JP2007314553A - Hyaluronidase activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellently safe hyaluronidase activity inhibitor effectively utilizing acerola seeds which have hitherto been mostly subjected to waste disposal and exhibiting an excellent hyaluronidase inhibitory activity by utilization in external preparations for skin, cosmetics, etc. <P>SOLUTION: The hyaluronidase activity inhibitor comprises a solvent extract of the acerola seeds containing quercitrin and having ≤25 wt.% content of the quercitrin as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an inhibitor of hyaluronidase activity containing an extract of acerola seed as an active ingredient, and can be used for an external preparation for skin and cosmetics.

In the case of including fats and oils in articles such as foods, cosmetics and pharmaceuticals, the most serious problem in the process of storage, storage and processing is oxidation and peroxidation of fats and oils components by oxygen in the air. In particular, it is known that unsaturated fatty acids such as linoleic acid and linolenic acid contained in fats and oils are easily peroxidized by oxygen to produce lipid peroxides and free radicals, and also carcinogenic substances. ing. When such oxidation or peroxidation occurs, not only coloring, discoloration, denaturation, off-flavor, or a decrease in nutritional value effectiveness, but also the production of toxic substances, etc., leading to deterioration of product quality.
Therefore, various antioxidants have been conventionally used in order to suppress the oxidation and peroxidation of the unsaturated fatty acid and prevent the quality of the product from deteriorating. Antioxidants act on peroxide radicals generated during oxidation and stop the chain reaction of oxidation, or act on free radicals to stop the oxidation reaction. Conventionally, synthetic antioxidants such as butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) are generally used as the antioxidant. However, in recent years, as the use of synthetic antioxidants has increased, its safety has become a problem, and consumer rejection has become stronger and the amount used has also decreased. Moreover, since these synthetic antioxidants are oil-soluble, they are difficult to use in aqueous solutions.
On the other hand, natural vitamin E (α-tocopherol), vitamin C and the like are known as highly safe antioxidants derived from natural products. However, since these natural-derived antioxidants have extremely fat-soluble or water-soluble properties, their use is limited. In addition, there is a disadvantage that the activity does not last stably for a long time.
Therefore, there is a strong demand for antioxidants derived from natural products that have strong antioxidant activity, are highly soluble in water, and are stable for a long time.
Further, collagen, hyaluronic acid, and the like are known as substances that affect skin moisture retention, flexibility, and elasticity. Collagen accounts for 90% of the dermis in the skin and is distributed throughout the dermis, allowing the skin to retain moderate elasticity and strength. Hyaluronic acid is widely distributed in living bodies such as skin, joint fluid, vitreous, and ligaments. In the skin, cell adhesion, cell protection, skin tissue formation, tissue moisture retention, flexibility maintenance, etc. Is responsible. Collagenase is known as an enzyme that degrades collagen in the living body, and hyaluronidase is known as an enzyme that degrades hyaluronic acid. However, when collagen and hyaluronic acid are degraded by these substances and the amount thereof is reduced, the skin becomes moisturized and firm. It is said that wrinkles and sagging are occurring.
Therefore, it has been proposed to add substances that inhibit the activity of these enzymes to prevent skin aging and wrinkle prevention, etc. in skin preparations and various cosmetics. Conventionally, various collagenase activity inhibitors, Hyaluronidase activity inhibitors have been developed.

By the way, acerola fruit has recently become known as a plant containing abundant vitamin C, and is now used as a drink or health food in various countries around the world. In addition, acerola fruit containing abundant vitamin C has come to be used in cosmetics and the like in anticipation of the antioxidant effect of vitamin C contained in the extract (see, for example, Patent Documents 1 to 4). ).
However, the fruits of acerola are used for cosmetics and foodstuffs only in the pulp containing a lot of vitamin C, and the seeds have hardly been found for effective use, and most of them are discarded. This is the current situation. Recently, a composition has been proposed in which steam distilled water obtained by treating plant seeds including acerola seeds by a steam distillation method is blended in cosmetics with the expectation of an effect of improving skin feel (for example, Patent Document 5). However, there is a need for further effective use.
In addition, such acerola seeds are hardly known in terms of their components and their action.
Patent No. 2814094 specification JP 2000-212026 A JP 2000-212027 A JP 2000-212032 A JP 2001-226218 A

  The object of the present invention is to enable effective use of acerola seeds, most of which have been disposed of in the past, and is excellent in safety and exhibits an excellent hyaluronidase activity inhibitory action when used in skin external preparations and various cosmetics. It is to provide a hyaluronidase activity inhibitor.

In order to solve the above-mentioned problems, the present inventors first studied diligently about the usefulness of acerola seeds that have been conventionally discarded after pressing fruit juice. As a result, the inventors have found that an extract obtained from acerola seeds has strong antioxidant ability, collagenase activity inhibition ability and hyaluronidase activity inhibition ability, and has completed the present invention.
That is, according to the present invention, there is provided a hyaluronidase activity inhibitor comprising, as an active ingredient, an acerola seed extract containing quercitrin and having a quercitrin content of 25% by weight or less as an active ingredient. Provided.

