CN107334718A - Collagen, which produces, to be reduced inhibitor, the composition for external application and cosmetics for reducing inhibitor is produced comprising the collagen - Google Patents

Abstract

It is that using the zymolyte of bird's nest as active ingredient, in dermal fibroblast the collagen as caused by irradiating ultraviolet produces reduced inhibitor that the collagen of the present invention, which produces and reduces inhibitor,.According to the present invention, there is provided can efficiently produce and collagen safe, in dermal fibroblast produces the excellent inhibitor reduced, and external preparation and cosmetics comprising the inhibitor.

Description

Collagen production reduction inhibitor, skin outer skin containing the collagen production reduction inhibitor Compositions and Cosmetics

This application is a divisional application of a patent application with an application date of June 21, 2010, an application number of 201080028249.3, and an invention title of "collagen production reduction inhibitor, skin external composition and cosmetics containing the collagen production reduction inhibitor" .

References to related applications

This application is based on Japanese Patent Application No. 2009-148772 (filing date: June 23, 2009 ) filed earlier, and claims the benefit of priority thereof, and the entire disclosure thereof is incorporated herein by reference.

technical field

The present invention relates to an inhibitor of collagen production reduction in dermal fibroblasts, which uses enzymatic hydrolyzate of bird's nest as an active ingredient. More specifically, the present invention relates to a collagen production decrease inhibitor that suppresses the decrease in type I collagen production that occurs in skin dermal fibroblasts due to ultraviolet irradiation. In addition, the present invention relates to a composition for external use on skin and cosmetics comprising an enzymatic hydrolyzate of bird's nest as an active ingredient thereof.

Background technique

Skin problems such as rough skin and/or wrinkles, sagging, and decreased elasticity of the skin are important concerns especially for women. These skin problems are generally thought to be caused by environmental factors, factors that accompany aging such as aging, and psychological factors such as stress. In particular, environmental factors that adversely affect the skin due to drying of the living environment, such as the increase in ultraviolet rays and the extensive use of air conditioners in recent years, are increasing.

Skin tissue includes epidermis, dermis, and subcutaneous tissue. The dermis contains a large amount of collagen and hyaluronic acid, which are closely related to the water retention and elasticity of the skin. Dermal fibroblasts present in the dermis are known to produce collagen and hyaluronic acid.

It is generally considered that collagen accounts for approximately 30% of proteins present in the human body, and 70% of which is present in the dermis. Collagens present in the dermis include type I collagen and type III collagen, but mainly type I collagen.

Conventionally, in order to improve the elasticity and luster of the skin and prevent and improve wrinkles, cosmetics containing collagen have been used externally to replenish collagen directly to the skin. However, in the case of topical collagen supplementation, it is difficult to increase the amount of collagen in the dermis of the skin, so it cannot be said that it is always sufficient for improving the elasticity and/or luster of the skin, and preventing and improving wrinkles.

It is known that when the skin is exposed to light including ultraviolet rays, the collagen occupying most of the extracellular matrix of the dermis is significantly reduced, and the fiber bundles thereof are scattered. Therefore, increasing the amount of collagen in the dermis is useful for the improvement and prevention of wrinkles, etc. (Non-document 1 (“Method for the development of functional cosmetic materials のための実験 method (experimental method for developing functional cosmetic raw materials)” (Yo Editor-in-Chief Pixuaner), published in 2007, Published by シーエムシー, pages 45-47)). In addition, this document shows that the collagen synthesis of dermal fibroblasts is greatly reduced by irradiation with ultraviolet rays (UVB). In this document, vitamin C derivatives, whey components, and the like are exemplified as examples of type I collagen synthesis accelerators.

On the other hand, it has been shown in literature (Akiko Kojima-Yuasa, et al., Fragrance Journal, April, 2009, No. 346 (Vol. 37, No. 4), pp. 103-) that inhibiting collagen production ability Reduction is useful for improvement of QOL, and reduction of the amount of collagen in the skin causes wrinkles, sagging, and the like.

