WO2011159098A2 - Method for preparing broussonetia kazinoki extract - Google Patents

Abstract

The present invention provides a method for preparing a Broussonetia kazinoki extract, comprising the following steps: solvent extracting Broussonetia kazinoki; isolating the Broussonetia kazinoki extract, extracted in the previous step; and crystallizing the Broussonetia kazinoki extract isolated in the previous step. It is possible to prepare a Broussonetia kazinoki extract having a remarkable skin whitening effect and excellent stability through the method for preparing a Broussonetia kazinoki extract.

Description

How to prepare the extract

The present invention relates to a method of preparing a mulberry extract.

Melanin is a black-brown pigment that has a function of blocking more than a certain amount of ultraviolet rays to protect the skin from ultraviolet rays. Therefore, when the skin is exposed to ultraviolet rays, a large amount of melanin is produced in the body to protect the skin. Since the skin color is determined by the amount of melanin and the amount of melanin increases, the skin becomes black. Therefore, the production of large amounts of melanin causes spots, spots, freckles, and blotch. Therefore, substances that inhibit melanin production may have a skin whitening effect.

Tyrosinase is an enzyme that converts tyrosine into dopa and oxidizes it to dopa quinine. Dopaquinine molecules continue to polymerize to form melanin. That is, since tyrosinase acts as a melanin-producing trigger, a substance that inhibits it may have a skin whitening effect.

The present invention is to provide a method for producing a mulberry extract showing an excellent stability while containing the active ingredient in high purity. In addition, it is prepared by the method of preparing the mulberry extract, to provide a high-purity skin whitening active ingredient, to provide a skin whitening mulberry extract having an excellent melanin production and tyrosinase activity inhibitory effect. And to provide an external composition for skin having an excellent skin whitening effect including the extract of the mulberry.

One aspect of the present invention comprises the steps of solvent extraction of the mulberry ( Broussonetia kazinoki ); Leaving the mulberry extract extracted in the step; And it provides a method of producing a mulberry extract comprising the step of crystallizing the mulberry extract neglected in the step.

Another aspect of the present invention provides a skin whitening mulberry extract prepared by the method of manufacturing the mulberry extract.

Another aspect of the present invention provides a skin external preparation composition comprising the extract of the mulberry.

According to one aspect of the present invention, the method of preparing the mulberry extract may include the active ingredient in high purity and at the same time produce a mulberry extract showing excellent stability.

The mulberry extract according to another aspect of the present invention is prepared by the method of extract of the mulberry extract exhibits excellent skin whitening effect and excellent stability at the same time.

The external preparation composition for skin according to another aspect of the present invention has an excellent skin whitening effect including the extract of the mulberry.

Figure 1 is a graph showing the relationship between the casinoin C content (%) and tyrosinase inhibitory effect (%) of the mulberry extract.

Figure 2 is a graph of HPLC analysis results showing that the HSCCC fraction contains the casinoin C.

As used herein, the term "extract" includes any material obtained by extracting a component therefrom from a natural product, regardless of the method of extraction or the kind of the component. For example, it is a broad concept that includes all the components which are dissolved in a solvent from a natural product using water or an organic solvent, and those obtained by extracting only a specific component such as an oil such as oil.

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a broad concept including not only tissues covering the body surface such as the face or body, but also the scalp and hair.

Hereinafter, the present invention will be described in detail.

It contains a skin whitening active ingredient called kazinol F, which is known to exhibit a skin whitening effect by inhibiting tyrosinase. Casinol C contained in the mulberry extract is also excellent in inhibiting tyrosinase, which is known to be superior to tyrosinase inhibition than kojic acid. However, the skin lightening active ingredients have an unstable titer, which makes it difficult to commercialize them. Therefore, if there is a method of preparing a mulberry extract showing excellent stability while containing the skin whitening active ingredients in high purity, it is possible to prepare a mulberry extract having a further improved tyrosinase inhibitory effect, it will be easier to commercialize.

One aspect of the present invention comprises the steps of solvent extraction of the mulberry; Leaving the mulberry extract extracted in the step; And it provides a method of producing a mulberry extract comprising the step of crystallizing the mulberry extract neglected in the step.

In the above, the mulberry ( Broussonetia kazinoki ) is a plant belonging to the nettle mulberry family. In one aspect of the present invention, the paper mulberry includes oak tree, hawthorn or tridar. In another aspect of the present invention, the mulberry may be 1 to 3 years old mulberry. In another aspect of the present invention, the mulberry includes one or more of all parts of the mulberry. For example, the mulberry may comprise one or more of the leaves, flowers, stems, branches, roots, fruits and seeds of the mulberry. In another aspect of the invention, the mulberry may be the root bark of the mulberry.

The method of preparing a mulberry extract according to an aspect of the present invention may further include preparing, by washing, drying, and cutting the mulberry prior to the solvent extraction of the mulberry. Specifically, the step of preparing the mulberry is a process of cutting off the root portion and peeling off the root after collecting the mulberry; Washing the bark of the peeled root with water and drying for 5 to 7 days in a well-ventilated place; The method may include cutting the dried mulberry root bark to about 10 to 20 cm in length.

The method for preparing a mulberry extract according to an aspect of the present invention includes the step of solvent extracting the mulberry. In one aspect of the invention, the solvent includes, but is not limited to, an organic solvent, specifically alcohol, ether, ethyl acetate, acetone or chloroform. The alcohol includes a C ₁ ~ C ₅ lower alcohol, C ₁ ~ C ₅ lower alcohol is any selected from the group consisting of methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol and isobutanol It includes, but is not limited to, one or more mixed solvents. In one aspect of the invention, the solvent may be included in a weight corresponding to 5 to 20 times the weight of the mulberry.

