WO2013042955A2

WO2013042955A2 - Cosmetic compositions including hydrolysate from silkworm gland

Info

Publication number: WO2013042955A2
Application number: PCT/KR2012/007539
Authority: WO
Inventors: Heui Sam Lee; Kang Sun Ryu; Mi Ja Kim; Jung Wook Hwang; Kyu Boem Han; Young Soo Choi; Ho Jin Kim
Original assignee: Republic Of Korea(Management : Rural Development Administration); Cha Bio & Diostech Co., Ltd.
Priority date: 2011-09-20
Filing date: 2012-09-20
Publication date: 2013-03-28
Also published as: WO2013042955A3; WO2013042955A9

Abstract

Disclosed is a cosmetic composition including a hydrolysate from a silkworm gland. The hydrolysate is prepared by separating a gland from a silkworm, dissolving the separated gland into water, centrifuging and obtaining a supernatant, adding an alkaline compound to conduct hydrolysis, neutralizing and desalting. The cosmetic composition is harmless to humans, and has a good antioxidant activity, a good dermal fibroblast proliferating property, a good skin affinity, etc.

Description

COSMETIC COMPOSITIONS INCLUDING HYDROLYSATE FROM SILKWORM GLAND

The present invention relates to cosmetic compositions including a hydrolysate from a silkworm gland, and more particularly, to cosmetic compositions including a hydrolysate from a silkworm gland, which are harmless to humans and has a good antioxidant activity, a good dermal fibroblast proliferating property, a good skin affinity, etc.

Most of generally used cosmetic ingredients use artificially synthesized compositions, and damage delicate skins or have an inferior regenerating effect to healthy and glowing skins.

In addition, the general cosmetic ingredients have serious defects of causing side effects such as a skin rash, allergy, etc. Recently, attemps on using natural products having a moisturizing effect and a good whitening effect in a beauty pack product are increasing. However, even though the natural products known until now are expected to provide the skin whitening effect, a moisture absorbing property and the moisturizing effect are not satisfactory.

Proteins composing a cocoon includes fibroin and sericin. Particularly, the sericin has a good cell proliferating effect and may be used as a serum substitute. In addition, the sericin is known as a promising main material in cosmetics because of the skin coating effect, the moisturizing effect, the antioxidant activity, the skin affinity, etc. thereof.

However, sericin obtained by a common method known until now, is a collagenic material and the material itself could not be used as the main material of the cosmetics. In addition, the common method of purifying and recovering the sericine uses a harmful solvent such as lithium bromide (LiBr), applies a heating process to a high temperature of 90℃ or over, etc. Thus, the producing process of the sericin is complicated and the yield thereof is low.

Related arts include Korean Registered Patent No. 10-0494229 (issue date: May 31, 2005, title: Manufacturing process of sericin and sericin mask pack) and Korean Registered Patent No. 10-0542266 (issue date: January 3, 2006, title: Face-pack composition comprising sericin protein extracted from cocoon).

The present invention provides a cosmetic composition including a hydrolysate from a silkworm gland, which may be obtained at a high yield by a simple extracting process without using a harmful solvent at room temperature, is harmless to human body, and has a high antioxidant activity, a good dermal fibroblast proliferating property, a good skin affinity, etc.

The technical problems of the present invention is not limited to the technical problems described above. Other technical problems will be readily understood by those skilled in the art from the following description.

According to an embodiment, a cosmetic composition includes a hydrolysate from a silkworm gland.

In example embodiments, the hydrolysate may be prepared by separating a gland from a silkworm, dissolving the separated gland into water, centrifuging and obtaining a supernatant, adding an alkaline compound to conduct hydrolysis, neutralizing and desalting.

In example embodiments, the hydrolysate may be included peptides obtained through the hydrolysis of sericin and fibroin.

In example embodiments, 0.001% to 50% by weight of the hydrolysate may be included based on a total amount of the cosmetic composition.

In example embodiments, the hydrolysis may be conducted at a temperatre of 15℃ to 30℃.

In example embodiments, the alkaline compound may be NaOH.

In example embodiments, the neutralizing may be conducted by using phosphoric acid.

In example embodiments, the cosmetic composition may have a formulation of an emollient (a skin lotion), a nutrition lotion, a nutrition cream, a massage cream or a nutrition pack.

