JP2011136968A - Hyaluronidase inhibitor produced by using silver skin extract and production method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hyaluronidase inhibitor which is prepared by effectively utilizing, as a functional material, silver skin of coffee traditionally treated mainly as an industrial waste. <P>SOLUTION: The silver skin is effectively utilized for the hyaluronidase inhibitor by administering a predefined extraction process. As an illustrative embodiment, the hyaluronidase inhibitor is produced by subjecting an extract extracted from the silver skin by using water as an extraction solvent to solid-liquid separation (filtration) and then utilizing a solid content obtained by drying a supernatant part as an active ingredient. As another illustrative embodiment, the hyaluronidase inhibitor is produced by subjecting an extract extracted from the silver skin by using water as an extraction solvent to solid-liquid separation (filtration), subjecting an object obtained by an enzyme treatment of a residual part to solid-liquid separation (filtration), and then utilizing a solid content obtained by drying a supernatant part as an active ingredient. In a case of obtaining a more powerful inhibitor, a polymer fraction is withdrawn from an extract and is made into the hyaluronidase inhibitor. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

TECHNICAL FIELD The present invention relates to effective use of silver skin that has been conventionally treated mainly as industrial waste (many are produced in large quantities in coffee roasting plants), and in particular, a hyaluronidase inhibitor using silver skin extract. About.

  Silver skin is a thin skin that wraps around coffee beans. The fruit of coffee is a drupe in botany, and as shown in FIG. 18, the coffee fruit has an outer skin, pulp, mucilage (mucus), parchment (inner skin), silver skin, seed as shown in FIG. Are arranged in the order. The seed has an embryo and an endosperm, and is arranged so as to face each other in one coffee fruit. As the coffee berries mature, the planar portions of the endosperm facing each other are recessed and folded into the endosperm inner side, and a groove called a center cut is formed in the center of the planar portion.

Raw beans are obtained by removing the outer part of the seeds, and the process of removing green beans from coffee fruits is called purification. In the refining process of green coffee beans, a part of the silver skin is removed, and most of the adhering ones are peeled off during roasting and usually become industrial waste.
Silver skin is partly used as a material such as psicose (Patent Document 1), paper (Patent Document 2), company envelopes, business cards, New Year's cards, wrapping paper, and toys. It has been.
However, in the past, silver skin, which has been treated mainly as industrial waste, has not been effectively used as a functional material such as a hyaluronidase inhibitor.

  Hyaluronidase is a hyaluronic acid-degrading enzyme, and hyaluronic acid exists in the extracellular matrix of living tissues and plays an important role in a wide variety of ways. For example, hyaluronic acid is abundant in synovial fluid and acts as a cushion to relieve the load on the joint, it exists between cells in the skin, maintains skin moisture and maintains healthy skin, Involved in the transportation of nutrition and waste. When the amount of hyaluronic acid in the body decreases, various symptoms such as arthritis, arthropathy, rheumatoid arthritis, dry skin, wrinkle formation, decreased infection prevention ability, atopic dermatitis worsening, cell aging, arteriosclerosis, etc. . Thus, it is hyaluronic acid which plays an important role in the living body, but its amount decreases with aging. Even if it is replenished from the outside, hyaluronic acid is present in food only in a small amount, and it is necessary to take a sufficient amount on a daily basis for reasons such as poor thermal stability and difficulty in being absorbed into the body due to high polymers. It is difficult.

  Hyaluronidase inhibitors are expected to contribute to the stabilization of the original hyaluronic acid by inhibiting the degradation of hyaluronic acid in vivo, and to alleviate various symptoms caused by a decrease in the amount of hyaluronic acid in the living body. .

  Conventionally known existing hyaluronidase inhibitors include sodium cromoglycate, tranilast, indomethacin, and aspirin, but side effects have been pointed out, and the development of naturally-occurring hyaluronidase inhibitors that are safer for the human body Is required.

  On the other hand, as hyaluronidase inhibitors obtained from natural extracts, chimpi, pheasant, Rahan fruit (Patent Document 3), Ursiaceae plant (Patent Document 4), Yaeyama Hirugi plant, Ohigi genus plant, Hazakuro genus plant (Patent Document 5) ), Those derived from willow, lime, loquat, oyster, onion (Patent Document 6), cashew nut shell oil (Patent Document 7), and the like. Thus, hyaluronidase inhibitors can be obtained from a wide variety of plants, but they are expensive because they use expensive materials, and there is a problem that the materials cannot be obtained constantly. Furthermore, in the case of these natural extracts, there are taste problems such as unique smell, bitterness and astringency.