In one aspect of the invention, the extraction may be made for 1 to 10 hours at 50 to 100 ℃. In another aspect of the present invention, the extraction may be made for 3 to 8 hours at 60 to 90 ℃. In another aspect of the invention, the extraction may be made for 4 to 6 hours at 60 to 80 ℃.

According to an aspect of the present invention, the method of preparing a mulberry extract may further include removing the insoluble component by filtering the mulberry extract after the solvent extraction of the mulberry. In one aspect of the invention, the filtration may be made using filter paper.

 The method of preparing a mulberry extract according to an aspect of the present invention may further include a step of concentrating the filtered mulberry extract after the filtering of the mulberry extract. In one aspect of the invention, the temperature at the time of concentration may not exceed 80 ° C, specifically 60 ° C, and may proceed until the volume of the extract is less than 1/10 to 1/20 of the volume before the concentration.

The method of manufacturing a mulberry extract according to an aspect of the present invention includes the step of leaving the extracted mulberry extract. In one aspect of the invention, the neglect may proceed at low temperatures. In another aspect of the invention, the leaving may be performed for 1 to 40 hours at -5-25 ℃. In another aspect of the invention, the neglect may be carried out for 5 to 30 hours at 0 to 20 ℃. In another aspect of the invention, the neglect may be performed at 0 to 15 ℃ for 10 to 25 hours. Impurities can be precipitated through such a low temperature, and the precipitated components are substances with low solubility in solvents, and do not affect the effects of the extracts of mulberry, such as inhibiting melanogenesis or tyrosinase. Are substances that do not. Through this, the effect of the extract of the mulberry, for example, melanin production inhibition or tyrosinase inhibitory effect may be further improved.

According to an aspect of the present invention, the method for preparing a mulberry extract may further include a step of removing the insoluble components precipitated by filtering the mulberry extract after leaving the extracted mulberry extract. In one aspect of the invention, the filtration may be made using filter paper.

The method of preparing a mulberry extract according to an aspect of the present invention may further include reconcentrating the filtered mulberry extract. In one aspect of the present invention, the temperature during reconcentration may not exceed 80 ° C, specifically 60 ° C, and may proceed until the volume of the extract is less than one-half to one-third the volume before reconcentration. .

The method for preparing a mulberry extract according to an aspect of the present invention includes the step of crystallizing the mulberry extract. In one aspect of the invention, the crystallization may proceed with stirring for 1 to 20 hours to put the mulberry extract in water of -5 to 15 ℃. In another aspect of the present invention, the crystallization may proceed with stirring for 1 to 15 hours to put the mulberry extract in water of 0 to 10 ℃. In another aspect of the invention, the crystallization may be carried out while stirring for 1 to 10 hours to put the mulberry extract in water of 0 to 7 ℃. In another aspect of the invention, the water may be purified water having a volume of 5 to 30 times, specifically 10 to 20 times the volume of the mulberry extract. The higher the amount of water added and the lower the temperature, the better the crystallization. Through this crystallization, the effect of the extract of the mulberry, for example, tyrosinase inhibitory effect can be further improved.

The method of preparing a mulberry extract according to an aspect of the present invention may further include the step of leaving the mulberry extract after crystallizing the mulberry extract. In one aspect of the invention, the leaving may be performed for 10 to 30 hours at 0 to 10 ℃. This process allows for better precipitation. In another aspect of the present invention, the method of preparing a mulberry extract may include the step of removing the solution of the upper layer when precipitation is formed and obtaining a crystallized mulberry extract.

The method of preparing a mulberry extract according to an aspect of the present invention may further include the step of drying the mulberry extract to prepare a powder after the step of obtaining the crystallized mulberry extract. In the drying, all conventional drying methods may be used, and in particular, a freezing drying method may be used.

According to an aspect of the present invention, the method of preparing a mulberry extract may further include, after crystallizing the mulberry extract, supporting the mulberry extract on a carrier. This step can improve the potency stability of the active ingredient in the extract of the mulberry. The active ingredient includes a component that exhibits a skin lightening effect. In particular, the active ingredient may be Casinol C.

In one aspect of the invention, the carrier is polylactic acid (polylactic acid, PLA), polyethylene adipate (polyethyleneadipate, PEA), polymethyl methacrylate (polymethylmethacrylate (PMMA), polybutyl methacrylate (polybutylmethacrylate, PBMA), Trimethylammoniumethylmethacrylate (TMAEMA), solid lipid nanoparticles (SLN) and silica (silica), one or more selected from the group consisting of two or more. In another aspect of the invention, the carrier is polylactic acid (polylactic acid, PLA), polylactic acid and polybutyl methacrylate / trimethylammonium ethyl methacrylate (polybutylmethacrylate / trimethylammoniumethylmethacrylate, PBMA / TMAEMA) copolymer, polyethylene adipate (polyethyleneadipate, PEA), polymethylmethacrylate (PMMA), polyethylene adipate and polymethyl methacrylate, solid lipid nano particles (SLN) or silica (silica). By supporting the mulberry extract on a carrier as described above, the stability can be increased efficiently and easily.

In one aspect of the invention, the mulberry extract may be 0.01 to 30% by weight based on the weight of the carrier. In another aspect of the present invention, the extract of Methanol may be 0.1 to 20% by weight based on the weight of the carrier. In another aspect of the present invention, the extract of Methanol may be 1.0 to 10% by weight based on the weight of the carrier. When used in the above range, it is not only suitable for showing the intended effect of the present invention, but also satisfies both the stability and safety of the extract, and may be appropriate to use in the above range in terms of cost-effectiveness.

In one aspect of the present invention, the method of supporting the mulberry extract on the carrier may be by a conventionally known method.

According to one aspect of the present invention, the method of preparing a mulberry extract further comprises the step of obtaining the skin whitening active ingredient from the mulberry extract using HSCCC (High speed counter current chromatography) of the biphasic solvent system after crystallizing the mulberry extract. It may include. In another aspect of the invention, the skin lightening active ingredient comprises kazinol C. The method can separate and obtain the skin lightening active ingredient by using the difference in the partition coefficient of each ingredient.

