specifically, when the total amount of the above components (A) to (F) is taken as 100%, the ratio (weight ratio) of each component is as follows:
Tangeretin: 40-60% or 50-60%
Nobiletin: 25-50% or 30-40%
Heptamethoxyflavone: 3-12% or 5-10%
Natudaidine: 0.5-8% or 1-5%
Sinensetin: 0.5-8% or 1-5%
Isothyme: 0.5-8% or 1-5%

In addition, if the content of tangeretin, which has the highest content, is taken as the standard (100%), the ratio (weight ratio) of other components to tangeretin is as follows.
Nobiletin: 55-99% or 60-75%
Heptamethoxyflavone: 8-25% or 10-20%
Natsudaidyne: 1-10% or 2-8%
Sinensetin: 1-10% or 2-8%
Isothyme: 1-10% or 2-8%

In addition to the above components (A) to (F), the kabuchi peel extract may contain pharma- ceutically acceptable salts thereof. Examples of pharma-ceutically acceptable salts include sodium salts, potassium salts, magnesium salts, calcium salts, barium salts, ammonium salts, monoethanolamine salts, diethanolamine salts, triethanolamine salts, monoisopropanolamine salts, triisopropanolamine salts, hydrochlorides, and sulfate salts.

The antioxidant composition may contain a solvent in addition to the kabuchi peel extract. Examples of the solvent are water, alcohol, and an aqueous alcohol solution. Preferred examples of the alcohol are ethanol and butylene glycol. As the butylene glycol, it is particularly preferable to use 1,3-butylene glycol. In addition, the solvent may contain a polyhydric alcohol used as a moisturizer in cosmetics and the like. Examples of polyhydric alcohols include glycerin, diglycerin, polyglycerin-3, polyglycerin-10, 1,3-butylene glycol, propylene glycol, 3-methyl-1,3-butanediol, 1,3-propanediol, 2-methyl-1,3-propanediol, trimethylolpropane, pentaerythritol, hexylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, dipropylene glycol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,2-octanediol, 1,2-decanediol, polypropylene glycol, sorbitol, maltitol, and trehalose. The solvent may be one of the above solvents alone, or
Two or more of them may be used in combination. The content of the solvent in the antioxidant composition can be appropriately adjusted, for example, to 10 to 99%, 30 to 99%, or 50 to 99%. Alternatively, the antioxidant composition may consist only of the kabuchi peel extract and the solvent, with the remainder of the kabuchi peel extract being the solvent.

The antioxidant composition can be used as an anti-aging agent. An anti-aging agent is a preparation that has the effect of preventing, inhibiting, or improving skin aging. More specifically, the antioxidant action of the antioxidant composition prevents, inhibits, or improves skin aging. For this reason, the anti-aging agent is suitably used as a cosmetic or quasi-drug for preventing skin aging.

In addition, the antioxidant composition according to the present invention has SOD activation activity, radical scavenging activity, and elastase inhibition activity, as shown in the examples described below. Therefore, the antioxidant composition can be used as an active oxygen inhibitor, a radical scavenger, and an elastase activity inhibitor. The active oxygen inhibitor has SOD (superoxide dismutase) activation activity, removes active oxygen generated on the skin or in the body, and promotes the improvement of spots and wrinkles. The radical scavenger removes free radicals with high oxidizing power generated on the skin or in the body. Examples of radicals that can be removed are the ABTS radical and the DPPH radical. The elastase activity inhibitor removes elastin decomposing enzyme (elastase) generated on the skin or in the body, and prevents aging such as wrinkles and sagging.

The antioxidant composition may use the kabuchi peel extract as is, but ingredients used in cosmetics and quasi-drugs may be added as appropriate within the range that does not impair the effects of the kabuchi peel extract. Examples of such ingredients include oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, and film-forming agents.

The antioxidant composition of the present invention can be incorporated into cosmetics or quasi-drugs, mainly for use as a skin topical agent. Examples of the dosage forms of cosmetics and quasi-drugs include lotions, creams, milky lotions, gels, aerosols, essences, packs, cleansers, bath agents, foundations, dusting powders, lipsticks, ointments, and poultices. In addition, the antioxidant composition of the present invention can also be incorporated into, for example, soaps, body soaps, facial cleansers, shampoos, rinses, treatments, and toothpastes.

[2. Method for producing antioxidant composition]
Next, a preferred embodiment of the method for producing an antioxidant composition will be described with reference to Fig. 1. As shown in Fig. 1, the method for producing an antioxidant composition according to this embodiment includes a washing and drying step (step S1), a crushing step (step S2), an extraction step (step S3), a solvent replacement step (step S4), and a solvent exchange step (step S5).
The method includes, in this order, a precipitating step (step S5), a concentration adjustment step (step S6), and a sterilizing filtration step (step S7). Each step will be described below.

The washing and drying step (step S1) is a step of washing the peel removed from the raw material kabuchi fruit and then drying it. Any method may be used for washing and drying, but it is preferable to use a method that does not deteriorate the components of the peel, for example, a method that does not apply ultrasound or heat. For example, the raw material kabuchi fruit is directly squeezed to obtain a residue, and the kabuchi peel is separated from this residue. The separated kabuchi peel is washed with water or hypochlorous acid water, and then dried at a low temperature of 30 to 50°C (preferably 40°C) under reduced pressure using, for example, a vacuum dryer, to obtain dried kabuchi peel. Alternatively, for example, the peel may be peeled from the kabuchi fruit, washed with water or hypochlorous acid water, and then placed on a colander or drying net to dry for 2 hours.
In the present specification, the dried pericarp is also referred to as "chenpi".