Therefore, from the viewpoint of preventing or improving the elasticity and/or luster of the skin caused by collagen reduction in skin exposed to ultraviolet rays, and preventing or improving wrinkles, it is possible to suppress or improve the growth of dermal fibroblasts that have been irradiated with ultraviolet rays. The phenomenon of decreased collagen synthesis is important.

However, although many attempts have been made to promote the production of type I collagen produced by dermal fibroblasts (for example, Japanese Patent Application Laid-Open No. 2008-105984, WO2004/085429), there has been no consensus on direct inhibition or improvement. As far as the inventors of the present invention are aware, almost no studies have been conducted on the reduction of collagen synthesis in dermal fibroblasts irradiated with ultraviolet rays itself to suppress the collagen in the dermis.

The bird's nest is a nest made by making its own saliva thread-like, and its components are known to contain a large amount of glycoprotein and contain almost no lipid. Conventionally, bird's nest has been used as an edible raw material of Chinese cuisine, as a raw material of traditional Chinese medicine, and the like. In recent years, health foods and the like using bird's nest as a raw material have also been developed, but only a few examples of application to external use such as external preparations and cosmetics are known.

JP-A-2003-95961 discloses an oral skin beautifying agent using an enzymatic hydrolyzate of bird's nest. However, in this document, only oral use is shown, and there is neither description nor suggestion about the possibility of utilization in external use. In addition, the effect here is to improve the moist feeling of the skin and obtain fine-textured skin. There is no description about the ability to promote the production of type I collagen accompanied by the improvement of skin elasticity and/or luster and the improvement of wrinkles. Inhibition of the decrease in collagen synthesis of dermal fibroblasts irradiated with ultraviolet light is neither described nor suggested.

In WO99/022709 a cosmetic product comprising an aqueous solvent extract, in particular a hot water extract, of bird's nest is disclosed. This cosmetic can be used for skin care and is a cosmetic used for external use. In addition, in this document, it is also disclosed that an aqueous extract of bird's nest shows a collagen synthesis promoting effect in skin cells. However, the extract used here is the extract obtained by extracting bird's nest with hot water and removing the solid content, and it is clearly different from the enzymatic hydrolyzate of bird's nest which is used as it is by enzymatically treating the whole bird's nest. In addition, this document neither describes nor suggests that the decrease in collagen synthesis of dermal fibroblasts irradiated with ultraviolet rays can be suppressed.

Contents of the invention

The present inventors unexpectedly found that the enzymatic hydrolyzate of bird's nest can effectively inhibit the reduction of type I collagen synthesis observed in skin dermal fibroblasts irradiated by ultraviolet rays. The present invention has been accomplished based on such knowledge.

In addition, the present inventors also found that the enzymatic hydrolyzate of bird's nest has an excellent effect of promoting the production of type I collagen in skin dermal fibroblasts.

Accordingly, an object of the present invention is to provide an excellent inhibitor of collagen production reduction in dermal fibroblasts that can be efficiently produced and is highly safe, and an external preparation and cosmetics containing the inhibitor.

The collagen production reduction inhibitor of the present invention uses bird's nest enzymatic hydrolyzate as an active ingredient, and is an inhibitor of collagen production reduction in dermal fibroblasts caused by ultraviolet irradiation.

According to a preferred mode of the present invention, the above-mentioned hydrolyzate is a protease hydrolyzate.

According to a more preferred aspect of the present invention, the molecular weight of the above-mentioned enzymatic hydrolyzate is 70-200,000.

According to a further preferred aspect of the present invention, the average molecular weight of the above-mentioned enzymatic hydrolyzate is 1,000-50,000.

According to a preferred aspect of the present invention, the collagen production decrease inhibitor of the present invention is used to suppress the decrease in type I collagen production in skin dermal fibroblasts due to ultraviolet irradiation.

According to another aspect of the present invention, there is provided a composition for external use on skin containing the collagen production reduction inhibitor of the present invention. The composition is preferably used for skin care before or after ultraviolet irradiation, and more preferably for preventing or improving the decrease in skin elasticity and luster and preventing or improving wrinkles that occur in ultraviolet-irradiated skin.