For more accurate separation, a two-phase solvent system in which the skin whitening active ingredient is separated into two phases upon mixing, but whose distribution coefficient of the skin whitening active ingredient is close to one, may be selected. At this time, the partition coefficient of other ingredients does not overlap with the partition coefficient of the skin whitening active ingredient.

In one aspect of the invention, the partition coefficient of the skin lightening active ingredient to the two-phase solvent system may be 0.90 to 1.10. In another aspect of the invention, the partition coefficient of the skin lightening active ingredient to the two-phase solvent system may be 0.95 to 1.05. In another aspect of the invention, the partition coefficient of the skin lightening active ingredient to the two-phase solvent system may be 0.97 to 1.03.

In one aspect of the invention, the two-phase solvent system may comprise hexane, ethyl acetate, methanol and water. In another aspect of the invention, the two-phase solvent system is hexane, ethyl acetate, methanol and water 1 to 10: 1 to 10: 1 to 10: 1 to 10, specifically 1 to 5: 1 to 5: 1 ˜5: 1 to 5, more specifically 1 to 3: 1 to 3: 1 to 3: 1 to 3, and the ratio may be included. The ratio of each solvent in the above may mean the weight ratio or volume ratio of each solvent.

One aspect of the present invention provides a skin whitening mulberry extract prepared by the method of manufacturing the mulberry extract. Unlike the conventional extraction method, the extract of M. hawthorn is prepared by the method according to an aspect of the present invention, which includes a step of leaving at low temperatures and a crystallization step of inducing crystals while stirring at a low temperature, thereby including a higher purity skin lightening active ingredient. can do. The skin lightening active ingredient includes kazinol C. The mulberry extract may have an improved melanin production inhibitory effect and tyrosinase inhibitory effect than substances known to exhibit the existing skin whitening effect, and may exhibit very excellent skin whitening effect.

Furthermore, the stabilizing process of supporting the extract of the mulberry on the carrier can increase the titer stability of skin whitening active ingredients, such as kazinol C, which have unstable titers, thereby improving melanin production and tyrosinase inhibitory effect. It can have a skin whitening effect. In addition, the mulberry extract supported on the carrier shows excellent skin safety, it can be used in the external preparation composition of the skin.

One aspect of the present invention provides a topical skin composition comprising the extract of the mulberry. The external preparation composition for skin includes a mulberry extract containing an active ingredient of skin whitening with high purity and potency stability, thereby exhibiting excellent melanin inhibition, tyrosinase inhibitory effect, and further skin whitening effect. In addition, the external preparation composition for the skin is safe to use because it does not cause abnormal skin reactions.

In one aspect of the present invention, the composition for external application of the skin containing the mulberry extract includes a cosmetic composition.

The cosmetic composition may be provided in any formulation suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol composition. Compositions of such formulations may be prepared according to conventional methods in the art.

The cosmetic composition may contain, in addition to the above-mentioned materials, other ingredients that can give a synergistic effect to the main effect within a range not impairing the main effect. The cosmetic composition according to the present invention may include a substance selected from the group consisting of vitamins, polymer peptides, polymer polysaccharides and sphingolipids. In addition, the cosmetic composition according to the present invention may include a moisturizer, an emulsifier, a surfactant, a ultraviolet absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, organic and inorganic pigments, flavoring agents, cooling agents or limiting agents. . The compounding quantity of the said component can be selected easily by those skilled in the art within the range which does not impair the objective and effect of this invention.

In one aspect of the present invention, the topical skin composition comprising the extract of Methanol includes a pharmaceutical composition.

Formulations of pharmaceutical compositions according to one aspect of the invention may be, but are not limited to, solids, solutions, suspensions, emulsions, gels, patches or sprays. The formulations can be readily prepared according to conventional methods in the art and include surfactants, excipients, hydrating agents, emulsifiers, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or Other commercially available auxiliaries can be used as appropriate.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Dosage determination based on these factors is within the level of one skilled in the art, and its daily dosage may be, for example, 0.001 mg / kg / day to 50 mg / kg / day, more specifically 0.1 mg / kg / day to 5 mg / kg / day, but is not limited thereto.

Hereinafter, the structure and effect of the present invention will be described in more detail with reference to Examples, Comparative Examples, Preparation Examples and Test Examples. However, these Examples, Comparative Examples, Preparation Examples and Test Examples are provided only for the purpose of illustration in order to facilitate understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

Test Example 1 Confirmation of Active Ingredients of Methanol Extract

1. Identification of Cazinol C

Kazinol C (molecular weight 464) was identified by mass spectrograph and NMR analysis of the mulberry extract extracted from the mulberry with ethanol, and it was confirmed that it was present in the mulberry extract as 8-15%.

2. Evaluation of Tyrosinase Inhibitory Effect

50 mg of the sample to be evaluated for the degree of tyrosinase inhibition is weighed and dissolved in ethanol to make 100 ml. Take 10 ml of the solution and add 100 ml of ethanol to make 100 ml. 500 μl of the buffer solution, 500 μl of substrate solution, 450 μl of purified water, and 50 μl of sample solution were mixed. Then, 50 μl of enzyme (tyrosinase) solution was added and mixed. After reacting at 37 ° C. for 10 minutes, the resultant is allowed to stand on ice for 5 minutes and the absorbance is measured at 490 nm. The tyrosinase inhibitory effect of the sample is evaluated using the absorbance of the sample relative to the absorbance of the blank sample.

3. Relationship between Casinol C and Tyrosinase Inhibitory Effect

The content of Cardinol C in the mulberry extract was analyzed by HPLC, and the degree of tyrosinase inhibition effect was compared according to its content. The result is shown in FIG. As shown in Figure 1, it can be seen that the higher the content of the casinoin C in the mulberry extract, the higher the tyrosinase inhibitory effect.

