KR20000056836A - The extracting method of sericin and use thereof - Google Patents

Abstract

PURPOSE: An extraction method of sericin is provided. The sericin has no unpleasant smell and can be used as cosmetic base supplement for moisturizing and elasticity of the skin as the sericin aqueous solution has excellent elastase inhibiting activity and hyaluronase inhibiting activity. CONSTITUTION: An extraction method of sericin comprises the following steps of: selecting and drying cocoon; adding 5-40 times, based on the volume of dried cocoon, of water or buffer solution; boiling at the temperature of 90-130°C for 1/2-1 hours under the condition that a cooling condenser is installed not to distill the solvent; precipitating by using the solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or mixture thereof to get the precipitate having no smell; filtering and drying the precipitate.

Description

Extraction method of sericin and its use {The extracting method of sericin and use kind}

The present invention relates to a method of extracting sericin and its use, and more particularly, to a method of extracting silk sericin from silkworm cocoon and to the use of silk sericin as an elastase inhibitor and a hyaluronase inhibitor.

In the skin aging mechanism, free radicals or free radicals are generated by ultraviolet rays or external environmental factors. Cell membranes are attacked by the generated free radicals or free radicals, and the skin is inflammated. Inflammated tissue causes skin aging to destroy proteins and genetic material and form wrinkles by physiological progression mechanisms. Inhibiting and preventing all steps involved in the skin aging mechanism can fundamentally reduce skin aging. The above operation is described in detail as follows.

In the skin, elastic fibers, along with collagen fibers, form crosslinks inside the dermis. As you age, the elasticity of the elastinase, an elastin-degrading enzyme, is secreted from fibroblasts and causes a sudden decrease in skin elasticity and sagging. Histologically, the infiltration of inflammatory cells is increased, the deficiency and aggregation of elastin fibers, and the reduction of collagen fibers are shown. Biochemically, the activity of elastase is significantly increased. Elastase is the only enzyme known so far to break down elastin, and its inhibition can fundamentally reduce skin aging. In order to delay skin aging, a method of combining nutrition and additives with cosmetics in tissues in which moisturizers, anti-inflammatory agents, elastin and collagen cross-links are broken is used, which is fundamentally limited in delaying aging. to be. Therefore, there is a need for inhibitors that fundamentally inhibit the degradation of elastin and collagen to maintain skin elasticity. Due to the disadvantage that the raw material having a conventional anti-aging effect is not fundamentally inhibited has been attempted to find new functional materials. In particular, synthetic peptide derivatives having a structure similar to that of collagen and elastin, which are conventional animal extracts, are used, but efforts to find new functional substances have been attempted due to the disadvantages of insufficient safety, stability, and effects.

The hyaluronicase action will be described in detail. The hyaluronate is an enzyme distributed in the living body and is also present in the skin and decomposes hyaluronic acid. Hyaluronic acid is a linear polymer polysaccharide in which β-D-N-acetylglucosamine and β-D-glucouronic acid bind to each other, and is a type of glucosamino glucan (GAG). In the action of hyaluronic acid in the connective tissue, the cells retain moisture, and the matrix forms within the tissue, thereby retaining the moisture. Hyaluronic acid decreases with age, and as a result, water retention decreases, causing aging. Therefore, inhibiting the activity of hyaluronic acid that decomposes hyaluronic acid contributes to the stability of hyaluronic acid used in the preparation and to the stability of hyaluronic acid and hyaluronic acid present in the skin in the formulation after application to the skin.

In view of the above, the present invention has searched for a material effective in preventing aging among various natural substances, and found that the extract extracted from silkworm cocoon exhibits very good elastase inhibitory effect and hyaluronase inhibitory effect. It solved the stability and safety issues at the same time

However, the silk sericin (Korean Patent Publication No. 95-5839) extracted from the existing cocoon has a big problem to use as a cosmetic additive due to a very hateful odor.

Moreover, the situation regarding the use of sericin as an antioxidant or a tyrosinase inhibitor has already been developed (Korean Patent Laid-Open Publication No. 98-042150), and its use as an elastase inhibitor or a hyaluronase inhibitor is not known.

Accordingly, it is an object of the present invention to provide a silk sericin extract that does not smell off and a preparation method thereof.