  The hyaluronidase activity inhibitor of the present invention uses an extract of acerola seeds as an active ingredient, is excellent in safety, and has a strong hyaluronidase inhibitory activity. The hyaluronidase activity inhibitor of the present invention can be used in an external preparation for skin and cosmetics to obtain an anti-aging action and an anti-wrinkle action. In addition, it is possible to effectively use the acerola seed that was an industrial waste.

Hereinafter, the present invention will be described in detail.
The hyaluronidase activity inhibitor of the present invention contains a solvent extract of acerola seed as an active ingredient.
Acerola seeds are seeds of Acerola (scientific name: Malpighia emarginata DC) native to the Caribbean West Indies of the Atlantic Ocean.
Acerola pulp is known to be rich in vitamin C and is used in foods, cosmetics, etc., but its seeds have hardly been found for effective use.

  The solvent extract of acerola seed is not particularly limited as long as it is extracted using an extraction solvent, for example, and has an antioxidant function such as a linoleic acid autooxidation inhibiting ability and a DPPH radical scavenging action. The extraction method and conditions are not particularly limited. Moreover, there is no restriction | limiting about the production place and kind of acerola seed, for example, Okinawa and Brazil are mentioned as a production place.

  The solvent extract of acerola seed is, for example, an extract obtained by processing raw acerola seed, its dried product or frozen product by pulverization, etc., and adding water and / or an organic solvent, and the obtained extract A concentrate obtained by concentrating the above-mentioned extract, a fraction obtained by further fractionating the extract or the concentrate by liquid-liquid extraction or column chromatography, etc., and a general term for these dried solids, containing quercitrin and containing quercitrin It means that the amount is 25% by weight or less, and its form includes any of liquid, paste, and solid.

  The organic solvent used for obtaining the extract may be either a hydrophilic organic solvent or a hydrophobic organic solvent. Examples of the hydrophilic organic solvent include alcohols such as methyl alcohol, ethyl alcohol, glycerin, propylene glycol, and 1,3-butylene glycol; acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, pyridine, dimethyl sulfoxide, N, N -Known organic solvents such as dimethylformamide and acetic acid. Examples of the hydrophobic organic solvent include known organic solvents such as hexane, cyclohexane, carbon tetrachloride, chloroform, dichloromethane, 1,2-dichloroethane, diethyl ether, ethyl acetate, benzene, and toluene. These organic solvents can be used alone or in combination of two or more. Among these, water and / or a hydrophilic organic solvent, particularly methanol, ethanol, 1,3-butylene glycol, water, or a mixture or combination thereof is preferable.

Although extraction conditions are not specifically limited, For example, temperature is 5-95 degreeC, Preferably it is 10-90 degreeC, More preferably, it is 15-85 degreeC, and it can extract suitably also at normal temperature. The higher the temperature, the higher the extraction efficiency. The extraction time is several hours to several days, and the amount of the solvent used for extraction is usually 1 to 20 times, preferably 2 to 10 times the mass of the raw material.
The extraction operation is not particularly limited, and may be performed according to a conventional method. In order to improve the extraction efficiency, the extraction can be performed using a shake extraction or an extractor equipped with a stirrer or the like. For example, the acerola seed is immersed in the extraction solvent, or without being immersed, the extraction solution is stirred and shaken with the extraction solvent, and the processing solution is separated into the extraction solution and the extraction residue by filtration, centrifugation, decantation, etc. Extraction processing can be performed by separating, and the extraction residue may be further subjected to similar extraction processing. Although the obtained extract may be used as it is, it can be further subjected to concentration treatment and / or fractionation treatment if necessary.

The concentration treatment is not particularly limited, for example, solvent removal, soluble content recovery processing utilizing solubility in water and / or organic solvent, insoluble content recovery processing, liquid-liquid distribution processing with water-hydrophobic organic solvent, Examples thereof include a recrystallization process, a reprecipitation process, a process for recovering precipitates generated by cooling, or a method of combining two or more kinds of processes selected from these.
The fractionation treatment is also not particularly limited, and examples thereof include normal phase and / or reverse phase chromatography treatment.

The solvent extract of acerola seeds as an active ingredient of the hyaluronidase activity inhibitor of the present invention contains quercitrin.
In the hyaluronidase activity inhibitor of the present invention, the amount of the solvent extract of acerola seed, which is an active ingredient, can be appropriately selected depending on the form of use.

The hyaluronidase activity inhibitor of the present invention can be used for external preparations for skin and cosmetics. The type of the cosmetic is not particularly limited, and examples thereof include skin care cosmetics such as skin lotions, emulsions, creams, packs, and cleaning agents; makeup cosmetics such as lipsticks and foundations; and cosmetics for hair. The type is not particularly limited and is arbitrary. Examples of the external preparation for skin include ointments and various skin drugs.
In the external preparation for skin and cosmetics, the mixing ratio of the hyaluronidase activity inhibitor of the present invention can be appropriately selected according to the type and the type, amount, form, etc. of other components to be mixed. It is 0.001-20 mass% in conversion of the dry substance of acerola seed extract with respect to cosmetics whole quantity, Preferably it is 0.01-10 mass%.