The crushing step (step S2) is a step of crushing the kaabuchi chin peel (dried peel). Any crushing method may be used, but it is preferable to adopt a method that can powder the peel in a short time so as not to deteriorate the peel components. For example, a known crusher used in the crushing processing of food products may be used to crush the kaabuchi chin peel. In addition, the crushed chin peel may be sieved to 1.0 mm or less. Sieving is an operation to separate the crushed chin peel particles into particles larger than the mesh opening of a mesh and particles smaller than the mesh opening. To sieve the chin peel particles to 1.0 mm or less, a mesh with a mesh opening of 1.0 mm is used to collect the chin peel particles that pass through this mesh. The collected chin peel particles have an average particle size of 1.0 mm or less. The average particle size is measured, for example, using a laser diffraction particle size distribution measuring device (SALD-2200 manufactured by Shimadzu Corporation), and the percentage is calculated by number percentage. It is preferable to crush and sift the Kabuchi tangerine peel just before (specifically, within one hour) the extraction process described below to prevent deterioration due to oxidation or light. In addition, if storage for more than one hour is required after crushing the Kabuchi tangerine peel, it is sufficient to store it in a sealed light-blocking container to prevent deterioration due to oxidation or light.

The extraction step (step S3) is a step of extracting components containing polymethoxyflavonoids from the kabuchi chinpi particles using an extraction solvent. As the extraction solvent, it is preferable to use ethanol, 1,3-butylene glycol, or an aqueous solution thereof. In particular, it is preferable to use an aqueous solution of 30 to 100 vol% (volume %) ethanol or an aqueous solution of 30 to 100 vol% 1,3-butylene glycol. As an extraction condition, for example, the kabuchi chinpi particles are immersed in an extraction solvent at 15 to 85°C, and then extracted for 2 to 24 hours under atmospheric pressure (about 1013 hPa) while gently stirring. The solution after extraction is filtered to separate the extraction residue, and an extract containing the extraction solvent and the extract from the kabuchi chinpi particles is obtained. The first filtration after the extraction step can be performed using, for example, filter paper or a filtration filter with a mesh size of 1 to 10 μm.

The solvent replacement step (step S4) is a step of replacing the extraction solvent contained in the extract obtained in the extraction step with another solvent. Specifically, first, the extract obtained in the extraction step is distilled under reduced pressure using an evaporator or the like to remove the extraction solvent from the extract. When the extraction solvent is removed from the extract in this way, the components of the Kabuchii Chimpi Extract remain. After the components of the Kabuchii Chimpi Extract are dried, the components are dissolved again in a solvent. The solvent used at this time is preferably ethanol, 1,3-butylene glycol, or an aqueous solution thereof, as with the extraction solvent described above. In particular, it is preferable to use an aqueous solution of 30 to 100 vol % ethanol or an aqueous solution of 30 to 100 vol % 1,3-butylene glycol. This results in a solvent replacement solution.

The precipitation step (step S5) is a step of removing insoluble components (sediment) from the above-mentioned solvent-replaced solution. Specifically, the solvent-replaced solution is first cooled to 1 to 10°C, more preferably 1 to 5°C, and left to stand for 6 to 10 hours to precipitate the insoluble components. Then, the kabuchi tangerine peel extract is suction-filtered through a first membrane filter to obtain a filtrate from which the insoluble components have been removed. The filtrate is again left to stand for 6 to 10 hours at 1 to 10°C, more preferably 1 to 5°C, to precipitate the insoluble components. Then, the filtrate is suction-filtered through a second membrane filter to further remove the insoluble components. It is preferable to perform the filtration process using this second membrane filter two or more times. Here, the second membrane filter has a finer mesh than the first membrane filter. For example, it is preferable that the mesh of the second membrane filter is half or less than that of the first membrane filter. Specifically, the first membrane filter may have a mesh size of, for example, 0.3 to 0.6 μm. The second membrane filter may have a mesh size of, for example, 0.15 to 0.3 μm.

The concentration adjustment step (step S6) is a step of adjusting the solid content concentration of the solution after the precipitation treatment to a predetermined amount. Specifically, the solid content concentration of the solution after the precipitation treatment is measured, and the same solvent as that used in the solvent replacement step (step S4) may be added so that the solid content concentration is a predetermined amount. For example, when a 30 vol% 1,3-butylene glycol aqueous solution is used in the solvent replacement step, the concentration of the solid content of the solvent replacement solution may be adjusted with the same aqueous solution. The solid content referred to here is polymethoxyflavonoid derived from kabuchi peel, more specifically, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin. In this way, kabuchi citrus fruit peel extract is obtained. However, when adjusting the solid content concentration of kabuchi citrus fruit peel extract, it is not necessary to use the same solvent as in the solvent replacement step, and kabuchi citrus fruit peel extract can also be adjusted with a different type of solvent. The predetermined amount of solid content is not particularly limited, but may be set to a concentration that adequately exhibits the antioxidant effect, anti-aging effect, active oxygen inhibitory effect, radical scavenging effect, and elastase activity inhibitory effect of the kabuchi peel, while ensuring stability and safety.
The upper limit of the solid content of the Kabuchi Citrus Fruit Peel extract is preferably 2 wt% or less, more preferably 1 wt% or less.

