KR20170067302A

KR20170067302A - Manufacturing method of Rhodiola sacra supercritical fluid extracts having whitening activits and cosmetic composition comprising the same

Info

Publication number: KR20170067302A
Application number: KR1020150173806A
Authority: KR
Inventors: 박영미
Original assignee: (주) 레비레스코
Priority date: 2015-12-08
Filing date: 2015-12-08
Publication date: 2017-06-16
Also published as: KR101757852B1

Abstract

The present invention relates to a cosmetic composition for skin whitening or wrinkle improvement comprising a hypercholesterolemic extract of Hongkongcheon as an active ingredient and a method for producing the supercritical extract of Hongkongchun, Toxic and can be used as a composition of skin care product.

Description

[0001] The present invention relates to a method for preparing a supercritical extract of Rhodiola having a whitening activity and a cosmetic composition comprising the same,

The present invention relates to a method for producing a supercritical extract of Hong Kyung Chun having a whitening activity and a cosmetic composition containing the same.

Skin can be divided into endogenous aging and extrinsic aging. Endogenous aging is mainly based on genetic causes, and exogenous aging is mainly caused by ultraviolet rays. Ultraviolet rays pass through the epidermis and dermis and break down the structures of collagen and elastin in the dermis and produce active oxygen. To protect the skin cells located in the basal layer of the epidermis, melanin cells generate melanin, which melanin absorbs ultraviolet light and protects cells from damage by ultraviolet rays.

The production of melanin is converted to dopaquinone by the action of tyrosinase in melanocytes, followed by enzymatic action and spontaneous oxidation. However, abnormal production of melanin causes skin lesions such as vitiligo. On the contrary, when melanin is synthesized excessively by ultraviolet rays, it not only damages the skin but also causes skin darkening, spots and freckles, do.

Conventionally, ascorbic acid, kojic acid, arbutin, hydroquinone, and extracts of natural plants have been used as a whitening raw material in order to inhibit the production process of melanin. However, kojic acid or arbutin, which is a representative whitening raw material, has a problem of low product safety despite good whitening effect.

Recently, interest in research on whitening materials using natural plant extracts, which have been used for a long time by folk remedies, is being paid attention.

Rhodiola sacra is a plant that is widely distributed in the high mountains of Europe and Asia. It is a traditional medicine effective for stimulation of the nervous system, reduction of depression, improvement of working ability, restoration of fatigue and prevention of altitude sickness. It has been used for a long time. Recently, it has been reported that the extract of Hongkong Stream is active in whitening. Korean Patent No. 0445404 discloses that Rhodiola sachalinensis A. Bor .) extracts. However, since the extract is obtained by sequentially extracting and fractionating Hong-Kyung Chun with ethanol, petroleum ether, chloroform, acetate and butanol, Korean Patent Publication No. 1999-0051310 discloses a feature relating to a cosmetic cosmetic composition containing Rhodiola sachalinensis A. Bor . Extract extracted with water or ethanol or water / ethanol, Despite the same effect, Hong Kyung Chun can cause skin irritation to sensitive skin due to cytotoxicity and its utilization is being limited.

The first problem to be solved by the present invention is to provide a cosmetic composition for improving whitening activity or wrinkle comprising a supercritical extract of Hong Kyung Chun as an active ingredient.

Another object of the present invention is to provide a method for preparing the supernatant extract of the present invention.

In order to accomplish the above object, the present invention provides a cosmetic composition for improving whitening activity or wrinkles, which comprises a supercritical extract of Hongkong Stream as an active ingredient.

The supercritical extract of Hong Kyung Chun is an extract obtained by extracting Hongkong Stream with supercritical extraction method.

According to one embodiment of the present invention, the above-mentioned Hong-Kyung Chun may be any one selected from the group consisting of a Hong-Kyung Chun enzyme hydrolyzate obtained by hydrolyzing Hong-Kyung Chun and Hong Kyung Chun with a cell wall degrading enzyme, and an ultrasonically treated Hong Kyung Chun.

According to an embodiment of the present invention, the cell wall degrading enzyme may be selected from the group consisting of β-glucanase, hemicellulase, cellulase, xylanase, pectinase, Glucosidase, arabinase, and the like. The enzyme may be one or more kinds of enzyme selected from the group consisting of glucosidase and arabinase.