Another object of the present invention is to provide novel antielastase and antihyaluronase.

The object of the present invention is to screen the silkworm cocoon and then completely dry and add 5 to 40 times the volume of water and a buffer solution as the extraction solvent with respect to dry weight, and then a cooling condenser is installed to prevent the solvent from evaporating. Sericin, characterized in that the solids extracted by heating 90-130 C, extracted for 30 minutes to 10 hours, precipitated in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof are filtered and dried. It is achieved by the extraction method.

The object of the present invention is also achieved by using sericin as an antielastase and an antihyaluronase.

Hereinafter, the present invention will be described in detail.

Silkworms are raised in large quantities for the purpose of supplying and receiving silk in Korea, China and Eastern Europe. In Korea, the silk has been produced in large quantities since the 1960s, but recently, most of the silk is dependent on imports. The silkworm cocoon used in the present invention is a purely domestic silkworm cocoon, which corresponds to a dog dog, which is sterilized and extracted in water or a buffered solution at a predetermined ratio and then precipitated in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof. To obtain solids. In the case of mixing with the cosmetic composition, it is used by dissolving a certain amount in purified water again. This extract is added to cosmetics, that is, nourishing cosmetics (lotion), nourishing cream, massage creams, essences, packs, emulsified foundations, and the like.

That is, the silkworm cocoon is first screened, and then dried completely, and then 5 to 40 times by volume of water and a buffered aqueous solution are used as the extraction solvent with respect to dry weight. Extraction method is performed by heating at 90-130 C for 30 minutes to 10 hours in a state where a cooling condenser is installed to prevent the solvent from evaporating. The extract is precipitated in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof, and the solids deodorized are filtered and dried. The extract obtained by dissolving the solid content again in the concentration of 0.1-10% in purified water is added to the cosmetic in an amount of 0.1-5% by weight, preferably 0.5-2%.

The following Examples and Experimental Examples illustrate the content of the invention, but the content of the invention is not limited thereto.

Example 1.

100 g of cocoon is completely dried and then placed in 20 kg of purified water and boiled for 5 hours in an extractor equipped with a cooling condenser. The extract is filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20 L of ethanol and filtered to obtain a solid of 10 g. Was dissolved in and filtered through a filter of 0.45 μm to obtain an aqueous solution of 1 kg.

Example 2.

100 g of cocoon is completely dried and then placed in 20 kg of purified water, extracted by boiling in an extractor equipped with a cooling condenser for 5 hours, filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20 L of methanol, filtered, and then filtered to obtain 10 g of solid content. Was dissolved in and filtered through a 0.45 µm filter to obtain an aqueous solution of 1 kg.

Example 3.

100 g of cocoon was completely dried and then placed in 20 kg of 0.2M phosphate buffer, boiled for 3 hours in an extractor equipped with a cooling condenser, filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20 L of ethanol, and filtered to obtain 15 g of solid content. After dissolving again in water, it filtered with a 0.45 micrometer filter and obtained 1.5 kg of aqueous solution.

Example 4.

100 g of cocoon was completely dried and then placed in 20 kg of 0.2 M phosphate buffer, boiled for 3 hours in an extractor equipped with a cooling condenser, filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20 L of methanol, and filtered to obtain 15 g of solid content. After dissolving again in water, it filtered with a 0.45 micrometer filter and obtained 1.5 kg of aqueous solution.

Example 5.

100 g of cocoon is completely dried and then placed in 20 kg of purified water, extracted by boiling in an extractor equipped with a cooling condenser for 3 hours, filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20 L of acetone, and filtered to obtain 8 g of solid content. Was dissolved in and filtered through a 0.45 µm filter to obtain an 8 kg aqueous solution.

Example 6.

After completely drying 100g of cocoon, put it in 20kg of purified water, boil it for 3 hours in an extractor equipped with a cooling condenser, filter it with 400 mesh filter cloth, cool it to room temperature, precipitate it in 20L of acetonitrile, and filter it to obtain 15g solids. The solution was dissolved in water and filtered through a 0.45 µm filter to obtain 1.5 kg of aqueous solution.

Example 7.