The sterile filtration process (step S7) is a process in which the kabuchi tangerine peel extract that has been through the concentration adjustment process described above is sterile filtered and filled into a sterilized container. For example, the kabuchi tangerine peel extract may be sterile filtered using a membrane filter with a mesh size of 0.22 um or less and filled into a sterilized container. The sterile filtration process is carried out in a clean environment such as a clean room or clean bench.

Next, the Kabuchii citrus fruit peel extract and its manufacturing method according to the present invention will be explained in more detail with reference to examples and comparative examples.

[Example 1]
In Example 1, the juice of kabuchi fruit was squeezed, and the peel separated from the juice residue was used as the raw material. The kabuchi peel was washed with hypochlorous acid water, and then dried at low temperature under reduced pressure at 40°C using a vacuum dryer to obtain kabuchi dried peel (Chinese citrus peel). The kabuchi dried peel was crushed with a crusher and sieved to 1.0 mm or less to obtain a powder. In order to prevent deterioration of the raw material, the crushing was performed immediately before extraction. 100 mL of 50 vol% ethanol aqueous solution was added to 10 g of kabuchi dried peel powder, and extraction was performed for 24 hours at room temperature of 20°C (liquid temperature 15°C) while stirring. After that, the mixture was suction filtered using 7 μm filter paper to separate the extract and the extraction residue. Next, the extract was evaporated to dryness at 40°C using an evaporator. Furthermore, the extract was dried under reduced pressure at 40°C overnight (12 hours) using a vacuum dryer to completely volatilize the ethanol aqueous solution, and the components of kabuchi chinpi extract were obtained. The components were redissolved by adding a 30 wt% 1,3-butylene glycol aqueous solution, and the concentration of the components was adjusted to 1.6 wt% by weight. The aqueous solution was then left to stand overnight (12 hours) at 4°C to precipitate and precipitate the insoluble matter, and then suction filtered and separated using a 0.45 μm membrane filter. The filtrate obtained here was left to stand overnight (12 hours) at 4°C, and then suction filtered using a 0.22 μm membrane filter twice. The concentration was adjusted with a 30 wt% 1,3-butylene glycol aqueous solution so that the final solid concentration was 1.0 wt%, and then sterilized and filtered in a clean bench, and filled into a sterilized container. In this way, kabuchi tangerine peel extract was produced.

[Example 2]
In Example 2, the extraction time in Example 1 was changed from 24 hours to 2 hours, and otherwise the same procedure as in Example 1 was used to produce Kabuchii Chenpi extract.

[Example 3]
In Example 3, the extraction time in Example 1 was changed from 24 hours to 4 hours, and otherwise the same procedure as in Example 1 was used to produce Kabuchii Chenpi extract.

[Example 4]
In Example 4, the extraction time in Example 1 was changed from 24 hours to 48 hours, and otherwise the same procedure as in Example 1 was used to produce Kabuchii Chenpi extract.

[Example 5]
In Example 5, the extraction temperature in Example 1 was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 1 hour, and otherwise the same procedure as in Example 1 was used to produce kabuchi tangerine peel extract.

[Example 6]
In Example 6, the extraction temperature in Example 1 was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 7]
In Example 7, the extraction temperature in Example 1 was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 4 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 8]
In Example 8, the extraction temperature in Example 1 was changed from room temperature (liquid temperature 15°C) to 80°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 9]
In Example 9, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 30 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 10]
In Example 10, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% ethanol aqueous solution, and the extraction time was changed from 24 hours to 2 hours. Otherwise, kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 11]
In Example 11, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 1 hour, and otherwise Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 12]
In Example 12, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 13]
In Example 13, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 4 hours, and otherwise Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 14]
In Example 14, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 78°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 15]
In Example 15, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% ethanol aqueous solution, and the extraction time was changed from 24 hours to 2 hours. Otherwise, kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 16]
In Example 16, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 1 hour, and otherwise Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 17]
In Example 17, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 18]
In Example 18, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 4 hours, and otherwise Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 19]
In Example 19, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% ethanol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 77°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 20]
In Example 20, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 30 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise, a kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 21]
In Example 21, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 50 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise a kabuchi citrus peel extract was produced using the same procedure as in Example 1.

[Example 22]
In Example 22, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 75 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise a kabuchi citrus peel extract was produced using the same procedure as in Example 1.

[Example 23]
In Example 23, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% 1,3-butylene glycol aqueous solution, and the extraction time was changed from 24 hours to 2 hours. Otherwise, kabuchi tangerine peel extract was produced using the same procedure as in Example 1.

[Example 24]
In Example 24, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise a kabuchi citrus peel extract was produced using the same procedure as in Example 1.

[Example 25]
In Example 25, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 80°C, and the extraction time was changed from 24 hours to 1 hour, and otherwise, a kabuchi citrus peel extract was produced using the same procedure as in Example 1.

[Example 26]
In Example 26, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 80°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise, a kabuchi citrus peel extract was produced using the same procedure as in Example 1.

[Example 27]
In Example 27, the extraction solvent in Example 1 (50 vol% ethanol aqueous solution) was changed to 100 vol% 1,3-butylene glycol aqueous solution, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 80°C, and the extraction time was changed from 24 hours to 4 hours, and otherwise, Kabuchii Chenpi extract was produced using the same procedure as in Example 1.