The supercritical extract of Hongcheon Stream according to the present invention can be prepared by carrying out the following steps.

(1) hydrolyzing the Rhizoctonia sp. With a cell wall degrading enzyme; And

(2) Supercritical extraction of the hydrolyzed Hongyeong Stream.

According to an embodiment of the present invention, before performing the step (1) And suspending and finishing the ground Hongqiao River in water to ultrasonically treat the suspension.

According to an embodiment of the present invention, the step (1) may be carried out at a temperature of 25 to 40 DEG C for 2 to 24 hours.

In the step (2), carbon dioxide may be extracted as an extraction solvent at 120 to 300 bar.

The step (2) may be carried out at 30 to 50 ° C for 40 to 150 minutes.

The cosmetic composition for skin whitening or wrinkle improvement comprising the hypercholesterolemic extract according to the present invention as an active ingredient is excellent in whitening activity, anti-aging and anti-wrinkle effect, and can be used safely in human body because of low cytotoxicity and skin irritation.

The inventors of the present invention studied various plant extracts in order to search for substances that can be safely used in the human body by improving the skin pigmentation and wrinkles while lowering the skin irritation. As a result, the extracts obtained by supercritical extraction of Hong- It was confirmed that the effect of wrinkle improvement was excellent and the cytotoxicity was low and skin irritation was greatly reduced, and the present invention was completed on the basis thereof.

Hereinafter, the present invention will be described in detail.

The present invention relates to a cosmetic composition for skin whitening or wrinkle improvement comprising a hypercholesterolemic extract of Hongkong Chun as an active ingredient.

According to one embodiment of the present invention, the supernatant extract of Hongcheon Stream can be obtained by extracting the supercritical fluid using a supercritical fluid.

According to another embodiment of the present invention, the supercritical extract of Hongcheong Stream can be obtained by hydrolysis of Hongkong stream into cell wall degrading enzyme and then by supercritical extraction.

According to another embodiment of the present invention, the supercritical extract of Hong Kyung Chun may be obtained by hydrolyzing the ultrasonically treated Hong-Kyung Stream with a cell wall degrading enzyme and then extracting it by a supercritical method.

The supernatant extract of Hongcheon Stream according to the present invention can be prepared by the following method.

(1) hydrolyzing the Rhizoctonia sp. With a cell wall degrading enzyme; And

(2) Supercritical extraction of the hydrolyzed Hongyeong Stream.

According to one embodiment of the present invention, it is preferable that the above-mentioned red ginseng is cut into a cutter or pulverized by a pulverizer to improve the extraction yield.

Specifically, firstly, the hydrolyzate of Rhodiola is hydrolyzed with a cell wall degrading enzyme to produce a hydrolyzate of Rhodiola. The cell wall degrading enzyme may be selected from the group consisting of beta-glucanase, hemicellulase, cellulase, xylanase, pectinase, glucosidase, Arabinase, and the cell wall degrading enzyme may be added in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of Rhodiola. If the content of the cell wall degrading enzyme is less than the above range, the effect of the enzyme treatment may be insignificant, so that an extract having reduced tyrosinase activity inhibition rate can be produced. If the content exceeds the above range, excessive hydrolysis of the Hong- An ingredient having a tyrosinase-inhibiting activity may be destroyed, and an extract having a reduced whitening function may be produced.

According to one embodiment of the present invention, the enzymes may be contained in an amount of 10 to 3,000 units / 100 g of the red blood cells, respectively, but are not limited thereto.

According to the present invention, in step (1), pH and temperature are preferably controlled so as to activate the cell wall degrading enzyme so as to facilitate hydrolysis of the Hongryong stream. The pH may be adjusted using a conventional pH adjusting agent available for food. The pH adjusting agent is not particularly limited, and the optimal range of the pH to be adjusted may be different depending on the type of enzyme to be added. For example, hydrolysis may be performed using citric acid at a pH range of 4.5 to 6, and the temperature may be adjusted at 30 to 50 ° C.

According to the present invention, when the hydrolysis reaction is completed, it is preferable to inactivate the enzyme. The method of inactivating the enzyme is not particularly limited, and conventional enzymatic deactivation methods can be used. For example, at 80 to 120 DEG C for 5 to 15 minutes.