100g of silkworm cocoons were completely dried and then poured into 20kg of purified water, extracted by boiling in an extractor equipped with a cooling condenser for 3 hours, filtered through a 400 mesh filter cloth, cooled to room temperature, precipitated in 20L of tetrahydrofuran, and filtered to obtain 15g solids. The solution was dissolved in water and filtered through a 0.45 µm filter to obtain 1.5 kg of aqueous solution.

Prescription Example 1.

A prescription example of a nourishing lotion (lotion) in cosmetics containing an aqueous solution of sericin is as follows.

Here, the aqueous solution of sericin was used in Example 1.

Raw material Weight part Sericin aqueous solution 1.0 Beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 5.0 Squalane 5.0 Caprylic / Capric Triglycerides 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxy Vinyl Polymer 0.1 Triethanolamine 0.2 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Remaining amount

Prescription Example 2

A prescription example of nutrient cream in cosmetics containing sericin aqueous solution is as follows.

Here, the sericin aqueous solution says the thing of Example 1.

Raw material Weight part Sericin aqueous solution 1.0 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 10.0 Squalane 5.0 Caprylic / Capric Triglycerides 5.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Remaining amount

Prescription Example 3.

A prescription example of a massage cream in cosmetics containing an aqueous solution of sericin is as follows.

Here, the sericin aqueous solution says the thing of Example 1.

Raw material Weight part Sericin aqueous solution 1.0 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 Caprylic / Capric Triglycerides 4.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Remaining amount

Prescription Example 4.

A prescription example of a pack in cosmetics containing an aqueous solution of sericin is as follows.

Here, the sericin aqueous solution says the thing of Example 1.

Raw material Weight part Sericin aqueous solution 1.0 Polyvinyl alcohol 13.0 Sodium Carboxymethylcellulose 0.2 Allantoin 0.1 ethanol 5.0 Nonylphenyl Ether 0.3 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Remaining amount

Prescription Example 5

Prescription examples of emulsified foundations in cosmetics containing sericin aqueous solution are as follows.

Here, the sericin aqueous solution says the thing of Example 1.

Raw material Weight part Sericin aqueous solution 1.0 Beeswax 2.0 Pyromethicone 2.0 Liquid paraffin 5.0 Squalane 5.0 Stearic acid 2.0 Caprylic / Capric Triglycerides 4.0 glycerin 4.0 Propylene glycol 3.0 Butylene glycol 3.0 Triethanolamine 1.0 Aluminum Magnesium Silicate 0.5 Pigment 12 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Remaining amount

Experimental Example 1.

The elastase inhibitory effect was measured about the said sericin aqueous solution 1-7.

1) Experiment Method

Elastase was extracted from porcine pancreas and used Sigma. An increase in the amount of 4-nitroaniline released per unit time at a wavelength of 410 nm was determined as the catalytic activity of elastase by a test based on the hydrolysis of synthetic peptide substrate (Sigma) by pancreatic elastase. First, 200 μl of substrate solution is added to the test tube, and Tris buffer solution and distilled water are added. 0.3 ml of each sericin aqueous solution was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat.

In this case, only 0.3 ml of solvent was added instead of each aqueous solution of silk sericin, and the absorbance at wavelength 410 nm was measured with a spectrophotometer. The elastase inhibitory effect of each extract is obtained by the following formula.

% Elastase Inhibition = [1-(Enzyme Activity of Each Silk Sericin Solution / Enzyme Activity of Control)] X 100

The results are shown in Table 1 below.

Table 1.Elastase Inhibitory Effects on Sericin Aqueous Solution

Example Final concentration of sericin aqueous solution contained in the reaction solution (㎍ / ㎖) Elastase Inhibition Rate (%) Example 1 157.5 10050 Example 2 157.5 10057 Example 3 157.5 10048 Example 4 157.5 10035 Example 5 157.5 10066 Example 6 157.5 10070 Example 7 157.5 10053

Experimental Example 2.

Hyaluronase inhibitory effect was measured with respect to the sericin aqueous solution of the sericin aqueous solution 1-7.

1) Experimental method.

