KR20040026272A - Cosmetic composition having whitening effect comprising extract of Pulsatilla Radix as main ingredient - Google Patents

Abstract

PURPOSE: Provided is a skin whitening cosmetic composition containing an extract of Pulsatilla Radix as main ingredient, as well as one of extracts of licorice root and Ulmus davidiana var. The composition has excellent skin whitening effect. CONSTITUTION: A skin whitening cosmetic composition is characterized by containing an extract of Pulsatilla Radix as main ingredient, one of ranunculin, deoxypodophyllotoxin, 3-O-α-L-rhamnopyranosyl(1→2)-£β-D-glucopyranosyl (1→4)|-α-L-arabino pyranoside(SB365). it further contains one of extracts of licorice root and Ulmus davidiana var.

Description

Cosmetic composition having whitening effect as a main ingredient {Cosmetic composition having whitening effect comprising extract of Pulsatilla Radix as main ingredient}

The present invention relates to a whitening cosmetic composition containing a baekduong extract as a main component.

Human skin consists of the epidermis and the dermis. The epidermis produces nails, hair and sweat glands. The dermis contains neural networks, blood vessels, hair glands and sweat glands. Melanocytes that produce melanin are present in the epidermis. The starting material for melanogenesis is that tyrosine, one of the essential amino acids, is oxidized by tyrosine hydroxylase (TH) to 3,4-dihydroxyphenylalanine (DOPA). The substance is converted into melanin, a black polymer, through various reaction steps. Congenital lack of TH function is manifested as albinism.

Therefore, the most important target of the development of whitening material in the melanin biosynthesis mechanism is TH, it is most important to find a substance that inhibits the activity of this enzyme.

Efforts have also been made to find tyrosinase inhibitors from natural products. According to Japanese Patent Application Laid-Open No. 2002-0023168, the extract of Gardenia jasminoides showed about three times stronger inhibitory effect than arbutin, which was simultaneously tested on TH. However, it is not possible to assess the adverse effects on human skin by not revealing which substances in gardenia contain TH inhibitory effects.

Conventional substances used as TH inhibitors are dihydroxybenzene (dihydroxybenzene) derivatives, retinoids, steroid hormones, and the like, all of which show side effects on the skin, so its use is limited.

The present invention is to make an extract preparation having a whitening effect by solvent extraction from Baekduong (root of Pulsatilla koreana), and to make a whitening effect of the whitening effect by mixing this extract with the extract of milky skin A first object thereof is to separate the whitening effect material from the baekduong extract and to mix it with milky skin to prepare a preparation having improved whitening effect.

Baekduong is called Radix Pulsatillae and includes all Pulsatilla genus plants such as Pulsatilla koreana, P..cerna and P. chinensis. It has a pharmacological effect.

Recently, anti-cancer effects have been recognized and are in clinical trials. Yubaekpi (blood of Ulmus macrocarpa) is a pharmacological effect and is effective for enteroparasites and plants, especially as a roundworm. It is also reported to be effective for various ringworms (Oreintal Materia Medica, H-Y Hsu et.al., Oriental Healing Arts Institute, pp 749).

1 is a chromatographic result of PT fractions through Sephadex LH20 column chromatography.

An object of the present invention relates to a whitening cosmetic composition containing an extract of baekduong as a main component.

Another object of the present invention relates to a whitening cosmetic composition containing the extract of Baekduong as a main component, and containing the dermis, ginseng and licorice extract as an auxiliary component.

Still another object of the present invention is to contain the extract of baekduong as a main ingredient, ranuculin (ranunculin), deoxypodophyllotoxin obtained from the extract of baekduong, (deoxypodophyllotoxin), 3- O- α-L-ramnopyranosyl ( It provides a whitening cosmetic composition containing at least one component selected from 1 → 2)-[β-D-glucopyranosyl (1 → 4)]-α-L-arabinopyranoside (SB365).

Still another object of the present invention is to contain the extract of baekduong as a main ingredient, ranuculin (ranunculin), deoxypodophyllotoxin obtained from the extract of baekduong, (deoxypodophyllotoxin), 3- O- α-L-ramnopyranosyl ( 1 → 2)-[β-D-glucopyranosyl (1 → 4)]-α-L-arabinopyranoside (SB365) containing at least one component selected from the dermis and licorice extracts It is to provide a whitening cosmetic composition containing at least one auxiliary component.

Deoxypodophyllotoxin isolated from the baekduong extract is a known compound having the following structural formula.

Deoxypodophyllotoxin

Ranunculin isolated from baekduong extract is a known compound having the following structural formula.

Ranunculin

3- O- α-L-lamnopyranosyl (1 → 2)-[β-D-glucopyranosyl (1 → 4)]-α-L-arabinofyranoside (SB365) isolated from the Baekduong extract It is a known compound which has the following structural formula as hederagenin.