  The skin external preparation or cosmetic can be blended with various other components that are usually used as skin external preparation raw materials or cosmetic raw materials. Other components include, for example, water, oils, surfactants, lubricants, alcohols, water-soluble polymer agents, gelling agents, moisturizers, buffers, preservatives, anti-inflammatory agents, thickeners, and fragrances. Vitamins, antioxidants, and the like. In use, one or more of these can be appropriately selected and blended.

EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to these.
Production Example 1
The washed acerola seeds were dried and then crushed and 6140 g of the pulverized product was added with 7 times mass of methanol and stirred overnight at room temperature. The whole amount was centrifuged and filtered, and the filtrate was concentrated to dryness to obtain 225.89 g of extract (A).
To this extract (A), 2000 ml of water was added, 1200 ml of hexane was further added and shaken, and then the separated aqueous layer was recovered. The same shaking using hexane was further repeated twice for this aqueous layer. To the aqueous layer obtained by removing the hexane layer, adding 1200 ml of ethyl acetate and shaking was repeated 10 times. The separated ethyl acetate layers were collected and concentrated to dryness, and the concentrate (A) was added to 11.48. g got. Next, the concentrate (A) was fractionated by silica gel column chromatography using a column packed with silica gel (Wakosil C-300: manufactured by Wako Pure Chemical Industries, Ltd.). Elution was successively performed with chloroform, chloroform: methanol (97: 3, 9: 1, 8: 2, 6: 4, 4: 6, 2: 8) and methanol. Divide the chloroform eluate into 15 fractions (fractions 1-15), and then separate the chloroform: methanol eluate into fractions 16 (97: 3), 17 (9: 1), 18 (8: 2), respectively. 19 (6: 4), 20 (4: 6).

Antioxidant activity of each fraction was confirmed by an evaluation method using thin layer chromatography (TLC). That is, a sample of each fraction was applied to a silica gel thin layer plate and developed with a developing solvent. At this time, a plate containing a fluorescent indicator (K5F Silica Gel 150 Gel: manufactured by Whatman) was used, and after development, the dried plate was irradiated with ultraviolet rays to detect a spot of the sample having UV absorption. Thereafter, a 6 × 10 ⁻⁴ M methanol solution of Diphenyl-p-picrylhydradil (DPPH), which is a stable radical, was sprayed on the plate. The DPPH solution is purple, but loses its color when radicals are eliminated. For this reason, in the spot portion of the substance having radical scavenging activity, the spot portion appears to be white as the DPPH radical is erased.
Fractions 1 to 20 were applied to a silica gel plate, developed to the tip of the plate with a solvent of chloroform: methanol = 9: 1, and then sprayed with a DPPH solution. The results are shown in Figure 1. As can be seen from FIG. 1, the acerola seed extract contains a large number of substances having antioxidant activity.

Among these, fraction 19 was further purified by reverse phase high performance liquid chromatography (HPLC). First, it was roughly purified under the following elution conditions, and further purified under the following conditions. The HPLC conditions are shown below.
Column: Hydrosphere C-18 [20 x 250 mm] (YMC), flow rate: 5 ml / min, temperature: 35 ° C
Detection: UV at 254nm, Eluent: 30% methanol (0-2min), 30-100% methanol (2-32min, linear), 100% methanol (32-40min), 30% methanol (40-50min) After crude purification, final purification was performed with acetonitrile / water (30/70) to obtain 72 mg of a purified fraction.
Various spectrum measurements were performed with respect to the obtained fraction purified product. The data obtained is shown below.
Mass spectrum: [MH] -447
UV absorption spectrum: (EtOH) 256.5 nm, 352.00 nm, H-NMR chemical shift: 500 MHz Solvent CD ₃ OD, 0.94 ppm (d: J = 6.1 Hz), 6.91 ppm (d: J = 8.2 Hz), 7.31 ppm ( d, d: J = 8.2,2.1Hz), 7.34 ppm (d: J = 2.1 Hz), 6.20 ppm (d: J = 2.1 Hz), 6.37 ppm (d: J = 2.1 Hz), 3.33 ppm (m: J = 9.5,9.3Hz), 3.41ppm (m: J = 9.5Hz), 3.74ppm (d, d: J = 9.3,3.3 Hz), 4.21ppm (d, d: J = 3.3Hz), 5.35ppm ( d: J = 1.7Hz), C-NMR Chemical shift: 125.8MHz Solvent CD ₃ OD, 17.7ppm, 72.0ppm, 72.1ppm, 72.2ppm, 73.3ppm, 94.8ppm, 99.9ppm, 103.6ppm, 106.0ppm, 116.4ppm 117.0ppm, 122.9ppm, 123.1ppm, 136.3ppm, 146.5ppm, 149.9ppm, 158.6ppm, 159.4ppm, 163.3ppm, 166.0ppm, 179.7ppm.
In the mass spectrum, an ion considered to be a deprotonated molecule was observed at m / z 447, and the molecular weight was considered to be 448. In H-NMR, 0.94 ppm is attributed to ¹ H of CH ₃ , 6.91 ppm, 7.31 ppm, 7.34 ppm, 1,2,4-substituted benzene, 6.20 ppm, 6.37 ppm, 1,2,3,5-substituted benzene It was done. Five peaks were observed between 3.33 and 5.35 ppm. In the ¹³ C-NMR spectrum, 21 peaks were observed, 4 peaks at 70-74 ppm were considered to be> CH—O—, and a peak attributable to conjugated carbonyl was observed at 179.7 ppm. Since these data suggested the possibility of quercetin glycosides, we further measured high-resolution mass spectra and compared them with the NMR spectra of quercitrin preparations with a molecular weight of 448 for quercetin glycosides. As a result of high-resolution mass spectrum measurement, an accurate mass of 447.0917 was obtained, and a composition calculation was performed using C, H, and O as elemental species. As a result, the composition formula C ₂₁ H ₁₉ O ₁₁ satisfying the carbon number 21 observed in the ¹³ C-NMR spectrum was calculated, and the molecular formula was determined to be C ₂₁ H ₁₉ O ₁₁ . Further, the NMR spectrum was compared with the spectrum of the quercitrin (quercetin-3-rhamnoside) preparation. As a result, it was found that the substance separated and purified from the acerola seed extract was identified as quercitrin.