Test Example 2 Evaluation of Tyrosinase Inhibitory Effect

1. Evaluation of Tyrosinase Inhibitory Effect by Extraction Solvents

Three samples of washed, dried, and cut mulberry roots were prepared, and then put into ethanol (Example 1), ethyl acetate (Comparative Example 1) and acetone (Comparative Example 2), respectively, for 4-6 hours at 60-80 ° C. After extraction, the yield was evaluated. Thereafter, the tyrosinase inhibitory effect of the three extracts was evaluated by the method of Test Example 1.2. The results are shown in the table below.

Table 1 menstruum Ethanol (Example 1) Ethyl Acetate (Comparative Example 1) Acetone (Comparative Example 2) Extraction yield (%) 6.7 5.5 5.6 Tyrosinase inhibitory effect (%) 25.1 22.8 26.8

As can be seen from the above results, when ethanol was used as the solvent, the extraction yield was the best, and the tyrosinase inhibitory effect was also excellent. In addition, it is judged to be preferable in that the use of ethanol as a solvent can facilitate the production process. Through this, it can be seen that when extracting using alcohol as a solvent, it is easy to obtain a mulberry extract excellent in tyrosinase inhibitory effect in a high yield.

2. Evaluation of Tyrosinase Inhibitory Effect by Extraction Method

After washing, drying and cutting the mulberry root bark, four samples prepared in ethanol were prepared and 4 hours at 80 ° C. (Example 2), 1 day at room temperature (Comparative Example 3), and 7 days at room temperature (Comparative Example 4). ) And 3 weeks (Comparative Example 5) at room temperature and evaluated the yield. The tyrosinase inhibitory effect of the four extracts was evaluated by the method of Test Example 1.2. The results are shown in the table below.

TABLE 2 Extraction method 4 hours extraction at 80 ℃ (Example 2) 1 day extraction at room temperature (Comparative Example 3) 7 days extraction at room temperature (Comparative Example 4) 3 weeks at room temperature (comparative example 5) Extraction yield (%) 6.7 2.9 4.5 5.2 Tyrosinase inhibitory effect (%) 25.1 19.2 25.4 18.2

As can be seen from the above results, when extracted at a high temperature (80 ℃) extraction yield is higher than when extracted at room temperature, tyrosinase inhibitory effect is also excellent. In addition, it is preferable in the case that extraction at high temperature can be prepared in a short time to extract the mulberry extract having an excellent tyrosinase inhibitory effect.

3. Evaluation of Tyrosinase Inhibition Effect by Low Temperature Neglect

Ethanol extract of Example 1 obtained in Test Example 2.1 was left at 4 ℃ for 12-24 hours. After the impurity was precipitated, this was removed to obtain Example 3. The tyrosinase inhibitory effect of Example 3 was evaluated by the method of Test Example 1.2. The results are shown in the table below.

TABLE 3 Example 1 Example 3 Tyrosinase inhibitory effect (%) 25.1% 31.6%

As can be seen from the results, when left at a low temperature (4 ℃) after extraction, the tyrosinase inhibitory effect increased. That is, it is possible to improve the tyrosinase inhibitory effect of the mulberry extract by leaving at low temperature after extraction. On the other hand, the yield was reduced from 6.7% to 5% level was found that impurities are effectively removed. This shows that due to the high temperature of the extraction process, substances with low solubility in ethanol were also extracted, and these impurities were effectively removed during low temperature. Impurities precipitated in this process showed little inhibitory effect on tyrosinase. This shows that almost no material having a tyrosinase inhibitory effect is lost due to low temperature.

4. Evaluation of Tyrosinase Inhibition Effect by Crystallization

The mulberry root bark was washed, dried and cut into ethanol and extracted at 60-80 ° C. for 4-6 hours. The extract was then left at 4 ° C. for 12-24 hours to precipitate impurities and remove them. The extract was placed in purified water having a temperature of 0-4 ° C. and 20 times the volume of the extract, and the crystals were induced by slowly stirring for 2-6 hours. Crystallized extract was obtained as Example 4, and the tyrosinase inhibitory effect was evaluated by the method of Test Example 1.2. The results of evaluating the tyrosinase inhibitory effect of kojic acid as a control group are shown in the table below.

Table 4 Example 4 Control Tyrosinase inhibitory effect (%) 95% 80%

As can be seen from the above results, it can be seen that the tyrosinase inhibitory effect is improved by inducing crystallization by stirring the mulberry extract in cold purified water to have a very excellent tyrosinase inhibitory effect. In other words, immersion of the mulberry extract at low temperature significantly increases the tyrosianase inhibitory effect due to crystallization. Meanwhile, 3% of the extract was finally obtained through the crystallization process, which shows that the crystallization process can also effectively remove impurities. The supernatant that was not crystallized in this process showed little tyrosinase inhibitory effect. This indicates that even in the crystallization process, the material showing the tyrosinase inhibitory effect is hardly lost.

Preparation Examples 1 to 7 Preparation of Carrier Composition

1. Preparation of Polylactic Acid (PLA) Capsules Containing 5% by Weight Methanol Extract

10 g of PLA and 0.2 to 0.3 g of stearyl alcohol were dissolved in 40 ml of methylene chloride, and 0.5 g of the green young mulberry extract powder was dissolved in 1 ml of polyvinyl alcohol (PVA). It was added to 400 ml of water and stirred. Methylene chloride was removed under reduced pressure at room temperature, followed by filtration and drying to prepare a PLA capsule (manufacture example 1) supporting the extract of the mulberry.

2. Preparation of PLA and Polybutylmethacrylate-trimethylammonium ethyl methacrylate (PBMA / TMAEMA) copolymer capsules containing 4 wt.