According to another more preferable aspect of the present invention, the external composition for skin of the present invention is a cosmetic.

According to another aspect of the present invention, there is provided a use of an enzymatic hydrolyzate of bird's nest for producing an inhibitor of collagen production decrease caused by ultraviolet irradiation in dermal fibroblasts of mammals including humans. According to another aspect of this invention, the use of the enzymatic hydrolyzate of bird's nest for manufacturing the external skin composition for skin care before or after ultraviolet-ray irradiation is provided.

According to another aspect of the present invention, there is provided a method for inhibiting collagen production reduction caused by ultraviolet irradiation in dermal fibroblasts, which comprises applying an effective amount of the enzymatic hydrolyzate as an active ingredient of the present invention to mammals including humans skin. According to another aspect of the present invention, there is provided a skin care method comprising applying an effective amount of the active ingredient of the present invention to the skin of mammals including humans before or after being irradiated with ultraviolet rays. Likewise, there is also provided a method for improving the elasticity and luster of the skin that has been irradiated with ultraviolet rays, preventing or improving wrinkles, which comprises applying an effective amount of the active ingredient of the present invention to mammals including humans. skin.

According to the present invention, by using the enzymatic hydrolyzate of bird's nest as an active ingredient, it is possible to effectively suppress and improve the decrease in collagen production that occurs in dermal fibroblasts derived from human skin. In particular, an effective suppression or improvement effect can be obtained on the decrease in the production of type I collagen observed in dermal fibroblasts of the skin irradiated with ultraviolet rays. In addition, based on the inhibitory effect on the reduction of collagen production in dermal fibroblasts, by using the enzymatic hydrolyzate for external skin use or cosmetic use, it is possible to improve the elasticity of the skin and the luster of the skin accompanying the reduction of collagen production in fibroblasts. Reduce, and prevent or improve wrinkles.

In particular, these effects are effective for skin in a state where collagen synthesis is reduced in sun-exposed skin. Thus, according to the present invention, it is possible to improve the QOL of consumers. In addition, since bird's nest, which is well-known as an edible raw material, is used as the enzymatic hydrolyzate, it is highly safe and easy to accept by consumers. Moreover, since the enzymatic hydrolyzate can be obtained by enzymatically treating bird's nest as it is, it can be manufactured efficiently, and manufacturing cost can also be suppressed low.

Description of drawings

Fig. 1 is a graph showing the measurement results of the amount of sialic acid in a bird's nest sample.

Fig. 2 is a graph showing the measurement results of the protein amount in bird's nest samples.

Fig. 3 is a graph showing measurement results of bird's nest enzymatic hydrolyzate 1 obtained by GPC-HPLC.

Fig. 4 is a graph showing measurement results of enzymatic hydrolyzate 2 of bird's nest obtained by GPC-HPLC.

Fig. 5 is a graph showing the measurement results of the ability to promote collagen production.

detailed description

Active ingredients

As mentioned above, the collagen production reduction inhibitor of this invention uses the enzymatic hydrolyzate of bird's nest as an active ingredient.

Bird's nest, which is a raw material of the enzymatic hydrolyzate, is a nest made by nest swallows by making their own saliva filamentous, and contains a large amount of glycoprotein as its component, and contains almost no lipid. Here, the cave swallow refers to the swallow class belonging to the swiftlet genus ( Collocalia ) belonging to the swift family ( Apodidae ), for example, swiftlet ( Apodidae Collocalia francica ), moss-nest swiftlet ( Apodidae Collocalia salangana ), swiftlet ( Apodidae Collocalia fuciphaga ) , swiftlet ( Apodidae Collocalia inexpectata ) , swiftlet ( Apodidae Collocalia vestita ) , swiftlet ( Apodidae Collocalia vestita ), white-bellied swiftlet ( Apodidae Collocalia esculenta) , Swift Swift ( Apodidae Collocalia maximus) , etc.