  In addition, hyaluronidase inhibitory activity has been reported in various other plants. For example, it is strong against extracts of various plants such as Rubiaceae plants, Asteraceae plants, Convolvulaceae plants, Rosaceae plants, Bambooceae plants, and Ilexaceae plants. It is known that it has hyaluronidase inhibitory activity, and its active body is a cinnamic acid derivative containing chlorogenic acid (Patent Document 8).

JP 2004-143062 A JP 2008-255519 A JP-A-4-255423 Japanese Patent Laid-Open No. 7-10765 JP 2008-24664 A JP 2006-104098 A JP-A-6-329526 JP 2007-161632 A

  In view of the above situation, an object of the present invention is to effectively use a silver skin of coffee that has been conventionally treated mainly as industrial waste as a useful functional material.

As a result of intensive research to make effective use of the silver skin of coffee, which has been treated mainly as industrial waste, as a useful material because of the global environment, the present inventors have determined that the silver skin of coffee is predetermined. The present invention was completed by obtaining the knowledge that it can be effectively used as a hyaluronidase inhibitor by performing the extraction step.
That is, the present invention can provide a hyaluronidase inhibitor using as an active ingredient an extract of silver skin of coffee that has been conventionally treated mainly as industrial waste.

  In the present invention, the silver skin refers to a thin skin surrounding the endosperm of a Rubiaceae plant (Cofia sp., Mascarocophia sp., Etc.), which may be raw or roasted.

It is preferable that the hyaluronidase inhibitor obtained from the above-described silver skin has a solid content obtained by solid-liquid separation of an extract extracted from silver skin using water as an extraction solvent and drying the supernatant portion as an active ingredient.
As shown in the Examples described later, the water extract has a high yield from silver skin of about 16% and has a hyaluronidase inhibitory function.
Here, as an extraction method, in the examples, extraction is performed using hot water at 121 ° C. to obtain a solid content (hereinafter referred to as a hot water extract in this specification) from the supernatant portion. Any of hot water extraction, room temperature water extraction, and cold water extraction may be used, and the water temperature is not particularly limited.

In addition, the hyaluronidase inhibitor obtained from the above silver skin is obtained by solid-liquid separation of an extract extracted from silver skin using water as an extraction solvent, and solid-liquid separation of the residue portion subjected to enzyme treatment. It is preferable to use a solid content (hereinafter referred to as an enzyme-treated product) obtained by drying the clear portion as an active ingredient.
For example, acetate buffer and cellulase can be preferably used as the enzyme treatment solution.
As shown in Examples described later, the enzyme-treated product has a high yield from silver skin of about 20% and has a hyaluronidase inhibitory function. In addition, the polymer fraction of the enzyme-treated product has an excellent hyaluronidase inhibitory function equivalent to sodium cromoglycate, which is known as an existing drug for hyaluronidase inhibitors.

Moreover, it is preferable that the hyaluronidase inhibitor obtained from said silver skin uses as an active ingredient the extract extracted from silver skin using the ethanol solution, the dilute acid, or the dilute alkali as the extraction solvent.
Extracts extracted with an ethanol solution, dilute acid, or dilute alkali also have a hyaluronidase inhibitory function.

The hyaluronidase inhibitor obtained from the above silver skin is a polymer fraction contained in the supernatant obtained by solid-liquid separation of an extract extracted from silver skin using water (hot water) as an extraction solvent. It is preferable that a minute (hereinafter referred to as a hot water extract polymer fraction) be an active ingredient.
The polymer fraction of hot water extract has an excellent hyaluronidase inhibitory function equivalent to or higher than sodium cromoglycate, which is known as an existing drug for hyaluronidase inhibitors.