  Quercitrin is one of the flavonoids and is widely distributed in nature, and is known to be particularly abundant in Dokudami. From the above results, it was found that quercitrin is contained in acerola seeds and that quercitrin is one of the substances involved in the antioxidant activity in the extract.

Production Example 2
100 g of the washed acerola seeds were crushed, 3 times the volume of water was added, and shaken overnight at room temperature. The whole amount was filtered through a glass filter, a 0.65 μm filter and a 0.22 μm filter, and the filtrate was concentrated to dryness to obtain 1 g of an extract.
Next, the antioxidant activity of the extract obtained by measuring the antioxidant activity using linoleic acid (the Rhodan iron method) was measured.
That is, 2 ml of 2.5% (w / v) linoleic acid (99.5% ethanol solution) and 4 ml of 0.05M phosphate buffer (pH 7.0), 0.4 mg of acerola seed extract, 2 ml of 99.5% ethanol and distilled A mixture of 2 ml of water was mixed and placed in a brown screw mouth bottle to prepare a 10 ml test solution. In addition, α-tocopherol or BHA was used instead of acerola seed extract, and the same operation was carried out so that the same amount of acerola seed extract was contained in the reaction solution. As a control. As a control, a test solution in which only 2 ml of 99.5% ethanol and 2 ml of distilled water were added to the reaction solution without using an acerola seed extract was used. Each test solution obtained was stored at 40 ° C. in the dark, and the test was stored at 4 ° C., and the test solution was taken out over time and measured by the following method. The test was conducted for 14 days.

First, add 0.1 ml of 2 × 10 ^-2 M ferrous chloride (3.5% hydrochloric acid solution) to a mixture of 0.1 ml of the test substance, 9.7 ml of 75% ethanol, and 0.1 ml of 30% aqueous rhodammonium solution. After 3 minutes, the absorbance at 500 nm was measured. The same measurement was performed for the blind test, and Δ absorbance = (absorbance of main test) − (absorbance of blind test) was set. As the sample begins to oxidize, the absorbance increases, and after reaching the highest point, the absorbance decreases as fewer samples are to be oxidized. Therefore, it can be said that the antioxidant peak becomes weaker as the peak of absorbance starts. Moreover, the antioxidant activity of each test solution was compared using the oxidation rate. The oxidation rate was determined by the following formula, assuming the control oxidation (Δ absorbance) as 100%. Higher oxidation rate means lower antioxidant activity.
Oxidation rate (%) = ([Δ absorbance of sample] / [Δ absorbance of control]) × 100
FIG. 2 shows the results of changes in absorbance over time, and FIG. 3 shows the oxidation rate of each sample solution 14 days after the start of the test.

Production Example 3
100 g of the washed acerola seeds were crushed, 3 times volume of aqueous ethanol aqueous solution with an ethanol content of 25% by volume was added and shaken overnight at room temperature. The whole amount was filtered through the same filter as in Production Example 2, and the filtrate was concentrated to dryness to obtain 1.69 g of an extract.
The antioxidant activity was measured in the same manner as in Production Example 2 using the obtained extract. FIG. 2 shows the results of changes in absorbance over time, and FIG. 3 shows the oxidation rate of each sample solution 14 days after the start of the test.

Production Example 4
100 g of the washed acerola seeds were crushed, 3 times volume of aqueous ethanol aqueous solution with 50% ethanol content was added and shaken overnight at room temperature. The whole amount was filtered through the same filter as in Production Example 2, and the filtrate was concentrated to dryness to obtain 2.27 g of extract.
The antioxidant activity was measured in the same manner as in Production Example 2 using the obtained extract. FIG. 2 shows the results of changes in absorbance over time, and FIG. 3 shows the oxidation rate of each sample solution 14 days after the start of the test.