[Example 28]
In Example 28, the extraction solvent (50 vol% ethanol aqueous solution) used in Example 1 was changed to pure water, the extraction temperature was changed from room temperature (liquid temperature 15°C) to 50°C, and the extraction time was changed from 24 hours to 2 hours, and otherwise Kabuchi Chenpi extract was produced using the same procedure as in Example 1.

[Example 29]
In Example 29, the number of times of suction filtration using a 0.22 μm membrane filter in Example 1 was changed from two to one, and otherwise a Kabuchi Citrus Peel extract was produced in the same manner as in Example 1. The extraction rate and polymethoxyflavonoids content in Example 29 were the same as in Example 1.

[Comparative Example 1]
In Comparative Example 1, a commercially available Unshu mandarin extract (Maruzen Pharmaceutical Co., Ltd.: Chinpi Extract BG) was used as the Unshu mandarin peel-derived Citrus unshiu peel extract.

[Comparative Example 2]
In Comparative Example 2, a commercially available Citrus depressa extract (NOF Corporation: Citrus depressa extract BG) was used as the Citrus depressa peel-derived extract.

[Test method]
For the above-mentioned Examples and Comparative Examples, component analysis, functionality test, stability test, and safety test were carried out by the methods described below.

[1. Extraction rate]
The solid content in each extract according to Examples 1 to 28 and Comparative Examples 1 and 2 was measured by the following procedure. 2 g of the extract was placed in an evaporating dish, dried on a hot plate at 105°C, and then vacuum After drying under reduced pressure at 105°C using a dryer, the weight was measured using a precision balance. Measurements were repeated until there was no change in weight, and the solid content was determined. The extraction rate was calculated from the solid content using the following formula: did.

[2. Component analysis method]
(Quantitative analysis of polymethoxyflavonoids)
The total amount of polymethoxyflavonoids in each extract according to Examples 1 to 27 and Comparative Examples 1 and 2 was measured by high performance liquid chromatography (HPLC) under the following measurement conditions. The amounts of sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin were quantified.
<Measurement conditions>
・HPLC equipment: NexeraX2 (Shimadzu Corporation)
Column: YMC-Pack ODS-AM (Φ4.6 × 150 mm, 5 μm)
Column temperature: 30°C
・Gradient conditions:
0-10 min; MeOH:water=50:50
10-35 min; MeOH:water=50-98:50-2
35-40 min; MeOH:water=98:2
40-50 min; MeOH:water=50:50
Flow rate: 1.0 mL / min
Injection volume: 10 μL
Detection: 330 nm (other than tangeretin), 367 nm (tangeretin)

[3. Functionality Test Method]
The ABTS radical scavenging activity and SOD inhibitory activity were measured as antioxidant activities of Kabuchii Chenpi extract and polymethoxyflavonoid standards (sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin). In addition, elastase inhibitory activity was measured as anti-aging activity. The methods for measuring each functionality are as follows.

(ABTS radical scavenging activity)
The ABTS radical scavenging activity of each extract and polymethoxyflavonoid standard according to the examples and comparative examples was measured by the ABTS radical scavenging test. The sample was reacted with the ABTS radical solution (37°C, 4 min), and the absorbance (734 nm) of the solution was measured by a plate reader. The IC50 (50% inhibitory concentration, 50% inhibitory activity) was determined for the polymethoxyflavonoid standard.

(SOD Inhibitory Activity)
The SOD inhibitory activity of each extract and polymethoxyflavonoid standard in the examples and comparative examples was measured using "SODAssayKit-WST" (Dojindo Laboratories). The sample and the reagent were reacted (37°C, 20 min), and the absorbance (450 nm) of the solution was measured using a plate reader. The IC50 (50% inhibitory concentration, 50% inhibitory activity) was determined for the polymethoxyflavonoid standard.

(Elastase inhibitory activity)
The elastase inhibitory activity of each extract and polymethoxyflavonoid standard product according to the examples and comparative examples was measured by an elastase inhibition test. A crude enzyme solution of human fibroblasts was reacted with a specimen and a pseudo-elastase substrate (Glutaryl-Ala-Phe-4-methoxy-β-nephthylamide), and the fluorescence of the substrate decomposition product (4-methoxy-β-nephthylamine) was measured (excitation wavelength 340 nm, fluorescence wavelength 425 nm) to calculate the elastase inhibitory activity. The IC50 (50% inhibitory concentration, 50% inhibitory activity) was determined for the polymethoxyflavonoid standard product.

4. Stability Test Method
A stability test was carried out on the kabuchi citrus peel extracts of Example 1 and Example 29. Each kabuchi citrus peel extract was placed in a 30 mL glass container and left to stand in a room (20°C) or in a thermostatic chamber (5°C, 40°C, 50°C, cycles of -10°C to 20°C, fluorescent light irradiation at 30°C) where the temperature was kept constant, and the changes over time were visually observed for 3 months.

5. Safety Testing
In order to confirm the safety to the skin, an in vitro skin sensitization test was conducted on the Kabuchii Chimpi extracts of Example 1 (50% EtOH, RT, 24h), Example 16 (100% EtOH, 50°C, 2h) and Example 25 (100% BG, 80°C, 2h).