In addition, the hyaluronidase inhibitor obtained from the above silver skin is obtained by solid-liquid separation of an extract extracted from silver skin using water as an extraction solvent, and by subjecting the residue portion to enzyme treatment to solid-liquid separation. The polymer fraction contained in the obtained supernatant portion (hereinafter referred to as an enzyme-treated polymer fraction) is preferably used as an active ingredient.
The enzyme-treated product polymer fraction has an excellent hyaluronidase inhibitory function equivalent to sodium cromoglycate, which is known as an existing drug for hyaluronidase inhibitors.

Since the above-described hyaluronidase inhibitor of the present invention is obtained from an extract of a natural coffee coffee silver skin, it can be suitably used as a cosmetic, food or drink, or pharmaceutical composition.
By using a hyaluronidase inhibitor in foods, drinks, cosmetics, pharmaceutical compositions, etc., it is possible to suppress skin aging, inflammatory reaction, allergic reaction, etc. caused by a decrease in the amount of hyaluronic acid.

Next, the manufacturing method of the hyaluronidase inhibitor of this invention is demonstrated.
The method for producing the hyaluronidase inhibitor of the present invention is characterized in that it is produced using a natural product of coffee silver skin. As described above, conventionally, silver skins are generated in large quantities in roasting plants and some of them have been used as materials, but most of them have been treated as industrial waste. However, the present inventors obtained a silver skin extract by subjecting the silver skin to a predetermined extraction step, and found a hyaluronidase inhibitory activity in the extract, whereby a method for producing a hyaluronidase inhibitor using the silver skin. Was completed.

Hereinafter, a specific method for producing a hyaluronidase inhibitor will be described.
First, one aspect of a method for producing a hyaluronidase inhibitor using silver skin is:
A1) An extraction step of extracting silver skin using water, for example, 121 ° C. hot water, as an extraction solvent;
A2) a solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
A3) a drying step of drying the supernatant obtained in the solid-liquid separation step;
A4) a recovery step for recovering the solid content obtained in the drying step;
It comprises.
This is a method for producing a hyaluronidase inhibitor comprising the above-mentioned hot water extract as an active ingredient among methods for producing a hyaluronidase inhibitor using silver skin.

Next, another aspect of the method for producing a hyaluronidase inhibitor using silver skin is:
B1) An extraction step of extracting silver skin using water, for example, 121 ° C. hot water, as an extraction solvent;
B2) a first solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
B3) an enzyme treatment step for carrying out an enzyme treatment on the residue obtained in the first solid-liquid separation step;
B4) a second solid-liquid separation step for separating the processed product obtained in the enzyme treatment step into a supernatant and a residue;
B5) a drying step of drying the supernatant portion obtained in the second solid-liquid separation step;
B6) a recovery step for recovering the solid content obtained in the drying step;
It comprises.
This is a method for producing a hyaluronidase inhibitor using the above-mentioned enzyme-treated product as an active ingredient among methods for producing a hyaluronidase inhibitor using silver skin.

Next, another aspect of the method for producing a hyaluronidase inhibitor using silver skin is:
C1) An extraction step of extracting silver skin using an ethanol solution, dilute acid, or dilute alkali as an extraction solvent;
C2) a solid-liquid separation step of separating the supernatant and the residue from the extract obtained in the extraction step of C1,
At least. A hyaluronidase inhibitor can be produced even when extracted with silver other than water.
Here, for the residue portion obtained in the solid-liquid separation step of C2) above,
C3) a water extraction step of extracting the residue part with water;
C4) a second solid-liquid separation step for separating the supernatant and the residue again from the extract obtained in the water extraction step;
C5) a drying step of drying the supernatant portion obtained in the second solid-liquid separation step;
C6) a recovery step for recovering the solid content obtained in the drying step;
Thus, a hyaluronidase inhibitor can be preferably produced.
These are methods for producing a hyaluronidase inhibitor using the above-described ethanol solution, dilute acid, or extract extracted with dilute alkali as an active ingredient, among the methods for producing a hyaluronidase inhibitor using silver skin.

  In the extraction step in the above production method, the extraction solvent is not particularly limited to water, and may be ethanol, dilute acid, or dilute alkali. However, the most efficient extraction method is to use water as an extraction solvent, and then perform polymer fractionation.