Production Example 5
100 g of the washed acerola seeds were crushed, and a 3-fold volume of aqueous ethanol solution containing 75% ethanol by volume was added and shaken overnight at room temperature. The whole amount was filtered through the same filter as in Production Example 2, and the filtrate was concentrated to dryness to obtain 2.50 g of an extract.
Antioxidant activity was measured in the same manner as in Production Example 2 using the obtained extract. FIG. 2 shows the results of changes in absorbance over time, and FIG. 3 shows the oxidation rate of each sample solution 14 days after the start of the test.
2 and 3, the acerola seed extract clearly has an inhibitory effect on the oxidation of linoleic acid, and its strength is equivalent to or better than that of typical antioxidants α-tocopherol and BHA. It turned out that. Furthermore, it turns out that the intensity | strength of antioxidant activity is controllable from the result of manufacture examples 2-5 by changing the ratio of ethanol in the water-containing ethanol used as an extraction solvent.

Production Example 6
The washed acerola seeds (760 g) were crushed, 5 times the mass of methanol was added, and the mixture was stirred overnight at room temperature. The whole amount was centrifuged and filtered, and the filtrate was concentrated to dryness to obtain 12.36 g of an extract. To this extract, 300 ml of water was added, and 100 ml of hexane was further added and shaken, and then the separated aqueous layer was recovered. The same shaking using hexane was further repeated 3 times for this aqueous layer. To the aqueous layer obtained by removing the hexane layer, adding 100 ml of ethyl acetate and shaking was repeated 6 times, and the separated ethyl acetate layers were collected and concentrated to dryness to obtain 0.41 g of solid content.
The antioxidant activity of the obtained acerola seed extract was measured by the rodan iron method in the same manner as in Production Example 2. At this time, the test period was 20 days. FIG. 4 shows the oxidation rate of each sample solution on the 20th day after the start of the test.
From Fig. 4, it was found that the ethyl acetate fraction of the extract of acerola seeds clearly has the effect of inhibiting the oxidation of linoleic acid as the extract itself, and its strength is stronger than α-tocopherol. .

Production Example 7
70 g of the washed acerola seeds were crushed and a 4-fold mass of 1,3-butylene glycol aqueous solution having a 1,3-butylene glycol content of 30 mass% was added and stirred overnight at room temperature. After centrifuging the whole amount, the supernatant was filtered through a 0.22 μm filter to obtain 124.12 g of an acerola seed extract as a solution.
Antioxidant activity of the obtained acerola seed extract was measured by the following DPPH radical scavenging method. The results are shown in Table 1.
1600 μl of 250 mM acetate buffer (pH = 5.5) was mixed with 1200 μl of ethanol and 400 μl of sample (prepared to an arbitrary concentration), and pre-incubated at 30 ° C. for 5 minutes. To this solution, 800 μl of 500 μM DPPH / ethanol solution was added and mixed, and after standing at 30 ° C. for 30 minutes, the absorbance at 517 nm was measured. The same operation was performed for α-tocopherol, and this was used as a positive control. As a control, a sample obtained by performing the same operation using the solvent instead of the sample solution was used. From the measured absorbance, the radical scavenging rate was calculated by the following formula.
Erase rate (%) = (1− [absorbance of sample] / [absorbance of control]) × 100
The above-mentioned erasure rate was measured by changing the sample concentration of the sample solution stepwise, and the concentration of the sample solution at which the DPPH radical erasure rate was 50% was determined to obtain the DPPH radical 50% erasure concentration. Therefore, the lower this value, the higher the radical scavenging ability.

表1より、製造例７の抽出物はα-トコフェロールと同等のラジカル消去能を有することが判った。

From Table 1, it was found that the extract of Production Example 7 had the same radical scavenging ability as α-tocopherol.

Production Example 8
1500 g of the washed acerola seeds were crushed, and a 2-fold mass of 1,3-butylene glycol aqueous solution having a 1,3-butylene glycol content of 30% by mass was added and stirred overnight at room temperature. After centrifuging the whole amount, the supernatant was filtered through a 0.22 μm filter to obtain 2327 g of an acerola seed extract as a solution.
The antioxidant activity of the obtained acerola seed extract was measured by the rodan iron method in the same manner as in Production Example 2. However, since the acerola seed extract is a solution, it is diluted with water and ethanol to the required concentration, and the acerola seed extract is controlled with the same solvent composition as the added acerola seed extract. What did not contain a seed extract was used. The test period was 7 days.
FIG. 5 shows the results of changes in absorbance with time, and FIG. 6 shows the oxidation rate of each sample solution on the seventh day after the start of the test.
From FIGS. 5 and 6, it was found that the acerola seed extract clearly has an inhibitory effect on the oxidation of linoleic acid, and its strength is equivalent to or higher than that of α-tocopherol and BHA.