The manufacturing method is a method of encapsulating the PBMA / TMAEMA copolymer once more on the outermost layer of the PLA capsule containing 5% by weight of mulberry extract. First, 10 g of PBMA / TMAEMA copolymer was dissolved in 250 ml of ethanol, and 55 g of the PLA capsule was evenly dispersed in 500 ml of water in which 1 wt% of PVA was dissolved. The ethanol phase was poured into the water phase and stirred at 5000 rpm with a homogenizer. After ethanol was removed at 40 degrees under reduced pressure, the resultant was filtered and dried to prepare a capsule (manufacture example 2) carrying the extract of the mulberry.

3. Preparation of Polyethyleneadipate (PEA) Capsules Containing 5% by Weight Methanol Extract

47.5 g of PEA was dissolved in 200 ml of methylene chloride, and 2.5 g of the mulberry extract powder of Example 4 was further dissolved and added to 450 ml of water in which 1% by weight of PVA was dissolved, followed by stirring at 5000 rpm. Methylene chloride was removed under reduced pressure at room temperature, followed by filtration and drying to prepare a PEA capsule (Preparation Example 3).

4. Preparation of solid lipid nanoparticles (SLN) comprising 2.5 wt.

15 g of Bee wax and 2 g of hydrogenated lecithin were added and melted by heating at 70 ° C., followed by adding 2 g of the young mulberry extract powder of Example 4. The wax phase was slowly added to 60 g of warmed water at 60 ° C. while stirring with a homogenizer to prepare SLN (Preparation Example 4).

5. Preparation of Polymethylmethacrylate (PMMA) Capsules Containing 5% by Weight Methanol Extract

10 g of PMMA was dissolved in 40 ml of methylene chloride, 0.5 g of the green powder of the mulberry extract of Example 4 was dissolved, and 0.5 wt% of poloxamer 407 was added to 400 ml of dissolved water and stirred. Methylene chloride was removed under reduced pressure at room temperature, followed by filtration and drying to prepare PMMA capsules (Preparation 5).

6. Preparation of Polyethylene Adipate-Polymethylmethacrylate (PEA / PMMA) Capsules Containing 5% by Weight Methanol Extract

23.75 g of PEA and 23.75 g of PMMA were dissolved in 200 ml of methylene chloride, 2.5 g of the mulberry extract powder of Example 4 was dissolved and added to 450 ml of water in which 1% by weight of PVA was dissolved, followed by stirring at 5000 rpm. Methylene chloride was removed under reduced pressure at room temperature, followed by filtration and drying to prepare a capsule in which PEA and PMMA were mixed (Preparation Example 6).

7. Preparation of Silica Powder Containing 5% by Weight Methanol Extract

After completely removing the mulberry extract in ethanol, medium-sized pore silica was mixed in the ethanol solvent in which the mulberry extract of Example 4 is dissolved, and stirred sufficiently for 1 to 3 hours. After filtration and drying in vacuo at room temperature to prepare a medium-porous silica powder (Preparation Example 7) on which the mulberry extract was carried.

[Examples 5 to 11 and Comparative Example 6]

O / w emulsion of Examples 5 to 11 and Comparative Example 6 having the composition shown in the following table in a conventional manner using the Preparation Examples 1 to 7 in order to evaluate the degree of stabilization of the active ingredient when the extract of the mulberry tree is supported on the carrier Formulations were prepared.

Table 5 ingredient Unit (wt%) Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Comparative Example 6 Paper mulberry 5% PLA 0.8 - - - - - - - Methanol Extract 4% PLA + (PBMA / TMAEMA) - One - - - - - - Takko Extract 5% PEA - - 0.8 - - - - - Takko Extract 2.5% SLN - - - 1.6 - - - - Takko Extract 5% PMMA - - - - 0.8 - - - Dokberry Extract 5% PEA + PMMA - - - - - 0.8 Paper mulberry 5% Silica - - - - - - 0.8 - Paper mulberry extract - - - - - - - 0.04 EDTA-2Na 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 glycerin 5 5 5 5 5 5 5 5 Butylene Glycol 5 5 5 5 5 5 5 5 Vegetable Cured Oil 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Stearic acid 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 Glyceryl Stearate One One One One One One One One Cetearyl Alcohol 2 2 2 2 2 2 2 2 Arachidil / Behenyl Alcohol & Arakidil Glucoside One One One One One One One One Cetylaryl Alcohol & Cetearyl Glucoside 2 2 2 2 2 2 2 2 Liquid paraffin 6 6 6 6 6 6 6 6 Caprylic / Carlic Triglycerides 6 6 6 6 6 6 6 6 Carbomer 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Triethanolamine 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Preservative, fragrance, coloring Quantity Quantity Quantity Quantity Quantity Quantity Quantity Quantity Purified water Remaining amount Remaining amount Remaining amount Remaining amount Remaining amount Remaining amount Remaining amount Remaining amount

Examples 5 to 11 and Comparative Example 6 both contain 0.04% by weight of the mulberry extract based on the total weight of the emulsion composition.

Test Example 3 Evaluation of Stabilization Degree of Active Ingredients of Methanol Extract

In order to evaluate whether the representative active ingredient Kazinol C is stabilized when the paper mulberry extract is supported on the carrier, the carrier composition of Preparation Examples 1 to 7, and the o / w emulsions of Examples 5 to 11 and Comparative Example 6 The content of Casinol C included was analyzed by HPLC.

Each Cazinol C content was measured at the beginning of the assay and at a monthly interval for 4 months. The result of calculating the casinoin C content at the start of the analysis as 100% and the relative content% thereof is shown in the table below.