Hyalurase (Sigma, Type 4) was used to measure the inhibitory activity of the activation step of the inactive enzyme by compound 48/80. A sample of sericin aqueous solution was dissolved in 100 ml of 0.1 M acetic acid buffer (pH 4.0), placed in a test tube, and 0.1 mg of the enzyme dissolved in 50 ml of acetic acid buffer was added and left to stand at 37 degrees for 20 minutes. Finally, 200 mg of sodium hyaluronate dissolved in 250 ml of acetate buffer was added and allowed to stand at 37 ° C for 40 minutes. After incubation for 20 minutes, absorbance is measured at 585 nm. As a control, acetic acid buffer is used as the sericin sample solution. The inhibitory effect was expressed as (%) by multiplying 100 by the ratio of the absorbance of the control test and the absorbance containing sericin aqueous solution.

The results are reported in Table 2 below.

Table 2. Effect of Hyaluronase Inhibition on Sericin Aqueous Solution

Example Final concentration of sericin aqueous solution contained in the reaction solution (㎍ / ㎖) Hyaluronicase Inhibition Rate (%) Example 1 2010 9042 Example 2 2010 8344 Example 3 2010 6832 Example 4 2010 9548 Example 5 2010 8140 Example 6 2010 8034 Example 7 2010 7735

Experimental Example 3.

1) Experiment Method

To test the effect of sericin aqueous solution on skin elasticity, it was applied twice a day to the left eye edge of 20 people. Untreated right eye edge was used as a control. The skin elasticity of the skin surface was measured by Corneometer CM80 and Cutometer SEM 474 before and 14 days after treatment with the cosmetic solution containing sericin solution. Skin moisture measurement by Corneometer CM 820 showed the increased moisturizing value in% compared to the control group and the average value of 20 subjects was shown in the results. Skin elasticity measurement by Cutometer SEM 474 is a measure of the depth of wrinkles, the smaller the value is the better elasticity. The elasticity value was shown as a percentage decrease compared to the control, and the average value of 20 subjects was shown in the results.

2) Experiment result

Table 3. Effect of Sericin Aqueous Solution on Skin Surface Moisturizing and Elasticity

Prescription Skin moisturizing effect (%) Skin elasticity effect (%) Prescription Example 1 30 37 Prescription Example 2 33 38 Prescription Example 3 40 49 Prescription Example 4 29 35 Prescription Example 5 34 40

The sericin aqueous solution obtained in the present invention was found to have an effect of improving the skin condition due to obvious skin elasticity and aging even in the experimental results of applying directly to human skin by combining a certain concentration or more in a cosmetic composition.

The sericin extract extracted by the method of the present invention did not smell off unlike the sericin extract extracted by the conventional method, the aqueous solution of sericin was excellent in the elastase inhibitory effect, hyaluronase inhibitory effect. Therefore, it can be used as a cosmetic additive effective for increasing the moisturizing and elasticity of the skin.

Claims (7)

Use of silk sericin extracted from silkworm cocoons as anti-elastase. The method of claim 1, wherein the extraction is selected after the cocoon and dried completely, 5-5 times the volume of water and a buffer solution as the extraction solvent with respect to dry weight, and then a cooling condenser is installed to evaporate the solvent. Extracted by heating at 90-130 C for 30 minutes to 10 hours in a prevented state, and then precipitated in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof to perform deodorizing solids by filtration and drying. Characterized in that the method. Use of silk sericin extracted from cocoon as antihyaluronase. The method of claim 3, wherein the extraction is selected for the cocoon and dried completely, and then 5-5 times the volume of water and a buffered solution as the extraction solvent with respect to dry weight, and then a cooling condenser is installed to evaporate the solvent. Extracted by heating at 90-130 C for 30 minutes to 10 hours in a prevented state, and then precipitated in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof to perform deodorizing solids by filtration and drying. Characterized in that the method. After silkworm cocoon is completely dried, add 5-40 times the volume of water and buffered solution as the extraction solvent with respect to dry weight, and then use a cooling condenser to prevent evaporation of the solvent at 90-130 C, Anti-aging cosmetic composition containing an aqueous solution of silk sericin obtained by heating and extracting for 30 minutes to 10 hours, and then filtering and drying the solid deodorized by precipitation in methanol, ethanol, acetone, acetonitrile, tetrahydrofuran or a mixed solvent thereof. . The cosmetic composition according to claim 5, wherein the concentration of the aqueous sericin solution is 0.1-10% by weight. 7. The cosmetic composition according to claim 6, wherein the sericin aqueous solution is contained in an amount of 0.1-5% by weight.