According to the present invention, the step (2) may be carried out by using supercritical carbon dioxide as an extraction solvent at a flow rate of 5 to 20 ml / min. The extraction may be performed at 30 to 50 ° C for 40 to 150 minutes , More preferably at a pressure of 120 to 300 bar.

Carbon dioxide has a property close to that of an organic solvent in a supercritical state. The higher the temperature and the pressure, the better the polarity property as well as the general nonpolar property, and the more amphoteric property, the better the extraction ability. According to the present invention, there is a problem in that the extraction ability is low at the extraction pressure of 100 bar under the supercritical extraction condition. When the extraction pressure is more than 350 bar, the extraction ability can be improved but the whitening activity or the components not involved in the wrinkle improvement are also extracted, Or the ratio of the functional material such as wrinkle reduction is lowered. Therefore, it was estimated that the extracting pressure of 120 bar or 300 bar, preferably 150 to 250 bar, is a range in which the whitening activity and the wrinkle reducing effect can be maximized. According to the present invention, the whitening active substance may be a substance exhibiting tyrosinase production inhibitory activity, and the wrinkle reducing substance may be a substance promoting collagen synthesis.

If the extraction time is less than the above range, the content of the whitening active ingredient or wrinkle improving ingredient to be extracted is low, and if the extraction time exceeds the above range, the extraction yield is improved but the ingredient which does not participate in whitening activity or wrinkle improvement There is a problem that is extracted together.

According to an embodiment of the present invention, before performing the step (1) And suspending the ground Hong-Kyung Stream in water and subjecting the suspension to ultrasonic treatment to thereby improve the extraction yield and obtain an extract having an enhanced wrinkle-reducing effect.

Specifically, when ultrasonication is performed on the Hongkong stream prior to the hydrolysis, the rigid structural matrix such as fibrin in the Hongryong stream is dissociated so that the physiologically active ingredient can be easily liberated, and the extraction yield is improved. In addition, the ultrasound - treated extracts showed a significantly enhanced collagen synthesis promoting effect, in particular, as compared with the unsterilized extracts.

According to the present invention, the ultrasonic treatment may be performed by irradiating ultrasonic waves at 20 to 35 ° C for 2 to 20 minutes under conditions of 15-25 kHz, 500-800 watt and 20 amplitude. It is difficult to expect an improvement in the extraction yield and the effect of promoting collagen synthesis is further difficult to obtain. The collagen synthesis promoting effect tended to increase as the ultrasonic irradiation time increased. However, when the ultrasonic irradiation time was longer than 20 minutes, the promoting effect of collagen synthesis was lower than that without ultrasonic irradiation.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are provided to further illustrate the present invention, and the scope of the present invention is not limited thereto.

Example

Example 1

Supercritical carbon dioxide was added at a flow rate of 10 ml / min at 250 bar and 40 ℃ for 2 hours.

Example 2

end. Rhodochrons were crushed and suspended in water, and citric acid was added to adjust the pH to 5.8. 1 g of a complex enzyme, Viscozyme L, which is a complex enzyme including cellulase, beta-glucanase, hemicellulase, arabinase and xylenase, was added to 100 g of the pH-regulated Rhodiola mill, The enzyme digestion reaction was carried out while stirring at a speed of 200 rpm at a temperature to homogenize. After completion of the reaction, the reaction mixture was heated at 100 ° C for 10 minutes to inactivate the enzyme, and the solvent was removed to prepare a hydrolyzate of the enzyme.

I. The hydrolyzate of Hongcheon Stream enzymes was added at a flow rate of supercritical carbon dioxide at a flow rate of 10 ml / min at 150 bar and 40 ° C for 2 hours to prepare a supercritical extract of Hong Kyung Chun.

Example 3

The ultrasonic extraction method used in the present invention was conducted by generating ultrasound at 20 kHz, 750 watt and 20 amplitude using an ultrasonic wave extractor (SEEC-SONIC II, UL-Tech, Uiwang, Korea) Circulating water bath (Circulator Water Bath) was connected and used.

The suspension was suspended in water and sonicated for 5 minutes. Then, the solvent was removed, and supercritical extraction was carried out by the method of Example 1 to prepare a supercritical extract of Hong Kyung Chun.