Table 6 carrier PLA PLA + (PBMA / TMAEMA) PEA SLN PMMA PEA + PMMA Silica No carrier Preparation Example 1 Example 5 Preparation Example 2 Example 6 Preparation Example 3 Example 7 Preparation Example 4 Example 8 Preparation Example 5 Example 9 Preparation Example 6 Example 10 Preparation Example 7 Example 11 Comparative Example 6 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 January 94 93 99 99 96 95 93 93 97 90 100 99 78 86 78 February 93 89 97 96 94 93 85 78 89 78 98 98 64 68 59 In March 91 85 96 94 93 90 73 59 94 63 96 96 53 47 42 April 88 80 93 90 89 84 71 46 87 46 95 95 44 25 23

As can be seen from the above results, in both Preparation Examples 1 to 7 and Examples 5 to 11, the content of Casinol C compared to Comparative Example 6 remained high even after time. In other words, when supporting the mulberry extract on the carrier, the degree of stabilization of the active ingredient is high, which can be seen that the same even when formulated in a cosmetic composition.

Test Example 4 Skin Safety Evaluation

To confirm the skin safety of the above embodiments, a patch test was conducted on 18 adult females and 12 males (average 32.5 years).

28 hours after the patch was attached, the patch was removed and the first reading was made 30 minutes later, and after 96 hours, the second reading was performed. In order to determine the intensity of skin irritation of Examples and Comparative Examples, weights were calculated according to the degree of skin positive response, and skin average reactivity was determined. . The results are shown in the table below.

TABLE 7 Test substance Average responsiveness Judgment grade Example 5 0 Non-irritating Example 6 0 Non-irritating Example 7 0 Non-irritating Example 8 0 Non-irritating Example 9 0 Non-irritating Example 10 0 Non-irritating Example 11 0 Non-irritating Comparative Example 6 0 Non-irritating

As can be seen from the results, all of Examples 5 to 11 did not irritate the skin. Thus, the cosmetic composition according to the present invention can be determined to be safe for the skin.

Test Example 5 Evaluation of Whitening Efficacy of Methanol Extract (Cell Level)

1. Cytotoxicity Measurement

(1) Cell culture

B16 melanoma cells, cell lines derived from melanoma of C57BL / 6J (black, a / a) mice, were obtained from the cell line bank. Cells were cultured at 37 ° C. under 10% CO ₂ conditions in DMEM medium containing 10% bovine serum serum (obtained from LONZA), 50 U / ml penicillin, and 50 μg / ml streptomycin 200 nM.

(2) cytotoxicity measurement (MTT assay)

B16 melanoma cells were allowed to be 1 × 10 ⁴ cells in 96-well plates and then incubated for one day. Arbutin and Preparation Example 6 were replaced with fresh medium containing each concentration, and then cultured again for 24 hours. 50 μl of the MTT solution dissolved in the cell culture medium at the concentration of 2 mg / ml was added to each well, and the medium was filled so that the final volume was 200 μl, followed by incubation for 3-4 hours. The medium was removed and dissolved in 150 μl of DMSO in the resulting formazan crystals, and the dissolved promazan stain was measured at 540 nm. The measured absorbance was compared based on the untreated material to evaluate the cytotoxicity. The results are shown in the table below.

Table 8 Concentration (㎍ / ml) % Of control Toxicity% Arbutin Control 100 ± 4.3 0.0 100 95.1 ± 4.8 4.9 Preparation Example 6 Control 100 ± 4.5 0.0 200 95.3 ± 2.6 4.7 150 95.5 ± 2.3 4.5 100 97.1 ± 2.3 2.9 50 98.6 ± 3.7 1.4

As can be seen from the results, in the case of Preparation Example 1, no cytotoxicity was observed at the concentration of 200 μg / ml or less, and in the case of arbutin, no cytotoxicity was observed at the 100 μg / ml concentration. Based on the above results, Preparation Example 6 was tested at a concentration of 100 μg / ml of arbutin at a concentration of 150 μg / ml or less.

2. Evaluation of inhibition of melanogenesis

Allow B16 melanoma cells to be 4 x 10 ⁴ cells / well in 24-well plates, incubate overnight to allow the cells to adhere well to the plate, and then contain 1 μM of α-MSH per well to promote melanin production in each well. And the medium containing the manufacture example 6 and arbutin which are test samples for each concentration was put. After treatment for 72 hours, 200 μl of the cell medium was taken and the absorbance was measured at 405 nm to calculate the amount of melanin. The difference in the calculated melanin amount was compared based on the untreated control material and the results are shown in the table below.

Table 9 Test Sample Concentration (µg / ml) % Melanin compared to the control % Melanin production inhibition P value (relative to control) Control 100 ± 4.9 0.0 1.0000 Arbutin 100 66.3 ± 4.7 33.7 0.0001 Preparation Example 6 150 59.6 ± 3.4 40.4 0.0000 100 68.1 ± 5.8 31.9 0.0002 50 79.7 ± 3.7 20.3 0.0006

As can be seen from the above results, when treated with 150 μg / ml of Preparation Example 6, melanin production was inhibited by 40.4% compared to the control, and melanin production was inhibited by 31.9% compared to the control when treated with 100 μg / ml. Treatment with 100 μg / ml of arbutin inhibited melanin production by 21.2%. It can be seen that the mulberry extract prepared according to the present invention is excellent in inhibiting melanin production.

3. Evaluation of Inhibition of Tyrosinase Activity

Reaction buffer (0.1 M potassium phosphate buffer, pH 6.8), 0.03% L-tyrosine substrate solution (0.3 mg / ml in reaction buffer), mushroom tyrosinase solution, 2 units / in reaction buffer A reaction solution containing [mu] l) was prepared. First, arbutin and preparation example 6 for each concentration were prepared as test samples, and an amount corresponding to 10 units of enzyme was added thereto, and then the final volume was 100 μl with 0.1 M phosphate buffer. Immediately after adding 100 µl of 0.03% L-tyrosine substrate solution, absorbance was measured at 475 nm, and reacted at 37 ° C. for 10 minutes. Then, the reaction was stopped on ice and the absorbance was measured again. The difference in absorbance changed for 10 minutes was compared with the control. The results are shown in the table below.