In accordance with exemplary embodiments, a cosmetic composition including a hydrolysate from a silkworm gland, which may be obtained by a hydrolysis and an extraction at room temperature without using a harmful solvent and so does not harm to humans, is provided.

In addition, a cosmetic composition including a hydrolysate from a silkworm glant, and having a good antioxidant activity, a good dermal fibroblast proliferating property, a good skin affinity, etc., is provided.

FIG. 1 is a graph illustrating an antioxidant activity of the hydrolysate in exemplary embodiments according to solvents;

FIG. 2 is a graph illustrating cell toxicity testing results according to the concentration of an added hydrolysate;

FIG. 3 is a graph illustrating proliferating effects of a hydrolysate on human dermal fibroblasts in a serum-free medium;

FIG. 4a illustrates cell adhering effects and proliferated cell shapes according to the concentration of a coated hydrolysate on a cell culture plate;

FIG. 4b is a graph illustrating cell adhering effects and proliferating effects according to the concentration of a coated hydrolysate on a cell culture plate; and

FIG. 5 illustrates results of skin irritating test of a hydrolysate, in which;

A: left portion of a rabbit head "healthy skin (without scratches) - test material (hydrolysate of Example 1)"

B: right portion of a rabbit head "scratched skin - test material (hydrolysate of Example 1)"

C: left portion of a rabbit head "scratched skin - not treated"

D: right portion of a rabbit head "healthy skin (without scratches) - not treated."

Unless otherwise defined, all terms, techniques, etc. used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs.

Hereinafter, a cosmetic composition including a hydrolysate from a silkworm gland in accordance with the present inventive concept will be described in detail.

1. Preparation of a hydrolysate

A hydrolysate obtained from a silkworm gland is prepared.

The hydrolysate is prepared by hydrolyzing silk proteins obtained by dissolving a gland having a mixture state of fibroin and sericin at room temperature. Particularly, the hydrolysate is water-soluble so as to be easily dissolved in water.

Thus, the hydrolysate has a mixture state of all the materials obtained after hydrolyzing the sericin and the fibroin, and includes peptides and amino acids.

The hydrolysate may include all materials separated from the silkworm gland and may preferably include materials separated from the hyperfine powder of the silkworm gland.

The method for preparing the hydrolysate will be described in more detail as follows.

First, the gland is separated from the silkworm. In this case, the silkworm is preferably freeze-dried to separate the gland.

The species of the silkworm is Bombix mori species, however, is not limited to this species. Generally, any species of the silkworm possibly providing the gland may be used. Particularly, a grown-up silkworm having a good cell proliferating property and decreased raising periods and personnel expenses, is desired for preparing the hydrolysate.

Secondly, the separated gland is dissolved in water and centrifuged. Thus obtained supernatant is used.

The gland is desirably processed to a hyperfine powder state. Preferably, the gland is freeze-dried and then pulverized to obtain the hyperfine powder state.

When the gland is the hyperfine powder state, the gland may easily dissolve in water at room temperature (15 to 30℃), and the ingredient change thereof may be minimized at this temperature.

In addition, when the gland dissolves in water in the hyperfine powder state, the contacting area of the gland with water molecules may be maximized to facilitate the dissolution, and only the gland proteins dissolved in water at room temperature may be separated. More preferably, the particle size of the hyperfine powder of the gland is in a range of 600 to 800 ㎛.

Third, an alkaline compound is added into the supernatant to conduct hydrolysis.

The alkaline compound desirably includes sodium hydroxide (NaOH). The supernatant may be degraded into peptides having lower molecular weights by sodium hydroxide (NaOH).

Preferred final concentration of NaOH may be 0.2 to 1 M, and more preferably, 1 M. After adding NaOH solution, the hydrolysis is conducted at room temperature (15 to 30℃) for 0.5 to 1 hour, more preferably, is conducted while heating for 30 minutes. In other words, all of the silkworm gland is degradable at room temperature. In this case, the modification of a portion of thus obtained hydrolysate generated through the common treatment at a high temperature, may be prevented. In addition, the separation of the hydrolysate is possible without using a harmful solvent such as LiBr. That means the hydrolysate includes hydrolyzed peptides from the sericin and the fibroin.