Example 4

After shaking the Hongyeongcheon, the suspension was suspended in water and irradiated with ultrasonic wave for 5 minutes under the condition of Example 3, and then pH was adjusted to 5.8 by adding citric acid. 1 g of Viscozyme L was added to 100 g of pH-regulated Rhodiola mill, and the mixture was stirred at a temperature of 40 ° C at a speed of 200 rpm for homogenization. After completion of the reaction, the reaction mixture was heated at 100 ° C for 10 minutes to inactivate the enzyme, and the solvent was removed to prepare a hydrolyzate of the enzyme.

The supernatant extract of Hongik Kyung Chun was prepared by supercritical extraction of the thus-prepared hydrolyzate of Hong Kyung Stream enzyme by the method of Example 1.

Comparative Example 1

After crushing Hongkong Stream, water was added and heated to 100 ℃ for 2 hours to reflux.

Comparative Example 2

After shredding Hongkryuncheon, 50% ethanol aqueous solution was added and the mixture was refluxed at 40 ℃ for 2 hours.

Test Example 1. Verification of inhibitory effect of hydrogen peroxide-induced cytotoxicity
Hydrogen peroxide produces hydroxyl radicals in the cells, leading to lipid peroxidation leading to cell membrane damage and cytotoxicity. It was confirmed that the extracts according to Examples 1 to 4 and Comparative Examples 1 and 2 can inhibit the cytotoxic action of hydrogen peroxide.
The experiments were performed by MTT assay using HaCaT cells. HaCaT cells in logarithmic growth phase were inoculated into 96 well plates at 8000 per well and cultured for 24 hours in 200 [mu] l DMEM medium. The extracts of Examples 1 to 4 and Comparative Examples 1 and 2 were each added to the medium at a final concentration of 50 占 퐂 / ml and reacted for 4 hours, and the cells were washed with PBS. The washed cells were incubated for 2 hours in medium containing hydrogen peroxide to a final concentration of 1 mM to induce oxidative stress, and the cells were washed with PBS. The washed cells were incubated for another 4 hours in a medium containing 0.5 mg / ml of MTT. At this time, the yellow MTT is reduced under the action of mitochondria in living cells and transformed into a purple Formazan which is not soluble in water. Since MTT is attached to the cells, the culture solution was dissolved with 200 μl of a dimethylsulfoxide (DMSO) solution to destroy the cells and liberate MTT. Since the amount of formazan was directly proportional to the number of living cells, the cytotoxicity was calculated by measuring the absorbance of the formazan when measured with an ELISA reader.
In addition, HaCaT cells treated with hydrogen peroxide but not containing the extract were used as a control, and the same procedure as described above was performed. Each of the samples dissolved in the above was measured for absorbance at 540 nm in an ELISA reader, and the cytotoxicity of each solution was calculated in accordance with the following formula 1,
[Formula 1]

division Cytotoxicity (%) Control group 62.4 Comparative Example 1 35.3 Comparative Example 2 33.1 Example 1 26.7 Example 2 22.4 Example 3 27.1 Example 4 23.6

Referring to Table 1, it can be confirmed that the supernatant extract of Hongcheon Stream of Examples 1 to 4 has lower cytotoxicity than the control group and Comparative Examples 1 and 2. In particular, the extracts of Rhodococcus spp. Degraded by cell wall degrading enzymes (Examples 2 and 4) were lower in cytotoxicity, and cytotoxicity was slightly increased in case of ultrasonic irradiation, but it was not significant.
Test Example 2. Verification of inhibition of tyrosinase activity
40 μl of L-tyrosine solution (Sigma) was added to 96 wells according to the method of Vanni, Gastaldi D, Giunata G et al., Annali Di Chimica, 1990; 80: The extracts of Examples 1 to 4 and Comparative Example 1 were added at concentrations of 0 占 퐂 / ml, 50 占 퐂 / ml and 100 占 퐂 / ml, respectively. 20 μl of 50 μg / ml tyrosinase (Sigma) was added to the mixed solution, followed by reaction at 37 ° C for 30 minutes. Tyrosine solution (Sigma) was subjected to the same procedure as described above except that arbutin, which is known to have a tyrosinase activity inhibitory function, was used as a control instead of the example. The absorbance of the mixed solutions was measured at 490 nm, and the inhibition rate (%) of tyrosinase activity of each solution was calculated in accordance with the following equation 2, and the results are shown in Table 2 below.
[Formula 2]

division Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Arbutin 5 ug / ml 21.13 23.82 21.15 23.47 13.26 4.81 25 [mu] g / ml 41.95 48.15 42.12 48.25 37.15 23.15 50 ug / ml 61.02 67.15 60.96 67.91 51.78 48.63 100 [mu] g / ml 67.99 73.12 68.46 73.46 59.13 60.28