Table 10 Concentration (㎍ / ml) % Tyrosinase activity compared to control % Tyrosinase activity inhibition IC ₅₀ (μg / ml) P value Control 100 ± 2.6 0.0 1.000 Arbutin 500 12.3 ± 0.6 87.7 209.6 0.000 250 43.5 ± 1.6 56.5 0.000 125 61.9 ± 2.2 38.1 0.000 Preparation Example 6 50 4.6 ± 0.5 95.4 23.7 0.000 25 22.4 ± 3.9 77.6 0.000 12.5 89.3 ± 9.1 13.0 0.017

As can be seen from the results, Preparation Example 6 showed a statistically significant inhibitory effect on tyrosinase activity compared to the control group without treatment, which was superior to arbutin known to be excellent in inhibiting tyrosinase activity. In addition, the concentration of inhibiting tyrosinase activity by 50%, that is, IC ₅₀ value was measured as 23.7 μg / ml in Preparation Example 6 and 209.6 μg / ml in Arbutin.

It can be seen that the mulberry extract prepared according to the present invention is very excellent in inhibiting tyrosinase activity.

The mulberry extract prepared according to the present invention can be confirmed to have an excellent whitening effect because it has an excellent effect of inhibiting melanin production and tyrosinase activity.

Test Example 6 Evaluation of Whitening Efficacy of Methanol Extract (Clinical Evaluation)

Twenty three adult women aged 18 to 60 years were selected (mean age 42.17 years). Skin pigmentation by irradiating 2.5MED of UV light to two areas of the subject's arm using a multi-pori Solar Simulator 601-300W Solar Light (USA) equipped with a 300W Xenon arc lamp. Deposition was induced. After 10 days, the two sites were each coated with a control group which did not include the o / w emulsion of Example 10 and the mulberry extract, to the UV irradiation site. After application, visual and mechanical evaluation by the first dermatologist was performed at 22-24 ° C and 40-60% humidity. Four weeks, six weeks, and eight weeks later, a dermatologist's visual evaluation, mechanical evaluation, and questionnaire were conducted.

1. Visual evaluation by specialist

Visual evaluation by a dermatologist was carried out by a dermatologist evaluating the degree of skin pigmentation in 8 steps of 0 to 7 points. The higher the score, the higher the degree of skin pigmentation. The results are shown in the table below.

Table 11 Example 10 Control Week 0 4 Weeks 6 Weeks 8 Weeks Week 0 4 Weeks 6 Weeks 8 Weeks Average 4.87 3.35 2.54 2.02 4.74 3.35 2.83 2.56 Standard Deviation 1.06 0.65 0.62 0.85 0.92 0.57 0.65 0.86

As can be seen from the above results, the application of Example 10 after 4 weeks, 6 weeks and 8 weeks of application was lower in the degree of skin pigmentation than when the control group was applied. In addition, in view of the fact that Example 10 had a higher degree of skin pigmentation immediately after application than the control group, the result means that Example 10 has a very excellent skin pigmentation inhibitory effect. Therefore, it can be seen that the composition comprising a mulberry extract prepared according to the present invention has an excellent skin whitening effect.

2. Instrumental evaluation

The mechanical evaluation was performed by measuring L * value of UV irradiation part with Chromameter CR-400 (Minolta, Japan). The results are shown in the table below. The result below is the average of three values after 5 measurements except the maximum and minimum values.

Table 12 Example 10 Control Week 0 4 Weeks 6 Weeks 8 Weeks Week 0 4 Weeks 6 Weeks 8 Weeks Average 57.69 61.76 63.22 64.30 57.66 61.61 62.49 63.26 Standard Deviation 3.32 2.29 2.29 2.31 3.81 1.87 1.90 1.85

In addition, mechanical evaluation was performed by measuring melanin index (M.I) using a Mexameter MX18 (Germany). The results are shown in the table below. The result below is the average of three values after 5 measurements except the maximum and minimum values.

Table 13 Example 10 Control Week 0 4 Weeks 6 Weeks 8 Weeks Week 0 4 Weeks 6 Weeks 8 Weeks Average 232.38 201.45 178.91 164.23 233.60 204.39 182.88 172.52 Standard Deviation 56.82 39.22 38.74 39.65 44.58 30.34 30.50 30.60

As can be seen from the above results, the application of Example 10 after 4 weeks, 6 weeks, and 8 weeks of application was higher than that of the control group. Therefore, it can be seen that the composition comprising a mulberry extract prepared according to the present invention has an excellent skin whitening effect.

3. Survey

Subjects were evaluated in five stages of 0 to 4 points to determine whether the skin color of the application site was lightened, whether the area of the pigmentation site was reduced, and whether the skin texture was soft over the test period. Higher scores mean more desirable. The percentage of subjects corresponding to each score is shown in the table below.