By the way, since it is known that vitamin C has an antioxidant effect, the amount of vitamin C in the acerola seed extract prepared above is measured, and the antioxidant effect in the acerola seed extract is contained. We examined whether it was due to vitamin C alone.
First, when the amount of vitamin C contained in 100 g of the acerola seed extract prepared above was measured, it was 57 mg / 100 g (oxidized vitamin C: 56 mg / 100 g + reduced vitamin C: 1 mg / 100 g). Therefore, when 100 g of the acerola seed extract prepared above was evaporated to dryness, a solid content of 975 mg was obtained. Subsequently, the antioxidant activity was measured by the rodan iron method in the same manner as in Production Example 2. At this time, since the acerola seed extract of this example was a solution, it was diluted and added so as to contain 0.4 mg as a solid content in the reaction solution as described above. In the same manner as described above, a control having the same solvent composition as the added acerola seed extract and containing no acerola seed extract was also used as a control. The measurement was performed for 7 days. As a control, the antioxidant activity was similarly measured using 0.023 mg (0.4 mg × (57 mg / 975 mg)) of vitamin C contained in 0.4 mg of the solid content.
The oxidation rate on the 7th day after the start of the test was 1.9% for the acerola seed extract, and 115.8% for the vitamin C alone.
From the above results, it was found that vitamin C contained in the acerola seed extract hardly contributed to the antioxidant action of the extract.

Further, in the acerola seed extract of the present invention, whether or not quercitrin, which is one of the substances showing the antioxidant action confirmed in Production Example 1, is contained in the acerola seed extract prepared as described above is a production example. Measurement was performed in the same manner as in 1. As a result, it was found that quercitrin was contained.
By the way, conventionally, for example, JP-A-7-300581 has proposed that quercitrin has an antioxidant action. Therefore, the following method was used to examine whether the antioxidant action in the acerola seed extract was solely due to quercitrin containing it.
First, since quercitrin is a kind of polyphenol, when the amount of polyphenol in the acerola seed extract prepared above was measured by the Folin-Denis method, the proportion of polyphenol in the solid content of 975 mg in 100 g of the extract was 25%. Met. Therefore, the amount of quercitrin contained in the solid content of the acerola seed extract prepared above is at most 25%. Based on this result, the antioxidant activity was measured by the rodan iron method in the same manner as in Example 2 in the same manner as in the comparative test with vitamin C. At this time, the measurement was performed for 7 days. As a control, the antioxidant activity was measured in the same manner using 0.128 mg of quercitrin, assuming that quercitrin was contained in 32 mg of the solid content of 0.4 mg (the maximum amount was 25%, which is more than that). .
As a result, the oxidation rate on the seventh day after the start of the test was 1.9% for the acerola seed extract, whereas it was 6.9% for quercitrin alone.
From the above results, quercitrin contained in the acerola seed extract is one of the active ingredients of the extract's antioxidant action, but the antioxidant action of the extract is not the action of quercitrin alone, Acerola seed extract was found to be superior to the antioxidant effect of quercitrin alone.

Test example 1
About each acerola seed extract prepared in Production Examples 2 to 5, the collagenase activity inhibitory action was measured by the following method.
Measurement method (reagent preparation)
Substrate solution: 0.39 mg of Pz-peptide (manufactured by BACHEM) was dissolved in 1 ml of 0.1 M Tris-HCl buffer (pH 7.1, containing 20 mM calcium chloride) and used (corresponding to 0.5 mM).
Enzyme solution: 5 mg of collagenase (TYPE IV, manufactured by Sigma) is dissolved in 1 ml of distilled water, dispensed in 100 μl aliquots, and stored at −20 ° C. At the time of use, it was diluted 50 times with distilled water and used for the reaction.

[Method for measuring collagenase activity inhibitory action]
The extract of Production Example 2 was prepared by dissolving in each extraction solvent so that the extract of Production Examples 3, 4 and 5 had a concentration of 0.5 mg / ml. did. These sample solutions (50 μl), collagenase solution (50 μl) and substrate solution (400 μl) were mixed and incubated at 37 ° C. for 30 minutes. The reaction was then stopped with 1 ml of 25 mM citric acid solution and extracted with 5 ml of ethyl acetate. After centrifugation (3000 rpm, 10 minutes), the absorbance of the ethyl acetate layer at a wavelength of 320 nm was measured using ethyl acetate as a control. For the control, each extraction solvent was used in place of the sample solution, and as each blank, distilled water was added instead of the enzyme solution, and the same operation was performed.
From these values, the collagenase activity inhibition rate was calculated by the following equation.
Inhibition rate (%) = [1− (A−B) / (C−D)] × 100
A: Absorbance at 320 nm of the sample solution, B: Absorbance at 320 nm of the sample solution blank, C: Absorbance at 320 nm of the control solution, D: Absorbance at 320 nm of the control solution blank.
The collagenase activity inhibition rate of the extracts of Production Examples 2 to 5 was determined by the above method. The results are shown in Table 2.

Test example 2
For the acerola seed extract prepared in Production Example 8, the collagenase activity inhibitory action was measured by the same method as in Test Example 1. However, since the extract of Production Example 8 was a solution, it was diluted with a 30% by mass 1,3-butylene glycol aqueous solution as an extraction solvent so that the acerola seed extract had a concentration of 0.5 mg / ml as a solid. This was used as a sample solution. Thus, as a result of calculating | requiring the collagenase activity inhibition rate of the extract of manufacture example 8, it was 71.5%.