As shown in Table 2, it was confirmed that the supercritical extract of Hongik Kyun Chun was excellent in inhibiting tyrosinase activity compared to the hot water extract of Hong Kyung Chun. In Example 2 and 3, which were degraded into cell wall degrading enzymes, And 4, the tyrosinase activity inhibitory effect was more excellent. On the other hand, it was confirmed that the ultrasonic irradiation had no effect on the inhibition effect of tyrosinase activity or was not significant.
Test Example 3. Collagen synthesis promotion effect
The effect of collagen synthesis at the cellular level was confirmed by adding the supernatant extract of Hong Kyung Chun of Examples 1 to 4 and the hot water extract of Hong Kyung Chun of Comparative Example 1 to the culture medium of human-derived fibroblasts. The synthesized collagen was quantitated using a PICP EIA kit (Procollagen Type I C-Peptide Enzyme Immunoassay KIT).
The extracts of Examples 1 to 4 and Comparative Example were added to the culture medium of human fibroblast cells to a final concentration of 0.05% and cultured for 1 day. Then, the culture broth was taken and cultured with PICP EIA Kit at each concentration with collagen The degree of synthesis was measured at 450 nm using a spectrophotometer. As a control group, vitamin C, a well known collagen synthesis promoter, was added to a final concentration of 0.005%, and the degree of collagen synthesis was measured.

division Growth rate (%) Control group (no addition) 100 Example 1 129.12 Example 2 131.52 Example 3 134.15 Example 4 135.89 Comparative Example 1 111.84 Vitamin C 137.12

As shown in Table 3, in Examples 3 and 4 irradiated with ultrasound before supercritical extraction, the degree of collagen synthesis tended to increase as compared with Examples 1 and 2, respectively.

Claims

A cosmetic composition for improving whitening activity or wrinkle comprising supercritical extract of Rhodiola sacra as an active ingredient.

The method according to claim 1,
The cosmetic composition for improving whitening activity or wrinkle according to any one of claims 1 to 3, wherein the hydrolyzate is hydrolyzed by a cell wall degrading enzyme.

3. The method of claim 2,
The cell wall degrading enzyme may be selected from the group consisting of beta-glucanase, hemicellulase, cellulase, xylanase, pectinase, glucosidase, Wherein the cosmetic composition is one or two or more kinds of mixed enzymes selected from the group consisting of arabinic acid and arabinose.

The method according to claim 1,
The above-mentioned Hong-Kyung Chun is a step of crushing Hong Kyung Chun; And suspending the ground Hong-Kyung Stream in water and ultrasonically treating the suspension. The cosmetic composition for improving whitening activity or wrinkles is characterized in that it is an ultrasound pretreatment produced by performing an ultrasonic treatment.

The method according to claim 1,
The cosmetic composition for improving whitening activity or wrinkles is characterized in that the above-mentioned Hongkong Stream is hydrolyzed with a cell wall degrading enzyme after ultrasonication.

(1) hydrolyzing the Rhizoctonia sp. With a cell wall degrading enzyme; And
(2) supercritically extracting the hydrolyzed Hongyeongcheon, and preparing a supercritical extract of Hongcheongcheon for skin whitening or wrinkle improvement.

The method according to claim 6,
Before performing the step (1); And suspending the ground Hong-Kyung Stream in water and subjecting the suspension to ultrasonic treatment.

The method according to claim 6,
The cell wall degrading enzyme may be selected from the group consisting of beta-glucanase, hemicellulase, cellulase, xylanase, pectinase, glucosidase, Wherein the enzyme is one or two or more kinds of mixed enzymes selected from the group consisting of arabinase.

The method according to claim 6,
Wherein the step (1) is performed at a temperature of 25 to 40 ° C for 2 to 24 hours.

The method according to claim 6,
Wherein the step (2) comprises extracting carbon dioxide as an extraction solvent at 120 to 300 bar.

6. The method of claim 5,
Wherein the step (2) is carried out at 30 to 50 ° C for 40 minutes to 150 minutes to extract the supercritical extract.