Table 14 Whether the skin tone is light score Example 10 (%) Control group (%) 4 weeks later 0 0.0 0.0 One 21.7 34.8 2 43.5 43.5 3 34.8 17.4 4 0.0 4.3 6 weeks later 0 0.0 0.0 One 17.4 13.0 2 30.4 39.1 3 43.5 39.1 4 8.7 8.7 8 weeks later 0 0.0 0.0 One 4.3 4.3 2 21.7 26.1 3 56.5 47.8 4 17.4 21.7

Table 15 Whether the area of the pigmentation site is reduced score Example 10 (%) Control group (%) 4 weeks later 0 13.0 13.0 One 17.4 17.4 2 43.5 52.2 3 26.1 17.4 4 0.0 0.0 6 weeks later 0 4.3 4.3 One 21.7 17.4 2 30.4 34.8 3 43.5 39.1 4 0.0 4.3 8 weeks later 0 4.3 0.0 One 17.4 4.3 2 39.1 26.1 3 39.1 47.8 4 0.0 21.7

Table 16 Whether the skin texture is smooth score Example 10 (%) Control group (%) 4 weeks later 0 8.7 8.7 One 21.7 26.1 2 60.9 56.5 3 8.7 8.7 4 0.0 0.0 6 weeks later 0 4.3 4.3 One 17.4 13.0 2 39.1 52.2 3 39.1 30.4 4 0.0 0.0 8 weeks later 0 0.0 0.0 One 4.3 8.7 2 30.4 30.4 3 60.9 52.2 4 4.3 8.7

As can be seen from the above results, the application of Example 10 had a better overall effect of thinning the skin color of the UV irradiation site, decreasing the area of the pigmentation site, and softening the skin texture than the application of the control group. .

4. Abnormal reaction

No contact dermatitis symptoms and other adverse reactions were observed at the application site of Example 10 over the entire test period. Based on this, it can be seen that the composition of Example 10 is suitable for use on the skin.

When applying the external composition for skin preparations containing the mulberry extract prepared according to the present invention to the human body it can be seen that the effect of improving skin pigmentation and further excellent skin whitening effect.

Test Example 7 Isolation of Cazinol C

1. Selection of two phase solvent system

Tests of hexane / ethyl acetate / methanol / water mixtures of various compositions showed that the partition coefficient of kazinol C was 0.997, very close to 1, when the solution was included at a ratio of 5/5/5/2. It was confirmed that it did not overlap with the partition coefficient of the component.

The method of measuring the partition coefficient in the above is as follows. First, 1 mg of Example 3 was placed in a 10 ml test tube, and 2 ml of the supernatant and the lower layer of the two-phase solvent system, which reached equilibrium, were shaken vigorously for 1 minute. After the two phases reached the equilibrium, 100 μl of the supernatant and the lower layer were taken, dried, and dissolved in 1 ml ethanol and analyzed by HPLC. The partition coefficient was determined by dividing the amount of Cazinol C present in the supernatant by the amount of Cazinol C present in the lower layer.

2. Separation using HSCCC

High speed counter current chromatography (HSCCC) TBE-1000A was used. The coil column was first filled with the supernatant used as the stationary phase at a flow rate of 10 ml / min, and the lower layer used as the mobile phase inside the coil was pumped at a flow rate of 5 ml / min while rotating the instrument at 400 rpm. It was judged that liquid-liquid equilibrium was reached when the mobile phase started flowing out. When equilibrium was reached, a 50 ml solution containing 0.5 g of Example 3 was injected. Separation temperature was maintained at room temperature. The liquid exiting the coil column exit was fractionated by passing through a 280 nm uv detector.

3. HPLC analysis

The HSCCC fractions were analyzed by HPLC and the results are shown in FIG. 2. As can be seen in Figure 2, the HSCCC fractions contain only very high purity Cazinol C, it can be confirmed that the Cazinol C has been separated.

Claims (15)

Solvent extraction of Broussonetia kazinoki ; Leaving the mulberry extract extracted in the step; And The method of manufacturing a mulberry extract comprising the step of crystallizing the mulberry extract neglected in the step. The method of claim 1, The mulberry is a method of manufacturing a mulberry extract, characterized in that it comprises a root bark of mulberry. The method of claim 1, In the step of extracting the solvent, the solvent is a method of manufacturing a mulberry extract, characterized in that containing alcohol. The method of claim 1, In the step of extracting the solvent, the extraction method of the mulberry extract, characterized in that made for 1 to 10 hours at 50 to 100 ℃. The method of claim 1, In the step of leaving the mulberry extract, leaving the mulberry extract, characterized in that proceeding for 1 to 40 hours at -5 to 25 ℃. The method of claim 1, In the step of crystallizing the mulberry extract, crystallization is carried out while stirring the mulberry extract in -5 to 15 ℃ water for 1 to 20 hours. The method of claim 1, After the step of crystallizing the mulberry extract, the method of producing a mulberry extract, characterized in that further comprising the step of supporting the mulberry extract on a carrier. The method of claim 7, wherein The carrier is polylactic acid (PLA), polyethylene adipate (polyethyleneadipate, PEA), polymethyl methacrylate (polymethylmethacrylate, PMMA), polybutyl methacrylate (polybutylmethacrylate, PBMA), trimethylammonium ethyl methacrylate ( Trimethylammoniumethylmethacrylate (TMAEMA), solid lipid nano particles (solid lipid nano particles, SLN) and silica (silica) is a method of producing a mulberry extract, characterized in that the mixture of one or more selected from the group consisting of. The method of claim 7, wherein The mulberry extract is a method of producing a mulberry extract, characterized in that 0.01 to 30% by weight based on the weight of the carrier. The method of claim 1, After the step of crystallizing the mulberry extract, the method of producing a mulberry extract further comprising the step of obtaining the skin whitening active ingredient from the mulberry extract using HSCCC (High speed counter current chromatography) of the two-phase solvent system. The method of claim 10, The skin lightening active ingredient is kachinol C (kazinol C) manufacturing method of the mulberry extract characterized in that it comprises. The method of claim 10, The biphasic solvent system hexane, ethyl acetate, methanol and water 1 ~ 10: 1 ~ 10: 1 ~ 10: 1 ~ 10 characterized in that the mulberry extract production method comprising a. Methanol extract for skin whitening prepared by the method of manufacturing a mulberry extract according to any one of claims 1 to 12. The method of claim 13, The mulberry extract inhibits melanin production, the mulberry extract, characterized in that it inhibits tyrosinase (tyrosinase). An external preparation composition for skin containing a mulberry extract according to claim 13.

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