Fourth, after completing the hydrolysis, a neutralizing process and a desalting process are conducted to prepare the hydrolysate from the silkworm gland.

In this case, the neutralizing process is conducted by adding an acid, preferably, phosphoric acid until the pH becomes 7.0 to 7.5.

Any common desalting process may be used as the desalting process. When the target material is a polymer having a molecular weight of several thousands or over, various methods utilizing a difference between molecular weights with the salt including may be used including a gel filtering, an ultrafiltration, a dialysis, etc.

Thus obtained hydrolysate in accordance with the present inventive concept, has a good serum-free addition effect and a good cell proliferating effect. Therefore, the hydrolysate may be used as a serum substitute and as a raw material having good properties such as a skin coating effect, a moisturizing property, an antioxidant activity, a skin affinity, etc.

2. Manufacture of the cosmetic composition in example embodiments

The hydrolysate prepared by the above-described method and having a good cell proliferating effect is dissolved to obtain a liquid state, and a cosmetic composition including the same is manufactured in accordance with the present inventive concept.

In this case, since the hydrolysate is readily water-soluble, the hydrolysate is preferably dissolved in water.

In addition, the cosmetic composition may most preferably include the hydrolysate exclusively as an effective ingredient, however, when considering economic aspects, the cosmetic composition may include the hydrolysate by 0.001 to 50% by weight, preferably, 0.1 to 15% by weight, and more preferably, 0.5 to 5% by weight based on the total amount of the cosmetic composition, and the remaining amount of acceptable materials as the cosmetic composition.

In this case, when the amount added of the hydrolysate is less than 0.001% by weight, the antioxidant activity, the dermal fibroblast proliferating property, the skin affinity, etc. may be insufficient, and when the amount of the hydrolysate exceeds 50% by weight, the same effect may be obtained as that obtained using 50% by weight.

The formulation of the cosmetic composition may be selected according to utilizing methods and objects.

Examples of the formulation include a lotion, a liquid formulation, an oil solution, an emulsion, a suspension, a tablet and a capsule.

The cosmetic composition may be used in cosmetics including basic cosmetics such as an emollient water, a milk lotion, a nutrition cream, a massage cream, an essence, a cleansing foam, a cleansing water, a pack, a body oil, etc. and cosmetics for makeup such as a foundation, a lip stick, a mascara, a makeup base, etc. The cosmetic composition may be mixed with common ingredients used for forming an external application of a skin such as an aqueous ingredient, an oily ingredient, a surfactant, a moisturizer, a viscosity increasing agent, an antiseptic, a flavoring, a pigment, etc. as occasion needs.

Example embodiments will be described more particularly by the following Examples and Experiments, however, the scope of the present inventive concept will not be limited thereto.

<Example 1> Preparing hydrolysate from silkworm gland

Grown-up silkworms were prepared as a silk raw material and were freeze-dried.

Glands were separated from the freeze-dried product, and the separated glands were added into a cryogenic mill and processed to obtain hyperfine powder.

Thus processes hyperfine powder of the silkworm gland was dissolved at room temperature (15 to 30℃) while stirring for 30 minutes. The solution was centrifuged and thus obtained supernatant was collected. Into the supernatant, NaOH was added so that the final concentration became 1 M and thus obtained product was stirred at 24℃ for 30 minutes.

Then, phosphoric acid was added to neutralize so as to adjust the pH of the product to 7, and a desalting process was conducted by means of an ultrafiltration.

After that, thus obtained product was freeze-dried again to obtain the hydrolysate in accordance with example embodiments.

<Example 2> Manufacturing cream type cosmetics in accordance with example embodiments

The hydrolysate according to Example 1 was dissolved in water and a cosmetic product having the following ingredients was manufactured.