Example 1
The hyaluronidase activity inhibitory action of the extract prepared in Production Example 8 was measured. Hyaluronidase activity inhibition was measured by the method of Yumi Maeda et al. Using the Morgan-Elson method (Eisho Journal, Vol. 31, 233-237, 1990).
Measurement method (reagent preparation)
Enzyme solution: Cow testicular hyaluronidase (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 0.1 M acetate buffer (pH = 4.0) to adjust the final enzyme activity to 400 units / ml.
Enzyme activation solution: Compound 48/80 (manufactured by Sigma) was dissolved in 0.1 M acetate buffer (pH = 4.0) to adjust the final concentration to 0.1 mg / ml.
Substrate solution: Potassium hyaluronate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 0.1 M acetic acid buffer (pH = 4.0) to adjust the final concentration to 0.4 mg / ml.
Boric acid solution: 50 ml of water was added to 4.95 g of boric acid, adjusted to pH = 9.1 with 1N sodium hydroxide solution, and adjusted to 100 ml with water.
p-Dimethylaminobenzaldehyde (p-DAB) reagent: 10 g of p-DAB (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in a mixture of 12.5 ml of 10N hydrochloric acid and 87.5 ml of acetic acid and stored in the refrigerator. Immediately before use, diluted 10-fold with acetic acid was used.

[Measurement of hyaluronidase activity inhibitory action]
Since the extract of Production Example 8 is a solution, a sample diluted with a 30% by mass 1,3-butylene glycol aqueous solution as an extraction solvent so that the acerola seed extract has a solid concentration of 1 mg / ml It was set as the solution. To 0.2 ml of this sample solution, 0.1 ml of enzyme solution was added and left at 37 ° C. for 20 minutes. Next, add 0.2 ml of enzyme activation solution and warm at 37 ° C for 20 minutes. Add 0.5 ml of substrate solution and react at 37 ° C for 40 minutes, then add 0.2 ml of 0.4N sodium hydroxide aqueous solution. The reaction was stopped by cooling with ice. Add 0.2 ml of boric acid solution and heat in a hot block bath (TOYO SEISAKUSHO, MODEL TPB-32) at 100-120 ° C for 5 minutes, then cool on ice, add 6 ml of p-DAB reagent, and warm at 37 ° C for 20 minutes. The color was developed and the absorbance at 585 nm was measured using distilled water as a control. For the control, an extraction solvent was used instead of the sample solution, and 0.1 M acetate buffer (pH = 4.0) was added instead of the enzyme solution as each blank, and the same operation was performed.
From these values, the inhibition rate of hyaluronidase activity was calculated by the following formula.
Inhibition rate (%) = [1− (A−B) / (C−D)] × 100
Where A: absorbance at 585 nm of the sample solution, B: absorbance at 585 nm of the sample solution blank, C: absorbance at 585 nm of the control solution, and D: absorbance at 585 nm of the control solution blank.
As a result of obtaining the hyaluronidase activity inhibition rate of the extract of Production Example 8 by the above method, it was 94.5%.

Test example 3
According to the Ministry of Health and Welfare Ordinance No. 21, “Ministerial Ordinance on Standards for Implementation of Non-clinical Studies on Drug Safety” dated March 26, 1997, the safety test of acerola seed extract was conducted. As the acerola seed extract, the acerola seed extract prepared in Production Example 8 was used. The results are shown in Table 3.
Single Oral Dose Toxicity Test Using Rats Two groups of rats (control group, administration group) were tested in males and females at 5 animals / group, and the administration group was administered at 2 g / kg body weight.
Skin primary irritation test using guinea pigs Three healthy guinea pigs were occluded for 24 hours, and the skin condition was observed at 24 hours, 48 hours, and 72 hours after administration to make a judgment.
14-day cumulative skin irritation test using guinea pigs Three healthy guinea pigs were applied once a day in an open system for 14 consecutive days, and each day during the test period, before application, and 24 hours after application. Judgment was made by observing the state of
Skin sensitization test using guinea pigs Five groups / group of guinea pigs (control group, application group, DNCB group) were tested according to the Adjuvant and Patch Test method, and 24 and 48 hours after application. In addition, each skin condition was observed and judged.
Skin phototoxicity test using guinea pigs The test was performed on the back skin of 10 guinea pigs according to the method of Fujikawa et al., And the skin condition was observed at 24 hours, 48 hours and 72 hours after UV irradiation, respectively. It was.

Skin photosensitization test using guinea pigs Three groups of guinea pigs (control group, administration group, TCSA group) were tested in 5 animals / group according to the Ajuvant and Strip method. At each time, the skin condition was observed and judged.
Ocular mucosal irritation test using rabbits Two rabbits (non-eyewash group, eyewash group) were tested at 3 animals / group. After instillation, leave the non-eyewash group as it is, wash the eyewash group with mild saline for about 1 minute, and then observe the condition of cornea, iris and conjunctiva after 1 hour, 24 hours, 48 hours and 72 hours, Judgment was made from the AFNOR category.
The reverse mutation test using bacteria was measured by the preincubation method in the case of no addition of S9mix and the addition of S9mix.
・ Strain used: Salmonella typhimurium TA100, TA98, TA1535, TA1537
・ Strain used: Escherichia coli WP2uvrA
Chromosome aberration test using mammalian cultured cells Three groups (negative control group, test substance group, positive control group) using mammalian cultured cells (CHL / IU cells) are treated for a short time (6 hours treatment) : S9mix non-added and S9mix added) and a continuous treatment method (24 hours and 48 hours treatment).