[Results and Discussion]
The extraction rate and the quantitative results of polymethoxyflavonoids of each extract according to Examples 1 to 28 and Comparative Examples 1 and 2 are shown in FIG. 2. In the Kabuchi Citrus Peel extract according to Examples 1 to 28, the polymethoxyflavonoids natudaidine, heptamethoxyflavone, isosinensetin, sinensetin, nobiletin, and tangeretin were confirmed, and the total amount was about 150 to 1500 mg/L. In comparison with existing products (Comparative Examples 1 and 2), the superiority of Comparative Example 1 was confirmed. On the other hand, the total amount of polymethoxyflavonoids in Comparative Example 2 was abundant at about 500 mg/L, but heptamethoxyflavone was hardly detected, suggesting the possibility that the pharmacological effect of heptamethoxyflavone is not exerted.

Using these results, the effects of solvent concentration, extraction temperature, and extraction time on the extraction rate, polymethoxyflavonoid content, and functionality (ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity) of Kabuchii Citrus Peel Extract were considered. Stability and safety tests were also conducted on the examples of the present invention.

1. Solvent concentration dependency Using the extraction rate and the quantitative results of polymethoxyflavonoids of each extract related to Examples 6, 9, 12, 17, 20, 21, 22, 24, and 28, the solvent concentration dependency of the extraction rate, the content of polymethoxyflavonoids, and functionality (ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity) was investigated.

[1. Extraction rate]
The dependence of the extraction rate on the solvent concentration in the extraction of Kabuchii Citrus Peel is shown in Figure 3. In both the 1,3-butylene glycol and ethanol extraction solvents, the extraction rate increased with the solvent concentration, but at 100 vol%, it decreased significantly.

[2. Content of polymethoxyflavonoids]
Figure 4 shows the solvent concentration dependence of the polymethoxyflavonoid content in Kabuchii Chimpi extract. In both extraction solvents, 1,3-butylene glycol and ethanol, the polymethoxyflavonoid content increased with the solvent concentration, reaching a maximum at 100 vol%. In comparison with existing products (Comparative Examples 1 and 2), both extracts were found to be superior to Comparative Example 1. On the other hand, it was found that the extract made from 100 vol% 1,3-butylene glycol and ethanol was superior to Comparative Example 2.

[3. Functionality test results]
(ABTS radical scavenging activity)
Figure 5 shows the solvent concentration dependence of ABTS radical scavenging activity in 1,3-butylene glycol extraction. The curves for the 50 vol% and 75 vol% extracts had steeper slopes than the existing products (Comparative Examples 1 and 2), confirming their superiority in ABTS radical scavenging activity.

Figure 6 shows the solvent concentration dependence of ABTS radical scavenging activity in ethanol extraction. In comparison with existing products (Comparative Examples 1 and 2), superiority was confirmed at all concentrations over Comparative Example 1. On the other hand, the curves for both extracts had similar slopes to Comparative Example 2, and it was considered that the ABTS radical scavenging activity was equivalent.

(SOD Inhibitory Activity)
Figure 7 shows the solvent concentration dependence of SOD inhibitory activity in 1,3-butylene glycol extraction. The curves of extracts with solvent concentrations of 50 to 100 vol% had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity. In particular, the extract extracted with 100 vol% 1,3-butylene glycol had excellent SOD inhibitory activity.

Figure 8 shows the solvent concentration dependence of SOD inhibitory activity in ethanol extraction. The curves for extracts with solvent concentrations of 50-100 vol% had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity. In particular, the extract extracted with 100 vol% ethanol had excellent SOD inhibitory activity.

(Elastase inhibitory activity)
Figure 9 shows the solvent concentration dependence of elastase inhibitory activity in 1,3-butylene glycol extraction. The curve for the extract with a solvent concentration of 100 vol% has a steeper slope than the existing products (Comparative Examples 1 and 2), confirming its superiority in elastase inhibitory activity.

Figure 10 shows the solvent concentration dependence of elastase inhibitory activity in ethanol extraction. Extracts with solvent concentrations of 50-100 vol% showed curves with a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity. In particular, the extract extracted with 100 vol% ethanol had excellent elastase inhibitory activity.

These results show that 100 vol% of 1,3-butylene glycol extraction had a high content of polymethoxyflavonoids and was excellent in SOD inhibitory activity and elastase inhibitory activity. Furthermore, 50-100 vol% of ethanol extraction had a high content of polymethoxyflavonoids and was excellent in SOD inhibitory activity and elastase inhibitory activity.

2. Temperature Dependence Using the extraction rate and the quantitative results of polymethoxyflavonoids for each extract in Examples 2, 6, 8, 10, 12, 14, 15, 17, 19, 23, 24, and 26, the temperature dependence of the extraction rate, the content of polymethoxyflavonoids, and functionality (ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity) was investigated.

[1. Extraction rate]
Figure 11 shows the temperature dependence of the extraction rate in the extraction of Kabuchii Chimaki peel. For both 100 vol% 1,3-butylene glycol and 100 vol% ethanol, the extraction rate increased with the extraction temperature, reaching a maximum at 80°C. For ethanol and 75 vol % ethanol, there was almost no change in the extraction rate depending on the extraction temperature.