Next, another aspect of the method for producing a hyaluronidase inhibitor using silver skin is:
D1) an extraction step of extracting silver skin using water, for example, 121 ° C. hot water, as an extraction solvent;
D2) a solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
D3) a fractionation step of fractionating the polymer fraction contained in the supernatant obtained in the solid-liquid separation step;
It comprises.
This is a method for producing a hyaluronidase inhibitor using the above-mentioned hot water extract polymer fraction as an active ingredient among the methods for producing a hyaluronidase inhibitor using silver skin. As the fractionation method after the extraction step, ethanol precipitation fractionation, gel filtration fractionation, fractionation using an ultrafiltration membrane, or the like is used.

Next, another aspect of the method for producing a hyaluronidase inhibitor using silver skin is:
E1) An extraction step of extracting silver skin using water, for example, 121 ° C. hot water, as an extraction solvent;
E2) a first solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
E3) an enzyme treatment step of performing an enzyme treatment on the residue obtained in the first solid-liquid separation step;
E4) a second solid-liquid separation step of separating the processed product obtained in the enzyme treatment step into a supernatant and a residue;
E5) a fractionation step of fractionating the polymer fraction contained in the supernatant obtained in the second solid-liquid separation step;
It comprises.
This is a method for producing a hyaluronidase inhibitor having the above-mentioned enzyme-treated polymer fraction as an active ingredient among methods for producing a hyaluronidase inhibitor using silver skin. As the fractionation method after the enzyme treatment step, ethanol precipitation fractionation, gel filtration fractionation, fractionation using an ultrafiltration membrane, or the like is used.

  The hyaluronidase inhibitor of the present invention has an effect that silver skin, which has been treated mainly as industrial waste, can be effectively used in a high yield as a functional material.

Moreover, since the silver skin used for the hyaluronidase inhibitor of the present invention is derived from a natural product, it is highly safe for the human body and should be applied, ingested, administered, etc. as cosmetics, foods, drinks, pharmaceuticals, etc. on a daily basis. Thus, it is expected that skin aging, inflammatory reaction, and allergic reaction due to a decrease in the amount of hyaluronic acid can be suppressed.
In the case of a conventional hyaluronidase inhibitor of a natural extract, problems of taste such as a unique odor, bitterness and astringency have been pointed out. By going through the process, odor and bitterness are improved.

Silver skin hot water extraction and enzyme treatment flow chart Manufacturing process flow diagram of hot water extract Manufacturing process flow chart of enzyme-treated product Silver skin ethanol extraction flow chart Manufacturing process flow diagram of ethanol extraction residue soluble fraction Ethanol precipitation flow diagram of hot water extract of silver skin Manufacturing process flow diagram of hot water extract polymer fraction Flow diagram of ethanol precipitation of enzyme-treated silver skin Manufacturing process flow chart of enzyme-treated polymer fraction Stepwise ethanol precipitation flow diagram of hot water extract of silver skin Graph 1 showing hyaluronidase inhibitory activity Graph 2 showing hyaluronidase inhibitory activity Explanatory diagram of utilization efficiency as a hyaluronidase inhibitor Graph showing heating conditions and solid yield Graph showing heating conditions and sugar yield Graph showing change over time in water ratio and solid yield Graph showing time-dependent change in water ratio and sugar yield Illustration of silver skin

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many changes and modifications can be made.

  FIG. 1 shows a hot water extraction and enzyme treatment flow of silver skin. Moreover, FIG. 2 and FIG. 3 have shown the manufacturing process flow of the hot water extract, and the manufacturing process flow of the enzyme processed material, respectively.

Here, the steps A1 to A4 in the flow of FIG. 2 are as follows.
A1) Hot water extraction step for extracting silver skin with hot water A2) Filtration step performed as a solid-liquid separation step for separating the extract obtained in the extraction step into a supernatant and a residue A3) Above obtained in the filtration step Drying process for drying the clear part A4) Recovery process for recovering the solid content obtained in the drying process

Further, the steps B1 to B6 in the flow of FIG. 3 are as follows.
B1) Hot water extraction process for extracting silver skin with hot water B2) Filtration process performed as a first solid-liquid separation process for separating the extract obtained in the extraction process into supernatant and residue B3) Obtained in the filtration process B4) Filtration step performed as a second solid-liquid separation step for separating the treatment product obtained in the enzyme treatment step into a supernatant and a residue B5) Above obtained in the filtration step Drying process for drying the clear portion B6) Recovery process for recovering the solid content obtained in the drying process