In this invention, a commercial item can be used for bird's nest. In general commercially available bird's nests, there are many kinds of bird's nests, such as bird's nests obtained by simply removing dirt such as hair and feces and washing, to bird's nests formed by collecting bird's nest debris and repeatedly bleaching and washing, but the bird's nest used in the present invention It is preferable to use the bird's nest which did not perform excessive washing, bleaching, etc. in the pretreatment.

The enzymatic hydrolyzate of bird's nest refers to a substance obtained by decomposing bird's nest by enzymatic reaction, and bird's nest or its processed product can be obtained by decomposing it with protease, a compound enzyme containing protease, or the like. As the enzyme used in the decomposition treatment here, protease is preferable, and trypsin is more preferable.

Generally, the enzymatic hydrolyzate of bird's nest can be obtained by first implementing heat sterilization treatment and then performing enzyme treatment in water. The conditions of the sterilization treatment can be appropriately set with reference to conventional heat sterilization conditions, and the conditions of the enzyme treatment can also be set appropriately according to the kind of enzyme to be used and the state of the bird's nest.

If a more specific example is given, the enzymatic hydrolyzate of bird's nest can be prepared as follows.

For example, 10 to 50 times the mass of water is added to the pulverized bird's nest to make it swell, and then heat and sterilize at 60 to 130° C. for 5 seconds to 30 minutes. Then, an appropriate amount of enzyme is directly added, and the enzyme reaction is carried out for 0.5 to 48 hours at the optimal pH and temperature of the enzyme. After the enzyme reaction is completed, the enzyme is inactivated by heat treatment or the like, and the reaction solution is filtered to remove insoluble matter, thereby obtaining an enzymatic hydrolyzate of bird's nest. In addition, this enzymatic hydrolyzate can be dried and powderized as needed. In addition, operations such as appropriate pH adjustment, decolorization, and deodorization can be performed in each of the above-mentioned steps.

As another example, when enzymatically hydrolyzing the processed product of bird's nest, water 10 to 100 times the mass is added to bird's nest crushed to a particle size of 2 mm or less, preferably 150 μm or less, and left to stand at 1 to 60° C. Or stir for 0.5 to 48 hours to make it swell, then heat and sterilize at 60 to 130°C for 5 seconds to 30 minutes, filter if necessary, and perform enzyme treatment on the resulting solution in the same manner as above to obtain enzymatic hydrolysis of bird's nest things.

In the enzyme hydrolyzate preparation method, the longer the enzymatic reaction time is, the more the decomposition of protein is promoted, and the enzyme hydrolyzate containing lower molecular weight protein is obtained. In the present invention, the enzyme reaction time is preferably about 0.5 to 48 hours, preferably about 3 to 36 hours, more preferably about 24 hours, and the enzymatic hydrolyzate is further subjected to the enzyme reaction.

The molecular weight of the enzymatic hydrolyzate of bird's nest obtained in this way is preferably 70-200,000, more preferably 70-180,000. More preferably, the molecular weight is 70 to 150,000, and still more preferably, it is 70 to 120,000.

Moreover, it is preferable that the average molecular weight (weight average molecular weight) of the enzymolyzate of bird's nest is 300-100,000, and it is more preferable that it is 500-70,000. The average molecular weight is more preferably 700 to 60,000, and still more preferably 1,000 to 50,000.

In addition, regarding bird's nest enzymatic hydrolyzate, the lower limit of the above-mentioned average molecular weight may be preferably 5,000 or more, more preferably 10,000 or more, and even more preferably It is 15,000 or more, and more preferably 20,000 or more.

skin topical composition

The collagen production reduction inhibitor of the present invention can be used alone as it is, but it can also be included as an additive in various skin external compositions such as cosmetics, pharmaceuticals, and quasi-drugs to obtain the effect of suppressing the reduction of collagen production in dermal fibroblasts Compositions. The obtained composition can be effectively used for skin care, preferably effective for skin care before or after being irradiated with ultraviolet rays, and more preferably effective for improving the elasticity and luster of skin occurring in skin irradiated with ultraviolet rays. Reduce, prevent or improve wrinkles.