[2. Content of polymethoxyflavonoids]
FIG. 12 shows the temperature dependence of the polymethoxyflavonoid content of Kabuchii Chimpi Extract. In both 100 vol% 1,3-butylene glycol and 100 vol% ethanol, the polymethoxyflavonoid content decreased with temperature. This is thought to be because the extraction rate increased with temperature, resulting in a decrease in the proportion of polymethoxyflavonoids per solid content. In both 50 vol% ethanol and 75 vol% ethanol, there was almost no change in the polymethoxyflavonoid content due to temperature. In comparison with existing products (Comparative Examples 1 and 2), both extracts were found to be superior to Comparative Example 1. On the other hand, in comparison with Comparative Example 2, both 100 vol% 1,3-butylene glycol and 100 vol% ethanol were found to be superior to the extract extracted at 50°C or less.

[3. Functionality test results]
(ABTS radical scavenging activity)
Figure 13 shows the temperature dependence of ABTS radical scavenging activity in 100 vol% 1,3-butylene glycol extraction. The curve of the extract extracted at room temperature had a steeper slope than the existing products (Comparative Examples 1 and 2), confirming its superiority. The curves of the other extracts had the same slope as the existing products, and it was considered that the ABTS radical scavenging activity was equivalent.

Figure 14 shows the temperature dependence of ABTS radical scavenging activity in 50 vol% ethanol extraction. Superiority was confirmed at all concentrations compared to Comparative Example 1. On the other hand, compared to Comparative Example 2, the curves for all extracts had similar slopes, and it was considered that the ABTS radical scavenging activity was equivalent.

Figure 15 shows the temperature dependence of ABTS radical scavenging activity in 75 vol% ethanol extraction. The curve for the extract extracted at 80°C had a steeper slope than the existing products (Comparative Examples 1 and 2), confirming its superiority in ABTS radical scavenging activity. The curves for the other extracts had the same slope as the existing products, and it was considered that the ABTS radical scavenging activity was equivalent.

Figure 16 shows the temperature dependence of ABTS radical scavenging activity in 100 vol% ethanol extraction. The curves for both extracts are close to those of existing products (Comparative Examples 1 and 2), and the ABTS radical scavenging activity is considered to be equivalent.

(SOD Inhibitory Activity)
Figure 17 shows the temperature dependence of SOD inhibitory activity in 100 vol% 1,3-butylene glycol extraction. The curves for both extracts had a steeper slope than the existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity. This shows that in 100 vol% 1,3-butylene glycol extraction, extracts with excellent SOD inhibitory activity can be obtained by extraction at room temperature or higher.

Figure 18 shows the temperature dependence of SOD inhibitory activity in 50 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superior SOD inhibitory activity. This shows that in 50 vol% ethanol extraction, extracts with excellent SOD inhibitory activity can be obtained by extraction at room temperature or above.

Figure 19 shows the temperature dependence of SOD inhibitory activity in 75 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superior SOD inhibitory activity. This shows that in 75 vol% ethanol extraction, extracts with excellent SOD inhibitory activity can be obtained by extraction at room temperature or above.

Figure 20 shows the temperature dependence of SOD inhibitory activity in 100 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superior SOD inhibitory activity. This shows that in 100 vol% ethanol extraction, extracts with excellent SOD inhibitory activity can be obtained by extraction at room temperature or above.

(Elastase inhibitory activity)
Figure 21 shows the temperature dependence of elastase inhibitory activity in 100 vol% 1,3-butylene glycol extraction. The curves for both extracts had steeper slopes than existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity. This shows that in 100 vol% 1,3-butylene glycol extraction, extracts with excellent elastase inhibitory activity can be obtained by extraction at room temperature or higher.

Figure 22 shows the temperature dependence of elastase inhibitory activity in 50 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity. This shows that in 50 vol% ethanol extraction, extracts with excellent elastase inhibitory activity can be obtained by extraction at room temperature or above.

Figure 23 shows the temperature dependence of elastase inhibitory activity in 75 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity. This shows that in 75 vol% ethanol extraction, extracts with excellent elastase activity can be obtained by extraction at room temperature or above.

Figure 24 shows the temperature dependence of elastase inhibitory activity in 100 vol% ethanol extraction. The curves for both extracts had a steeper slope than existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity. This shows that in 100 vol% ethanol extraction, extracts with excellent elastase inhibitory activity can be obtained by extraction at room temperature or above.

These results show that the polymethoxyflavonoid content and ABTS radical scavenging activity of both extracts were superior to those of Comparative Example 1, and that the SOD inhibitory activity and elastase inhibitory activity were superior to those of Comparative Examples 1 and 2. Furthermore, the polymethoxyflavonoid content of the 100 vol% 1,3-butylene glycol extract and the 100 vol% ethanol extract was superior to those of Comparative Examples 1 and 2.

3. Time dependency Using the extraction rate and the quantitative results of polymethoxyflavonoids of each extract according to Examples 1 to 7, 11 to 13, 16 to 18, and 25 to 27, the time dependency of the extraction rate, the content of polymethoxyflavonoids, and the functionality (ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity) was investigated.

[1. Extraction rate]
Figure 25 shows the time dependence of the extraction rate in the Kabuchi Citrus Peel extraction. 100 vol% 1,3-butylene glycol extraction (80°C), 50 vol% ethanol extraction (50°C), 75 vol% ethanol extraction (50°C), and 100 vol% In the case of ethanol extraction (50°C), the change in extraction rate due to extraction time was small in all cases. In addition, in the case of 50 vol% ethanol extraction (RT), the extraction rate hardly changed from 2 hours to 48 hours.