Next, the manufacturing process of a hot water extract and the manufacturing process of an enzyme processed material are demonstrated by a specific example.
First, as shown in FIG. 1, 180 L of distilled water was added to 4938 g of silver skin, and heated extraction was performed at 121 ° C. for 15 minutes. The obtained extract was filtered through a mesh and separated into a supernatant and a residue (primary filtration). And the supernatant of primary filtration was filtered using the filter aid silica (secondary filtration). Next, the supernatant was sterilized by passing through a 0.45 μm filter, concentrated, sterilized at 121 ° C. for 20 minutes, and spray-dried to obtain 831.6 g of a solid.
Let the obtained solid substance be a hot-water extract.

On the other hand, to 11 kg (weight including water) of the extraction residue obtained by the above primary filtration, 180 mL of 0.05 M acetate buffer (pH 5.0) and 60 mL of commercially available cellulase were added, and The enzyme treatment was performed for 16 hours and 30 minutes. Next, the enzyme-treated liquid was filtered and passed through a 0.45 μm filter for sterilization. After concentration, sterilization was performed at 121 ° C. for 15 minutes to obtain 1027.2 g of liquid.
Let the obtained liquid be an enzyme processed material.

FIG. 4 shows a 20% ethanol extraction flow of silver skin as one of extraction methods using an extraction solvent other than water. Moreover, FIG. 5 has shown the manufacturing process flow of an extraction residue soluble fraction among the refinements obtained by 20% ethanol extraction. Here, the steps C1 to C6 in the flow of FIG. 5 are as follows.
C1) Ethanol extraction step of extracting silver skin with ethanol solution C2) Filtration step performed as the first solid-liquid separation step of separating the extract obtained in ethanol extraction step into supernatant and residue C3) Obtained in filtration step C4) Filtration step performed as a second solid-liquid separation step of separating the extract obtained in the water extraction step into a supernatant and a residue C5) Above obtained in the filtration step Drying process for drying the clear portion C6) Recovery process for recovering the solid content obtained in the drying process

Next, the manufacturing process of an ethanol extraction residue soluble fraction is demonstrated with a specific example.
As shown in FIG. 4, 60.5 g of silver skin and 3000 mL of 20% (v / v) ethanol were added to a 20 L-volume plastic tank, and a shaking incubator (AT-24R, manufactured by Thomas Scientific Instruments Co., Ltd.) was added. ) For 16 hours under conditions of 25 ° C. and 50 rpm.
The obtained extract was filtered with a filter paper (Toyo Filter Paper No. 51), and separated into a supernatant and a residue. To this residue, 3000 mL of distilled water was added, and extraction was performed again with a shaking incubator under conditions of 25 ° C. and 50 rpm. The obtained extract was filtered with filter paper and separated into a supernatant and a residue. The supernatant was concentrated with a rotary evaporator and then lyophilized to obtain 0.85 g of a solid.
Let the obtained solid matter be a 20% ethanol extraction residue soluble fraction.

FIG. 6 shows an ethanol precipitation flow of the above-described silver skin hot water extract. FIG. 7 shows a manufacturing process flow of the hot water extract polymer fraction. Here, the steps D1 to D3 in the flow of FIG. 7 are as follows.
D1) Hot water extraction process for extracting silver skin with hot water D2) Filtration process performed as a solid-liquid separation process for separating the extract obtained in the hot water extraction process into supernatant and residue D3) Obtained in the filtration process Fractionation process for fractionating the polymer fraction contained in the supernatant

FIG. 8 shows an ethanol precipitation flow of the above-mentioned silver skin enzyme-treated product. FIG. 9 shows the manufacturing process flow of the enzyme-treated product polymer fraction. Here, the steps E1 to E5 in the flow of FIG. 9 are as follows.
E1) Hot water extraction step for extracting silver skin with hot water E2) Filtration step performed as a first solid-liquid separation step for separating the extract obtained in the hot water extraction step into supernatant and residue E3) In the filtration step Enzyme treatment step of subjecting the resulting residue to an enzyme treatment E4) Filtration step performed as a second solid-liquid separation step of separating the treatment product obtained in the enzyme treatment step into a supernatant and a residue E5) Obtained in the filtration step Fractionation process for fractionating the polymer fraction contained in the supernatant