The enzymatic hydrolyzate of bird's nest as an active ingredient of the present invention has the activity of effectively suppressing the decrease in the production of type I collagen observed in human skin dermal fibroblasts irradiated with ultraviolet rays (test results in Examples described later) .

Therefore, in the present invention, the inhibitory activity of collagen production reduction refers to the effect of suppressing or improving the collagen production ability reduction that occurs in fibroblasts of the human skin dermis, thereby meaning that the collagen production ability in the human skin dermis can be suppressed or improved. Quantitative phenomenon. By inhibiting or improving the decrease in the amount of collagen in the dermis, it is possible to obtain the improvement and preventive effect of wrinkles, and it is well known to those skilled in the art that the decrease in skin elasticity and skin luster accompanying the decrease in collagen production can be prevented or improved. It can be easily understood that this point is found in (for example, the above-mentioned literature ("Functional Cosmetic Materials の开発のための実験法 (Experimental Method for Developing Functional Cosmetic Raw Materials)" (Edited by Yupi Xuaner), 2007 Published in 2009, published by シーエムシー, pages 45-47 (Non-Patent Document 1)), literature (Akiko Kojima-Yuasa, et al., Fragrance Journal, April, 2009, No.346 (Vol.37, No.4), pp.103-(Non-Patent Document 2)) and the like are clearly shown.

In particular, these effects can be said to be effective for skin in a state where collagen synthesis is reduced in sun-exposed skin. Therefore, since it was confirmed that the active ingredient of the present invention has an inhibitory activity for reducing collagen production in dermal fibroblasts, it became clear that the active ingredient of the present invention is effective in skin care. In particular, it has been clarified that the active ingredient of the present invention is effective in improving skin care before or after exposure to ultraviolet rays, and more preferably in improving skin elasticity and luster reduction that occurs in skin exposed to ultraviolet rays, and preventing or improving wrinkles. It is vaild. In addition, the active ingredients of the present invention are also effective in improving the decrease in skin elasticity due to aging, preventing wrinkles, and the like.

In addition, in this specification, "prevention or improvement" of skin or skin conditions such as reduction of skin elasticity and/or luster, wrinkles, etc., includes adjustment of the conditions, delay of progress, alleviation, prevention of onset, prevention of recurrence, suppression, etc. mean to use.

Therefore, as described above, according to another aspect of the present invention, there is provided a composition for external use on skin comprising the collagen production reduction inhibitor of the present invention. Preferably, a composition for external use on skin is provided as a drug, a quasi-drug, or a cosmetic, and it is more preferable to provide a composition for external use on skin as a cosmetic.

In the composition for external use on skin (especially cosmetics) of the present invention, the content (in terms of dry matter) of bird's nest enzymolyzate as an active ingredient is not particularly limited, and varies depending on the form of the composition, but in general, as a solid Components are in the range of 0.001 to 20% by weight, preferably in the range of 0.005 to 5% by weight, and can be appropriately changed depending on the form of the composition.

Alternatively, the bird's nest enzymatic hydrolyzate content (in terms of dry matter) in the external skin composition of the present invention is preferably 0.01 mg or more per day, more preferably 0.01 mg to 2 mg, and can be applied per 1 square cm of skin.

The composition for external use on skin of the present invention may further contain other arbitrary components as long as the composition for external use on skin of the present invention contains an active ingredient and does not impair the effect of inhibiting the reduction of collagen production in dermal fibroblasts. Such optional components include, for example, various components commonly used in skin external preparations and cosmetics, such as humectants, lower alcohols, polyhydric alcohols, sugars, surfactants, buffers, emulsifiers, stabilizers, enhancers, etc. Thickeners, antioxidants, antiseptic and antibacterial agents, chelating agents, pH regulators, fragrances, pigments, ultraviolet absorbers, ultraviolet scattering agents, vitamins, amino acids, anti-inflammatory agents, water, peptides, sugar alcohols, enzymes , plant extracts, antioxidant substances, talc, clay, pollen, pearls, etc.