[2. Content of polymethoxyflavonoids]
Figure 26 shows the time dependence of polymethoxyflavonoid content in Kabuchii Chimaki Peel Extract. There was little change in polymethoxyflavonoid content with extraction time in all of the following extractions: 100 vol% 1,3-butylene glycol extraction (80°C), 50 vol% ethanol extraction (RT), 50 vol% ethanol extraction (50°C), 75 vol% ethanol extraction (50°C), and 100 vol% ethanol extraction (50°C). From this, it is believed that extraction of polymethoxyflavonoids from Kabuchii Chimaki Peel can be completed in about 1 to 2 hours.

[3. Functionality test results]
(ABTS radical scavenging activity)
27 shows the time dependence of ABTS radical scavenging activity in 100 vol% 1,3-butylene glycol extraction (80°C). The curves for both extracts had the same slope as the existing products (Comparative Examples 1 and 2), and it was considered that the ABTS radical scavenging activity was equivalent.

Figure 28 shows the time dependence of ABTS radical scavenging activity in 50 vol% ethanol extraction (R.T.). The curves for both extracts were slightly steeper than those for existing products (Comparative Examples 1 and 2), confirming the superiority of ABTS radical scavenging activity.

Figure 29 shows the time dependence of ABTS radical scavenging activity in 50 vol% ethanol extraction (50°C). The curve for the extract extracted for 1 hour has a steeper slope than the existing products (Comparative Examples 1 and 2), confirming the superiority of ABTS radical scavenging activity.

Figure 30 shows the time dependence of ABTS radical scavenging activity in 75 vol% ethanol extraction (50°C). The curve for the extract extracted for 1 hour has a steeper slope than the existing products (Comparative Examples 1 and 2), confirming the superiority of ABTS radical scavenging activity.

Figure 31 shows the time dependence of ABTS radical scavenging activity in 100 vol% ethanol extraction (50°C). The curve for the extract extracted for 1 hour has a steeper slope than the existing products (Comparative Examples 1 and 2), confirming the superiority of ABTS radical scavenging activity.

(SOD Inhibitory Activity)
32 shows the time dependence of SOD inhibitory activity in 100 vol% 1,3-butylene glycol extraction (80°C). The curves for both extracts were slightly steeper than those for the existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity.

Figure 33 shows the time dependence of SOD inhibitory activity in 50 vol% ethanol extraction (R.T.). The curves for both extracts were steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity.

Figure 34 shows the time dependence of SOD inhibitory activity in 50 vol% ethanol extraction (50°C). The curves for both extracts were slightly steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity.

Figure 35 shows the time dependence of SOD inhibitory activity in 75 vol% ethanol extraction (50°C). The curves for both extracts were slightly steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity.

Figure 36 shows the time dependence of SOD inhibitory activity in 100 vol% ethanol extraction (50°C). The curves for both extracts were steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in SOD inhibitory activity.

(Elastase inhibitory activity)
37 shows the time dependence of elastase inhibitory activity in 100 vol% 1,3-butylene glycol extraction (80°C). The curves for both extracts were slightly steeper than those for the existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity.

Figure 38 shows the time dependence of elastase inhibitory activity in 50 vol% ethanol extraction (RT). The curves for both extracts were steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity.

Figure 39 shows the time dependence of elastase inhibitory activity in 50 vol% ethanol extraction (50°C). The curves for both extracts were slightly steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity.

Figure 40 shows the time dependence of elastase inhibitory activity in 75 vol% ethanol extraction (50°C). The curves for both extracts were slightly steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity.

Figure 41 shows the time dependence of elastase inhibitory activity in 100 vol% ethanol extraction (50°C). The curves for both extracts were steeper than those for existing products (Comparative Examples 1 and 2), confirming their superiority in elastase inhibitory activity.

From the above results, 100 vol% 1,3-butylene glycol extraction (80°C) was superior to Comparative Example 2 in polymethoxyflavonoid content and ABTS radical scavenging activity when the extraction time was 1 hour or more, and superiority was confirmed over Comparative Examples 1 and 2 in SOD inhibitory activity and elastase inhibitory activity. 50 vol% ethanol extraction (room temperature) was superior to Comparative Example 1 in polymethoxyflavonoid content and ABTS radical scavenging activity when the extraction time was 2 hours or more, and superiority was confirmed over Comparative Examples 1 and 2 in SOD inhibitory activity and elastase inhibitory activity. In addition, ABTS radical scavenging activity was superior to Comparative Examples 1 and 2 when the extraction rate was 4 hours or more. 50 vol% and 75 vol% ethanol extraction (50°C) was superior to Comparative Example 1 in polymethoxyflavonoid content when the extraction time was 1 hour, and superiority was confirmed over Comparative Examples 1 and 2 in ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity. With 100 vol% ethanol extraction (50°C), the polymethoxyflavonoid content, ABTS radical scavenging activity, SOD inhibitory activity, and elastase inhibitory activity were all superior to those of Comparative Examples 1 and 2 after an extraction time of 1 hour.

Figure 42 shows the results of the polymethoxyflavonoids (PMFs) content of Kabuchii Citrus Peel Extract, and the superiority of various functional solvents in terms of concentration dependency, temperature dependency, and time dependency.