Formulation Example 1
Purified by mixing 0.20 parts by mass of dipotassium glycyrrhizinate, 0.10 parts by mass of citric acid, 0.30 parts by mass of sodium citrate, 5.00 parts by mass of the acerola seed extract prepared in Production Example 7 and 5.00 parts by mass of 1,3-butylene glycol. Water was added to make a total amount of 80.0 parts by mass, and the mixture was dissolved with stirring at 50 ° C. to prepare an extract-containing aqueous solution.
Next, 0.90 part by mass of POE (60) sorbitol tetraoleate, 0.10 part by mass of sorbitan monooleate, an appropriate amount of preservative and 10.00 parts by mass of ethanol were mixed and dissolved at 50 ° C. with stirring. Subsequently, the obtained solution was added little by little to the initially prepared extract-containing aqueous solution and mixed and stirred at 50 ° C. When uniformly mixed, the liquid temperature was lowered from 50 ° C. to 30 ° C. with further stirring. When 30 ° C. was reached, stirring was stopped, and appropriate amounts of fragrance and purified water were added to make the total amount 100.00 parts by mass. The mixture was stirred again and mixed uniformly to prepare a lotion.

Formulation Example 2
10.00 parts by mass of squalene and an appropriate amount of an antiseptic were mixed, purified water was added to adjust the total amount to 70.00 parts by mass, and the mixture was heated to 80 ° C. to prepare solution (1). Further, 0.10 parts by mass of carboxyvinyl polymer and 0.20 parts by mass of xanthan gum were stirred and dissolved in an appropriate amount of purified water at room temperature to prepare a solution (2). Furthermore, 0.10 parts by mass of triethanolamine and 5.00 parts by mass of 1,3-butylene glycol were stirred and dissolved in an appropriate amount of purified water at room temperature to prepare a solution (3). Furthermore, 2.00 parts by mass of sodium hyaluronate and 5.00 parts by mass of the acerola seed extract prepared in Production Example 8 were stirred and dissolved in an appropriate amount of purified water at room temperature to prepare solution (4).
Next, the solution (1) was added little by little to an appropriate amount of purified water, mixed and stirred at 80 ° C., the solution (2) was added with further stirring, and then the solution (3) was added. When uniformly mixed, the solution was lowered to 50 ° C. while stirring, and when it reached 50 ° C., the solution (4) was added, and purified water was further added to adjust the total amount to 100 parts by mass. Stirring was continued until the solution reached 30 ° C. When the temperature reached 30 ° C, stirring was stopped and a uniformly mixed emulsion was prepared.

Formulation Example 3
POE (20) sorbitan monostearate 2.00 parts by mass, POE sorbitan tetraoleate 0.50 parts by mass, glyceryl monostearate 0.50 parts by mass, stearic acid 7.00 parts by mass, cetyl alcohol 3.00 parts by mass, cetyl palmitate 3.00 parts by mass, jojoba oil 7.00 parts by mass, 3.00 parts by mass of paraffin and an appropriate amount of an antiseptic were mixed and dissolved at 80 ° C. with stirring to prepare a solution (1). On the other hand, 5.00 parts by mass of the acerola seed extract prepared in Production Example 8, 7.00 parts by mass of 1,3-butylene glycol and 62 parts by mass of purified water were mixed and dissolved with stirring at 80 ° C. to obtain a solution (2). Prepared.
Next, the solution (1) was added little by little to the solution (2), emulsified, cooled with stirring and cooled to 40 ° C., and the stirring was stopped to prepare a uniformly mixed cream.

It is a copy of the photograph which shows the result of having measured the antioxidant activity of each fraction which fractionated the acerola seed extract prepared in manufacture example 1 by column chromatography using thin layer chromatography. It is a graph which shows the time-dependent change of the light absorbency for the oxidation rate measurement of the acerola seed extract prepared by manufacture examples 2-5. It is a graph which shows the oxidation rate of the 14th day of the acerola seed extract prepared by manufacture examples 2-5. It is a graph which shows the oxidation rate on the 20th day of the acerola seed extract prepared in Production Example 6. It is a graph which shows the time-dependent change of the light absorbency for the oxidation rate measurement of the acerola seed extract prepared in manufacture example 8. 6 is a graph showing the oxidation rate on day 7 of an acerola seed extract prepared in Production Example 8.

Claims (2)

A hyaluronidase activity inhibitor containing an acerola seed extract as an active ingredient, which contains a solvent extract of acerola seeds containing quercitrin and having a quercitrin content of 25% by weight or less. The hyaluronidase activity inhibitor according to claim 1, wherein the solvent is water, a hydrophilic organic solvent, a hydrophobic organic solvent, or a combination thereof.

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