Next, the manufacturing process of the hot water extract polymer fraction and the manufacturing process of the enzyme-treated product polymer fraction will be described as specific examples.
As shown in FIG. 6, 50 mL of distilled water was added to 5 g of hot water extract of silver skin and dissolved by heating at 90 ° C. To this solution, 200 mL of 99.5% ethanol was added and stirred, allowed to stand at −80 ° C., and then separated into a precipitate and a supernatant by centrifugation.
The obtained precipitate is freeze-dried to obtain a hot water extract polymer fraction. Hyaluronidase inhibitory activity was measured for this hot water extract polymer fraction.

On the other hand, as shown in FIG. 8, 820 mL of the enzyme-treated product was centrifuged, and the resulting supernatant was concentrated with a rotary evaporator. Thereafter, 99.5% ethanol was added and stirred so that the final concentration would be 80%, and the mixture was allowed to stand at −80 ° C. and then separated into a precipitate and a supernatant by centrifugation.
The precipitate obtained by freeze-drying is used as the enzyme-treated product polymer fraction. The hyaluronidase inhibitory activity was measured for this polymer-treated polymer fraction.

  FIG. 10 shows a flow obtained by subjecting the silver skin hot water extract to ethanol precipitation with stepwise ethanol concentration and fractionation depending on the molecular weight. 150 g of distilled water was added to 5 g of hot water extract of silver skin, and heated and dissolved at 90 ° C. To this solution, 30 mL of 99.5% ethanol was added and stirred, allowed to stand at −80 ° C. for 1 hour, and then separated into a precipitate and a supernatant by centrifugation. The resulting precipitate was lyophilized to give a 0-20% ppt fraction.

The supernatant was further stirred by adding 99.5% ethanol to a final concentration of 40%, and allowed to stand at −80 ° C. for 1 hour, and then separated into a precipitate and a supernatant by centrifugation. The resulting precipitate was lyophilized to give a 20-40% ppt fraction.
Further, the same operation was repeated two more times for the obtained supernatant, and lyophilized precipitates were obtained as a 40-60% ppt fraction and a 60-80% ppt fraction, respectively.
The finally obtained supernatant was concentrated by a rotary evaporator and then dried at 103 ° C. to obtain a sup fraction.
Using each of the obtained fractions, hyaluronidase inhibitory activity was measured.

  FIG. 11 shows a graph showing hyaluronidase inhibitory activity. Evaluation of the hyaluronidase inhibitory effect was performed according to a method applying the Morgan-Elson method. This is because N-acetylhexosamine is produced by the hydrolysis of hyaluronic acid by hyaluronidase, and the amount thereof is measured as an index.

  First, 40 μL of 10 mg / mL hyaluronidase (derived from bovine testicles, manufactured by Nacalai Tesque) dissolved in the same buffer solution is added to 200 μL of a test sample solution dissolved in 100 mM acetate buffer (pH 4.0), and the temperature is 37 ° C. For 20 minutes.

Next, 160 μL of an activator in which 0.1% (w / v) Compound 48/80 (manufactured by Nacalai Tesque), 2.5 mM CaCl ₂ , and 150 mM NaCl was dissolved was added to the same buffer, Incubate for 20 minutes at 0 ° C. to activate hyaluronidase. To this solution, 400 μL of 1 mg / mL potassium hyaluronate (derived from human umbilical cord, manufactured by Nacalai Tesque) was added and incubated at 37 ° C. for 40 minutes, followed by enzyme reaction, and then 100 μL of 0.5 N NaCl was added and cooled with water. Was stopped.

  Furthermore, 100 μL of 100 mM borate buffer (pH 9.1) was added to this solution, and the mixture was heated at 95 ° C. for 3 minutes, and then water-cooled again. 300 μL of p-dimethylaminobenzaldehyde reagent was added to 360 μL of this reaction solution, reacted at 37 ° C. for 5 minutes, and then the absorbance was measured at a wavelength of 550 nm with a microplate reader (manufactured by Corona Electric Co., Ltd.). Hyaluronidase inhibitory activity was calculated as an inhibition rate by the following formula (1).