Functionality tests were performed using standard polymethoxyflavonoids sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidin, and tangeretin. The results of the functionality tests are shown in FIG. 43. The IC50 of SOD inhibitory activity was low for heptamethoxyflavone and nobiletin, and the activity was high, so these heptamethoxyflavone and nobiletin are considered to be the components that contribute to SOD inhibitory activity. Note that natudaidin and tangeretin did not dissolve in the reagent, and SOD inhibitory activity could not be measured. The IC50 of ABTS radical scavenging activity was low for natudaidin, and the activity was high, so this natudaidin is considered to be the component that contributes to ABTS radical scavenging activity. In addition, the IC50 of elastase inhibitory activity was low for sinensetin and nobiletin, and the activity was high, so these sinensetin and nobiletin are considered to be the components that contribute to elastase inhibitory activity.

From the above results, it is believed that the Kabuchi Citrus Unshiu Peel Extract has the functionality of SOD inhibitory activity, ABTS radical scavenging activity, and elastase inhibitory activity by containing all of the polymethoxyflavonoids contained in the Kabuchi Citrus Unshiu peel, such as sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin. Therefore, the Kabuchi Citrus Unshiu Peel Extract of the present invention can be used as an antioxidant composition. More specifically, the Kabuchi Citrus Unshiu Peel Extract can be used as an anti-aging agent, active oxygen inhibitor, radical scavenger, and elastase activity inhibitor.

4. Stability Test Results A stability test was carried out on the kabuchi tangerine peel extracts of Example 1 and Example 29. Example 1 was subjected to suction filtration twice using a 0.22 μm membrane filter, and Example 29 was subjected to suction filtration once using a 0.22 μm membrane filter. The rest of the procedure is the same as Example 1 and Example 13. Example 29 produced white crystalline precipitates after one week under all conditions (5° C., room temperature, 40° C., 50° C., cycles of −10° C. to 20° C., and fluorescent light irradiation at 30° C.). On the other hand, Example 1 was stable, with no precipitates like those in Example 29 even after two months. Therefore, as a means for separating and purifying the extracted components, the stability of the kabuchi tangerine peel extract can be reliably guaranteed by filtering the kabuchi extract components at least twice using a membrane filter or filter paper of 0.22 μm or less. However, since the stability of the extract can be guaranteed by other known methods, Example 29 can also be adopted as an example of the present invention.

5. Safety Test Results An in vitro skin sensitization test was carried out on the kabuchi tangerine peel extracts of Examples 1, 16, and 25. The test results are shown in FIG. 44. In Examples 1 and 16, the kabuchi tangerine peel extract was used in its original form, diluted 2-fold, and diluted 10-fold, respectively. The solid content concentrations of each were 1.0 wt% (original form), 0.5 wt% (diluted 2-fold), and 0.1 wt% (diluted 10-fold). In Example 25, the kabuchi tangerine peel extract was used with its solid content adjusted to 0.3 wt% and 0.1 wt%. The kabuchi tangerine peel extract of Example 1 was negative at all concentrations, and it was confirmed that it was a safe kabuchi tangerine peel extract. The kabuchi tangerine peel extracts of Examples 16 and 25 were negative when the solid content concentration was adjusted to 0.1 wt%, and it was confirmed that a safe kabuchi tangerine peel extract could be obtained by adjusting the concentration. Example 16 uses 100 vol% ethanol aqueous solution as the extraction solvent, and Example 25 uses 100 vol% 1,3-butylene glycol as the extraction solvent, but even with these 100% concentration solvents, safety can be ensured by adjusting the concentration, so it can be said that the safety of the other Examples can also be ensured at least by adjusting the concentration. Moreover, Example 28 uses pure water as the extraction solvent, so it can be said to be safe without even needing to conduct a test. From these results, it is recognized that Kabuchii Chenpi Extract has sufficient safety to be incorporated into cosmetics or quasi-drugs, mainly for use as a skin topical agent.

Claims (9)

An antioxidant composition containing polymethoxyflavonoid, an extract from the peel of kabuchi, as an active ingredient. The antioxidant composition according to claim 1,
The antioxidant composition, wherein the kabuchi peel extract contains sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin, or pharma- ceutically acceptable salts thereof. The antioxidant composition according to claim 1,
The antioxidant composition, wherein the kabuchi peel extract contains sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, natudaidine, and tangeretin, and pharma- ceutically acceptable salts thereof, in a total amount of 0.01 wt% or more and 1 wt% or less. The antioxidant composition according to claim 1, which is an anti-aging agent.
Antioxidant composition. The antioxidant composition according to claim 1,
An active oxygen inhibitor, a radical scavenger, or an elastase activity inhibitor.
Antioxidant composition. The antioxidant composition according to claim 1,
As a solvent, it contains ethanol or butylene glycol.
Antioxidant composition. A method for producing an antioxidant composition having polymethoxyflavonoid as an active ingredient, which is an extract from Kabuchi peel, comprising the steps of:
The method includes an extraction step of extracting the kabuchi peel with a solution containing ethanol or butylene glycol;
Method. A method for producing the antioxidant composition according to claim 7, comprising: a drying step of drying the kabuchi peel;
The method further comprises a grinding step of grinding the dried kabuchi peel, and the extracting step is carried out after the grinding step. The method for producing an antioxidant composition according to claim 7 further comprises a step of filtering the extracted component obtained by the extraction step at least twice using a filter paper or a filtration filter having a mesh size of 0.05 μm or more and 0.22 μm or less.
Method.

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