Here, examples of humectants include acidic mucopolysaccharides such as polyethylene glycol, polypropylene glycol, glycerin, propylene glycol, sorbitol, hyaluronic acid, and chondroitin sulfate, amino acids, collagen, and elastin.

As a lower alcohol, ethanol, propanol, isopropanol etc. are mentioned, for example.

As a polyhydric alcohol, glycerin, pentaerythritol, dipentaerythritol, ethylene glycol, propylene glycol, polypropylene glycol, 1, 3- butanediol etc. are mentioned, for example.

Examples of sugars include sugars such as glucose, maltose, lactose, D-glucuronic acid, sucrose, D-sorbitol, sorbitan, cellulose, and starch, oligosaccharides, and polysaccharides. and their derivatives etc.

The skin external composition of the present invention can be prepared as a lotion, emulsion, or cream by appropriately blending and adjusting the collagen production reduction inhibitor containing bird's nest enzymatic hydrolyzate and these optional ingredients according to known methods. It is the usual product form in various cosmetics such as moisturizing liquid, mask agent, lotion, body water, body cream and so on. The composition for external use on the skin of the present invention can also be used as makeup cosmetics such as foundations, lipsticks, eye shadows, and blushes, medicinal cosmetics such as sunscreen products and deodorant cosmetics, hair cosmetics such as shampoos, hair conditioners, and hair oils, and skin washing products. Agents, bath cosmetics for bath preparations, etc.

According to another aspect of the present invention, as described above, there is provided the use of an enzymatic hydrolyzate of bird's nest for producing an inhibitor of collagen production decrease caused by ultraviolet irradiation in dermal fibroblasts of mammals including humans. Here, the suppression of decreased collagen production in dermal fibroblasts by ultraviolet irradiation is preferably suppression of decreased production of type I collagen in skin dermal fibroblasts due to ultraviolet irradiation.

According to another aspect of this invention, as mentioned above, the use of the enzymatic hydrolyzate of bird's nest for manufacturing the external skin composition for skin care before or after ultraviolet irradiation is provided. Here, the composition for external use on the skin is preferably used for preventing or improving the decrease in skin elasticity and luster and wrinkles that occur in skin irradiated with ultraviolet rays. In addition, the composition for external use on skin is preferably a cosmetic.

According to another aspect of the present invention, as described above, there is provided a method for inhibiting collagen production reduction caused by ultraviolet irradiation in dermal fibroblasts, which comprises applying an effective amount of an enzymatic hydrolyzate as an active ingredient of the present invention to Human mammalian skin. In addition, the "effective amount" here refers to an amount sufficient for exhibiting the inhibitory activity of reducing collagen production in a desired region in the body depending on the application. Here, the suppression of decreased collagen production in dermal fibroblasts by ultraviolet irradiation is preferably suppression of decreased production of type I collagen in skin dermal fibroblasts due to ultraviolet irradiation.

In addition, according to another aspect of the present invention, there is provided a skin care method comprising applying an effective amount of the active ingredient of the present invention to the skin of mammals including humans before or after being irradiated with ultraviolet rays. Likewise, it also provides a method for improving the elasticity and luster of the skin that has been irradiated by ultraviolet rays, preventing or improving wrinkles in the skin that has been irradiated by ultraviolet rays, which comprises an effective amount of the effective amount of the present invention. The ingredients are applied to the skin of mammals including humans. In addition, the "effective amount" here refers to an amount sufficient to obtain the desired effect.

In addition, in this specification, the expression using the value of "about" and "approximately" means that, in order to achieve the purpose of setting the value, changes in the value that can be tolerated by those skilled in the art are included. It means, for example, that a variation within 20%, preferably within 10%, and more preferably within 5% of the specified value or range can be tolerated.

Example

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

(1) Preparation of bird's nest samples

(1-1) Bird's nest enzymatic hydrolyzate 1

Commercially available unbleached bird's nest is pulverized with a pulverizer to obtain bird's nest powder with a 100-mesh pass (below particle size 150 μm), add about 50 times the amount (mass) of water to the bird's nest powder, and swell at 5°C for 20 hours, and then sterilized at 121°C for 15 minutes.