(Equation 1)
Hyaluronidase inhibition rate (%)
= [(A−B) − (C−D)] / (A−B) × 100 (1)

Here, A indicates the absorbance (control) of the reaction solution to which the test sample is not added. Moreover, B has shown the light absorbency (blank) of the test sample and the solution which has not added the enzyme. C indicates the absorbance of the reaction solution to which the test sample is added. Moreover, D has shown the light absorbency (sample blank) of the solution which has not added the enzyme.
As a positive control, it was compared with a known component of cromoglycate sodium. Both the hot water extract polymer fraction and the enzyme-treated product polymer fraction exhibited almost the same inhibitory activity as sodium cromoglycate.

  FIG. 12 shows samples obtained by fractionating a hot water extract of silver skin in a molecular weight-dependent manner by ethanol precipitation (0-20% ppt, 20-40% ppt, 40-60% ppt, 60-80% ppt, sup. ) Hyaluronidase inhibitory activity. There was a tendency that the higher the molecular weight, the stronger the hyaluronidase inhibitory activity.

The results of calculating IC ₅₀ from the inhibitory activity of each sample are shown in Tables 1 and 2 below. In the table, the smaller the value, the stronger the hyaluronidase inhibitory effect.

  FIG. 13 shows the yield of each sample. As described above, 831.6 g of hot water extract having a hyaluronidase inhibitory activity function was obtained from 4938 g of silver skin. From this, the hot water extract was recovered from silver skin as a functional material with a high yield of 16.84%. Furthermore, as described above, the hot water extract can be further divided into samples fractionated in a molecular weight-dependent manner by the ethanol precipitation method, and the higher the molecular weight, the stronger the hyaluronidase inhibitory activity. That is, in order to obtain a stronger hyaluronidase inhibitor, it is preferable to recover the polymer fraction from an extract using water as an extraction solvent.

  As described above, 1027.2 g of an enzyme-treated product having a function of inhibiting hyaluronidase was obtained from 4938 g of silver skin. This means that the enzyme-treated product was recovered from silver skin as a functional material at a high yield of 20.80%.

  That is, from a silver skin that has been treated as an industrial waste, a hot water extract and an enzyme-treated product are recovered in a very high yield of 37.64% in total as a functional material having a hyaluronidase inhibitory activity function. It was done.

In Example 6, in the extraction using water as the elution solvent performed in the above Example, the influence on the recovery yield and component composition of the extract as a functional material due to the difference in temperature, time, and water addition ratio. It has been investigated.
14 to 17 show the solid yield and sugar yield of the extract when the extraction temperature and the extraction time are changed.

  The heating conditions will be described. First, 1.5g of silver skin and 150mL of distilled water are put into a heat-resistant bottle, and using a preheated autoclave, conditions of 105 ° C for 1 minute, 105 ° C for 20 minutes, 121 ° C for 1 minute, 121 ° C for 20 minutes And heated. After heating, when the temperature dropped to 100 ° C., the bottle was taken out from the autoclave and the residue was removed with Kim Towel (registered trademark).

  The obtained extract was measured for solid content and total sugar content. In the solid content measurement, first, insoluble matters in the extract were removed by centrifugation, and 3 mL of the supernatant was placed in an aluminum dish and dried at 99 ° C. for 4 hours. The weight after drying was measured and the solid yield was calculated. The total sugar amount was measured by the phenol sulfuric acid method. 0.5 mL of 5% (v / v) phenol and 2 mL of sulfuric acid are reacted with 0.5 mL of the diluted sample of the extract, and 200 μL of the reaction solution is collected and 450 nm using a microplate reader (Corona Electric Co., Ltd.). The absorbance was measured. Glucose was used to create a calibration curve, and the amount of sugar in the diluted sample was calculated from the calibration curve to determine the sugar yield. The respective measurement results are shown in FIGS.

Moreover, it experimented as follows about extraction time and water ratio.
Silver skin and distilled water were placed in an Erlenmeyer flask at a ratio of 3 g: 300 mL, 6 g: 300 mL, or 9 g: 300 mL, and stirred at 25 ° C. in a shaking incubator. After hydration, the time immediately after the silver skin was completely immersed in water was 0 hour, and the supernatant was collected after 4 hours, 8 hours, 14 hours, 18 hours, and 24 hours.
The recovered solution was freed from insolubles by centrifugation to obtain an extract.
The obtained extract was calculated for solid yield and sugar yield by the same method as described above. The respective measurement results are shown in FIGS.