Hydrolysate solution of Example 1 --- 5.0% by weight

Ethanol --- 2.0% by weight

PEG-20 --- 1.0% by weight

Sorbitan monostearate --- 1.0% by weight

Mineral oil --- 10.0% by weight

Trioctanoate --- 5.0% by weight

Triethanolamine --- 0.5% by weight

Carbomer --- 0.2% by weight

Glycerine --- 5.0% by weight

Propylene glycol --- 3.0% by weight

Antiseptic, flavoring --- moderate amount

Purified water --- remaining amount of the total of 100% by weight

<Example 3> Manufacturing ointment type cosmetics in accordance with example embodiments

Hydrolysate solution of Example 1 --- 5.0% by weight

Diethyl sebacate --- 8.0% by weight

Cetaceum --- 5.0% by weight

Polyoxyethylene oleyl ether phosphate --- 6.0% by weight

Sodium benzoate --- moderate amount

Vaseline --- remaining amount of the total of 100% by weight

<Example 4> Manufacturing essence type cosmetics in accordance with example embodiments

Hydrolysate solution of Example 1 --- 5.0% by weight

Propylene glycol --- 10.0% by weight

Glycerine --- 10.0% by weight

Aqueous sodium hyaluronate (1%) --- 5.0% by weight

Ethanol --- 5.0% by weight

Polyoxyethylene cured castor oil --- 1.0% by weight

Methyl paraoxy benzoate --- 0.1% by weight

Carbomer --- 0.4% by weight

Flavoring --- moderate amount

Purified water --- remaining amount of the total of 100% by weight

<Experiment 1> Analysis on antioxidant activity according to purifying process of the hydrolysate

In order to evaluate the antioxidant activity of the hydrolysate according to Example 1, a DPPH assay was conducted and the antioxidant activity (free radical scavenging activity) was compared with a vitamin C solution as a control.

The results are illustrated in the following Table 1.

Table 1

antioxidant activity (%)
number of assay	mean	1	2	3
15% vitamin C	97	97	97	97
hydrolysate of Example 1	82	82	81	84

As illustrated in Table 1, the hydrolysate of Example 1 showed similar results as the control.

Vitamin C is known as one of trace elements for maintaining the function of the human body and for keeping in good health. Vitamin C is resistant to infection, heals the wounds, and helps the tissues in keeping health. Vitamin C is one of the antioxidants and is known to prevent the cell damage due to free radicals.

Since the hydrolysate of example embodiments showed similar results as those of the control, the hydrolysate was acknowledged to have a high antioxidant activity.

From the comparison results on the antioxidant activity as illustrated in FIG. 1, it was confirmed that the hydrolysate according to Example 1 showed the highest oxidant activity when comparing with the extracts from the silkworm gland obtained by using harmful lithium bromide (LiBr) and Urea (organic compound of CO(NH₂)₂).

<Experiment 2> Evaluation on cell toxicity of the hydrolysate

In order to evaluate the cell toxicity of the hydrolysate, stem cells were cultured and the hydrolysate was added to confirm the cell toxicity by a CCK-8 assay.

According to the CCK-8 assay, tetrazolium salt in the treated cell through the culturing is degraded into formazan illustrating dark red by a mitochondrial succinate-tetrazolium-reductase system in the cell, and through detecting the formazan at 450 nm, the amount of viable cells may be quantitatively expressed.

Based on IC₅₀ concentration, the hydrolysate having the concentration of 0 to 5 mg/㎖ was treated by the culturing cell, and then, additional culturing was performed for two days. Then, the CCK-8 assay was conducted.

The suppression on the cell proliferation was not conducted as illustrated in FIG. 2. From the result, the hydrolysate according to Example 1, was confirmed to show no toxicity.

<Experiment 3> Measuring proliferating effect of human dermal fibroblast using the hydrolysate

The hydrolysate according to Example 1 was dissolved into DPBS (WelGENE, cat. LB 001-02) to a concentration of 20 mg/㎖, and then a serum removed medium was treated with the solution of the concentration of 0 mg/㎖ and 0.1 mg/㎖.

In order to compare the growth with a 10% serum added cell (10% FBS), a WST-1 assay (Daeillab) was conducted for four days and was measured once a day.

WST-1 assay reagent was added by 10% of the medium and was reacted for 3 hours at the same condition as the culturing. Then, absorbance at 450 nm was measured.

As illustrated in FIG. 3, an excellent cell proliferating effect was confirmed when 1 mg/㎖ of the hydrolysate from the silkworm gland was added into the serum-free medium, when comparing with other company's cocoon-derived sericin having the same concentration. From the result, the hydrolysate from the silkworm gland according to example embodiments was acknowledged to have an excellent effect in proliferating human dermal fibroblast. Therefore, when the hydrolysate is used as an ingredient of cosmetics, a good effect may be obtained.