After cooling the obtained treatment solution, the pH was adjusted, and an enzyme containing protease (trade name "Pancleachin F", manufactured by Amano Pharmaceutical Co., Ltd.) was added in an amount of 2% by mass relative to the bird's nest powder, and reacted at 45° C. for 3 hours.

The enzyme reaction solution was adjusted to pH 7.0, then heated at 90° C. for 5 minutes to inactivate the enzyme, filtered, and the filtrate was recovered. The filtrate was concentrated and then freeze-dried to obtain "bird's nest hydrolyzate 1".

(1-2) Bird's nest enzymatic hydrolyzate 2

Except having made the time of enzyme reaction 24 hours, it carried out similarly to the enzymatic hydrolyzate 1 of said (1-1) term, and obtained "bird's nest enzymatic hydrolyzate 2".

(1-3) Hot water extract of bird's nest (comparative example)

40 g of commercially available unbleached bird's nest (dried product) was pulverized with a pulverizer, the pulverized product was added to 1000 ml of distilled water, and extraction treatment was performed for 2 hours under heating and reflux. After heating to reflux, let stand, then separate the supernatant and filter, and recover the filtrate. The residue was further added to 1000 ml of distilled water in the same manner as above, followed by re-extraction under heating and reflux, and the solid residue was removed by filtration, and the filtrate was recovered. The obtained filtrates (extracts) were combined, concentrated under reduced pressure, and the obtained concentrate was freeze-dried to obtain a yellow-white "hot water extract of bird's nest" (comparative example).

(2) Analysis of bird's nest samples

The amount of sialic acid and the amount of protein in each of the obtained bird's nest samples (bird's nest enzymatic hydrolyzate 1, bird's nest hydrolyzate 2, and bird's nest hot water extract) were measured as follows.

The amount of sialic acid was measured as free N-acetylneuraminic acid by high performance liquid chromatography after acid hydrolyzing each sample.

The amount of protein was measured using a Bio Rad protein assay kit (Protein Assay Kit) based on the Brad Ford method.

The results are shown in Figure 1 and Figure 2.

Next, in order to measure the molecular weight and distribution of the protein contained in the enzymatic hydrolyzate of bird's nest 1 and the enzymatic bird's nest 2, it analyzed by GPC under the following conditions.

[Measurement conditions of HPLC]

■Column: Shodex Asahipak GS320HQ (φ7.6×300mm)

■Column temperature: 35℃

■Mobile phase: 50mM CH ₃ COONH ₃ pH6.7

■Flow rate: 0.4ml/min

■Sample injection volume: 10μl

■Detector: UV spectroscopic detector (measured under UV220nm)

[Molecular weight markers used]

■BSA MW 66,000

■Gly-gly MW 132.1

■Gly-gly-gGly MW 189.17

■L-Aspartic Acid MW133.1

■DL-Aspartic Acid MW 150.1

■N-Acetylneuraminic acid MW 309.3

The measurement results are shown in Fig. 3 and Fig. 4 .

Moreover, the molecular weight of the protein contained in the bird's nest hydrolyzate was evaluated using the Western blotting method. Specifically, first, the amount of protein in the enzymatic hydrolyzate of bird's nest was confirmed according to the above-mentioned measuring method. Protein (100 μg) derived from bird's nest hydrolyzate was separated using 10-15% polyacrylamide using SDS-PAGE. The protein developed on the gel was transferred to an immunoblotting PVDF membrane (polyvinylidene fluoride, available from Bio-Lad Lab). PVDF membranes were blocked in PBS containing 3% skim milk and 0.1% Tween-20 for 1 hour, judged visually.

In addition, when evaluating the molecular weight of the protein, the data shown in the known literature (Guo, C.T., Suzuki Y., et al., Antiviral Res. 70, pp. 140-146 (2006) was also referred to.