From the measurement results, it can be seen that even if the extraction time, the extraction temperature, and the water ratio are different, there is no significant difference in the extraction efficiency. From this, it can be understood that the temperature, time, and water addition ratio are not particularly limited during extraction. Moreover, it turns out that there is no difference in a yield and the component composition of an extract by the difference in conditions of temperature, time, and a water addition ratio.
That is, in extraction using water as the extraction solvent, there is no significant difference in the hyaluronidase inhibitory activity of the extract between room temperature water and hot water, and there is no significant difference in yield and inhibitory activity.

  ADVANTAGE OF THE INVENTION According to this invention, the silver skin of the coffee conventionally processed mainly as industrial waste can be effectively utilized as a functional raw material, such as a hyaluronidase inhibitor.

Claims (15)

  Hyaluronidase inhibitor containing silver skin extract as an active ingredient.   The hyaluronidase inhibitor according to claim 1, wherein the active ingredient is a solid content obtained by solid-liquid separation of an extract extracted from silver skin using water as an extraction solvent and drying the supernatant.   The extract extracted from the silver skin using water as the extraction solvent is solid-liquid separated, the residue part is subjected to enzyme treatment, the solid part is separated, and the supernatant part is dried and the solid content is the active ingredient Item 5. A hyaluronidase inhibitor according to item 1.   The hyaluronidase inhibitor according to claim 1, comprising an extract extracted from silver skin using ethanol solution, dilute acid, or dilute alkali as an extraction solvent.   The hyaluronidase inhibitor according to claim 1, comprising a polymer fraction contained in a supernatant obtained by solid-liquid separation of an extract extracted from silver skin using water as an extraction solvent.   Effective extraction of the polymer fraction contained in the supernatant obtained by solid-liquid separation of the extract extracted from silver skin using water as the extraction solvent, and the residue with enzyme treatment The hyaluronidase inhibitor of claim 1 as a component.   Cosmetics containing the hyaluronidase inhibitor according to any one of claims 1 to 6.   Food-drinks containing the hyaluronidase inhibitor in any one of Claims 1-6.   The pharmaceutical composition containing the hyaluronidase inhibitor in any one of Claims 1-6.   A method for producing a hyaluronidase inhibitor using silver skin. An extraction step of extracting silver skin with water as an extraction solvent;
A solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
A drying step of drying the supernatant obtained in the solid-liquid separation step;
A recovery step of recovering the solid content obtained in the drying step;
A method for producing a hyaluronidase inhibitor according to claim 10. An extraction step of extracting silver skin with water as an extraction solvent;
A first solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
An enzyme treatment step for carrying out an enzyme treatment on the residue obtained in the first solid-liquid separation step;
A second solid-liquid separation step of separating the processed product obtained in the enzyme treatment step into a supernatant and a residue;
A drying step of drying the supernatant portion obtained in the second solid-liquid separation step;
A recovery step of recovering the solid content obtained in the drying step;
A method for producing a hyaluronidase inhibitor according to claim 10. An extraction step of extracting silver skin using an ethanol solution, dilute acid, or dilute alkali as an extraction solvent;
A solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
The manufacturing method of the hyaluronidase inhibitor of Claim 10 which comprises at least. An extraction step of extracting silver skin with water as an extraction solvent;
A solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
A fractionation step of fractionating the polymer fraction contained in the supernatant portion obtained in the solid-liquid separation step;
A method for producing a hyaluronidase inhibitor according to claim 10. An extraction step of extracting silver skin with water as an extraction solvent;
A first solid-liquid separation step of separating the extract obtained in the extraction step into a supernatant and a residue;
An enzyme treatment step for carrying out an enzyme treatment on the residue obtained in the first solid-liquid separation step;
A second solid-liquid separation step of separating the processed product obtained in the enzyme treatment step into a supernatant and a residue;
A fractionation step of fractionating a polymer fraction contained in the supernatant obtained in the second solid-liquid separation step;
A method for producing a hyaluronidase inhibitor according to claim 10.