<Experiment 4> Test on adherence of cell by the hydrolysate

The hydrolysate obtained according to Example 1 was dissolved in DPBS (WelGENE, cat. LB 001-02) to a concentration of 0.2 mg/㎖, 0.5 mg/㎖ and 1.0 mg/㎖, respectively. Then, the surface of a culture plate (SPL, 10035) was treated using the solution and stood for 1 hour at 37℃ in a culture incubator.

After that, the hydrolysate was removed, and then, the culture plate was washed using a phosphate buffered saline solution two times, and completely dried at room temperature.

The test on adherence of the cell by the hydrolysate was conducted by inoculating cells according to coated concentrations of the hydrolysate, culturing for two days and directly counting the number of proliferated cells.

As illustrated in FIG. 4A, it was confirmed that the amount of the cells adhered to the culture plate increased as the coating concentration of the hydrolysate increased after 30 minutes from the cell inoculation.

In addition, as illustrated in FIG. 4B, it was confirmed through verifying the total number of the proliferated cells after two days of the culturing that as the concentration of the coated hydrolysate increased, the amount of the proliferated cells increased.

Therefore, the hydrolysate obtained in accordance with example embodiments was acknowledged to function as an additive to promote the proliferation of the cells and to have an effect of promoting the proliferation of the cells when being coated on the culture plate.

<Experiment 5> Test on skin irritation of the hydrolysate

The test was conducted according to 'Standard for toxicity test of drugs, etc.' announced by Korea Food & Drug Administration.

In order to examine the skin irritation of the hydrolysate according to Example 1, 1% (w/v) of the hydrolysate of Example 1 was applied to a normal skin (skin without scratch) and a scratched skin of a New Zealand white male rabbit, and the results were compared and evaluated with those of not treated normal skin (skin without scratch) and scratched skin of the rabbit.

24 hours before the test, the hair on back portion of the rabbit was carefully removed using a depilator to prepare a healthy skin without scratches. The skin was divided into left and right to prepare four sections having a size of 2.5 cm x 2.5 cm, respectively.

A sufficient amount of the hydrolysate according to Example 1 was coated onto the normal skin and the scratched skin. The results were scored and recorded at 24, 48 and 72 hours after the coating according to the standard of skin reaction.

As illustrated in FIG. 5, the hydrolysate of Example 1 was evaluated as non-irritant (evaluation index 0 to 0.5) at the final irritation test. Thus, the hydrolysate was confirmed as a raw material causing no irritation to the skin.

As described above, a hydrolysate may be obtained through an extraction at room temperature, and a cosmetic composition using a hydrolysate from a silkworm gland, which is harmless to human, and having a good antioxidant activity, a good skin cell proliferating property, a good skin affinity, etc. may be provided in accordance with example embodiments.

The present inventive concept has been described with reference to the specific embodiments and experiments. It will be readily understood by those skilled in the art that various modifications and changes can be made thereto without departing from the spirit and scope of the present invention defined by the appended claims. Many modifications are possible in the example embodiments without departing from the present inventive concept. Accordingly, all such modifications are intended to be included within the scope of the present inventive concept as defined in the claims.

Claims

A cosmetic composition comprising a hydrolysate from a silkworm gland.
The cosmetic composition of claim 1, wherein the hydrolysate is prepared by separating a gland from a silkworm, dissolving the separated gland into water, centrifuging and obtaining a supernatant, adding an alkaline compound to conduct hydrolysis, neutralizing and desalting.
The cosmetic composition of claim 1, wherein the hydrolysate includes peptides obtained through the hydrolysis of sericin and fibroin.
The cosmetic composition of claim 1, wherein 0.001 to 50% by weight of the hydrolysate based on a total amount of the cosmetic composition is included.
The cosmetic composition of claim 2, wherein the hydrolysis is conducted at a temperature of 15 to 30℃.
The cosmetic composition of claim 2, wherein the alkaline compound is NaOH.
The cosmetic composition of claim 2, wherein the neutralizing is conducted by using phosphoric acid.
The cosmetic composition of any one of claims 1 to 7, wherein the cosmetic composition has one formulation among an emollient water, an essence, a lotion, a cream, a pack, a foundation, a gel, an ointment and a spray.