From these results, it was found that the protein molecular weight contained in the enzymatic hydrolyzate of bird's nest exists in the range of about 70 to 120,000 (in addition, depending on evaluation conditions, the upper limit may be about 200,000).

In addition, from the obtained results, it can be seen that the average molecular weight of the protein in the bird's nest hydrolyzate 1 is about 55,000. Moreover, it turned out that the average molecular weight of the protein in the bird's nest hydrolyzate 2 was about 33,000.

(3) Inhibition effect on reduction of collagen production in dermal fibroblasts associated with ultraviolet irradiation

Normal human skin dermal fibroblasts (KF-4109, supplied by クラボウ) were seeded at a cell density of ² × 104 cells on a 96-well plate using DMEM medium (obtained from SIGMA) containing 0.5% calf serum (FBS). obtained by the society). 24 hours after the inoculation, it was exchanged with the DMEM medium containing 0.5% FBS containing each bird's nest sample of the following predetermined concentration, and cultured for another 24 hours.

In addition, regarding the sample concentration, it is as follows:

(i) Concentration of the hydrolyzate (0, 78.1, 312.5, 1250, 5000 μg/ml),

(ii) Concentration of hot water extract (0, 1.56, 6.25, 25, 100 μg/ml).

After culturing, a part of the culture supernatant was recovered, and the amount of type I collagen in the supernatant was measured by the ELISA method.

It can be confirmed from the measurement results that the enzymatic hydrolyzate of bird's nest can significantly increase the production of type I collagen in human skin dermal fibroblasts. This is a significant difference compared with the case of the hot water extract which is a comparative example.

After culturing, further in Hank's buffer solution containing each bird's nest sample at a predetermined concentration, after irradiating UVB (ultraviolet rays) (using an ultraviolet irradiator (TL20W/12RS manufactured by Phillips Corporation), UV short-wavelength cut filter (cut on wavelength 300nm))), exchanged with DMEM medium containing 0.5% FBS, and cultured for another 24 hours.

After culturing, the culture supernatant was recovered, and the amount of type I collagen in the supernatant was measured by the ELISA method.

Cells were lysed with 0.1% Triton X-100 solution, and then the protein was quantified.

In the ELISA method, the primary antibody uses anti-human type I collagen antibody (rabbit), the secondary antibody uses HistofinePO (rabbit), and the chromogenic reagent uses 2,2'-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS), using a microplate reader to measure the absorbance at 405 nm.

The amount of protein was quantified by the BCA method (bicinchoninic acid method) (a method of reacting copper ions reacted with a peptide bond with BCA (bicinchoninic acid) and measuring absorption at a wavelength of 562 nm).

The amount of type I collagen in the medium was calculated as the amount of type I collagen produced per unit protein amount by dividing the amount of type I collagen in the medium by the protein amount of whole cells.

The result is shown in Figure 3.

From the results, it was confirmed that the enzymatic hydrolyzate of bird's nest significantly suppressed the reduction of type I collagen production in dermal fibroblasts caused by UVB irradiation. Furthermore, in the case of the hot water extract (comparative example), such an inhibitory effect was not found.

Claims (5)

1. An application of trypsin hydrolyzate of bird's nest for the manufacture of sunscreen products, The average molecular weight of the hydrolyzate is 33000-55000, Said applications do not include those for moisturizing, anti-wrinkle or whitening. 2. An application of a trypsin hydrolyzate of bird's nest, for the manufacture of an inhibitor of collagen production reduction caused by ultraviolet irradiation in dermal fibroblasts of mammals including humans, The average molecular weight of the hydrolyzate is 33000-55000, Said applications do not include those for moisturizing, anti-wrinkle or whitening. 3. The use according to claim 2, the inhibition of decreased collagen production in dermal fibroblasts caused by UV irradiation is the inhibition of decreased collagen production of type I occurring in skin dermal fibroblasts due to UV irradiation. 4. The application according to claim 2 or 3, which is used to manufacture an external skin composition for skin care before or after being irradiated with ultraviolet rays. 5. The application according to any one of claims 2 to 4, wherein the composition for external use on skin is a cosmetic.