SB365

These materials have been identified by the present invention as having an excellent whitening effect.

In the present invention, an excipient commonly used in creams, liquids, injections, tablets, capsules, and the like may be added to form a conventional formulation.

In the present invention, the extraction solvent may be water, methanol, methanol, propanol, lower alkanols such as butanol, methylene chloride, acetone or a mixed solvent thereof.

Next, the present invention will be described in more detail as Examples and Experimental Examples.

Example 1

General Baekduong Extract Manufacturer

The fine baekduong was ground to 40-100 mesh, and then a predetermined amount was taken into an extractor and extracted with a low alcohol solution. The lower alcohols used were methanol, methanol, propanol, butanol, etc. Among them, aqueous ethanol solution showed the best selectivity for active substance extraction. In the case of using 50% aqueous ethanol solution, very polar substances and high molecular weight polymers were not extracted. The extraction temperature was 15 ° C. to 35 ° C. and the most suitable temperature ranged from 20 ° C. to 25 ° C. The extraction time was 1 hour to 3 hours was appropriate bar extraction. The extract extracted at 25 ° C. for 3 hours using 50% ethanol was labeled as an ET fraction and used as it was for the whitening effect.

Example 2

1. Separation of substances with whitening effect

On the other hand, the whitening effect was confirmed in clinical trials in humans in the following experimental examples. The separation of the whitening effect material was not possible for humans, so it was performed by the method of measuring tyrosinease inhibitory effect commonly used.

Acetone was added to the ET fraction to extract low-molecular and moderate-polar substances. The remaining portion was labeled as PT fraction and acetone extract as PA.

The PT and PA fractions inhibited 10% and 73% of their activities against tyrosinase at 5 mg / ml, respectively.

2. Active substance of PT fraction 1

The fraction 3 obtained when the PT fraction was re-fractionated in Sephadex was purified to obtain an active substance. The active substance was found to be hederagenin 3- O- α-L-lamnopyranosyl (1 → 2)-[β-D-glucopyranosyl (1 → 4)]-α-L-arabinofyranoside. . It is already patented as an anticancer substance (patent pending). This substance does not show activity against tyrosinase, but it shows excellent whitening effect in clinical practice. Considering the relationship between the saponin derivatives and the cell surface, it is assumed that the action of melanin is prevented from accumulating in keratinocytes by interfering with the movement of melanosomes.

3. Whitening substance of PT fraction 2

The SPX2 fraction obtained when the PT fraction was fractionated with Sephadex showed 48% inhibition of tyrosinase. This fraction was passed through a silica gel column again to identify a substance showing Rf = 0.3 in the silica gel thin film (developing solvent; methylene chloride / methanol = 4: 1). This Rf value was consistent with that of ranunculin. His NMR was the same as that of ranunculin, as the material was isolated in a known manner.

ranunculin is a substance that is widely distributed in ranunculus plants and is known to have mitotoxicity (Vonderbank, Pharmazie 5, 21 (1950)). Pure ranunculin showed 51% inhibitory effect on tyrosinase.

4. Active substance of PA fraction

Deoxypodophyllotoxin is contained in several kinds of plants such as Jeonho and shows mitotoxicity. In addition, foreign bodies have been reported to inhibit blood vessel formation in human navel endothelial cells (Yong Kim, Song-Bae Kim, byung-Zun Ahn, Deoxypodophyllotoxin; the cytotoxic and antiangiogenic component from Pulsatilla koreana Nakai, Planta Medica, 68, 271- 274 (2002) This substance inhibited the activity of tyrosinase by 38% at 0.03 ug / ml It is difficult to test at higher concentrations due to its low solubility in water.

Example 3

50 g of finely ground baekduong and 500 ml of 50% ethanol are added to the extractor, stirred at room temperature for 3 hours, and filtered. The filtrate is stored, and 50 ml of 50% ethanol is added to the plant residue, and extracted at room temperature for 3 hours. After filtration, the same extraction is repeated once again for the plant residue. The extract is dried under reduced pressure. Yield 23 g.

Example 4

Preparation of Tyrosinase Inhibitory Fractions PT and PA Fractions

Take 20g of this extract, add 200ml of acetone, suspend and shake for 10 minutes. Filtration gives acetone water content and dissolved content. Acetone insolubles are suspended in 200 ml of aceto, shaken for 10 minutes, and filtered. The insolubles are dried and labeled as PT fractions (17.4 g), and the acetone dissolved fractions are dried and labeled as PA fractions (3.5 g).

Example 5

Isolation of the Tyrosinase Inhibitor PTpur from PT Fraction

Fraction PT 560 mg was extracted using a Sepadex LH20 column (200 g, 60 × 4 cm) with methanol: water = 80: 20 at a flow rate of 1 ml per minute and a fraction of 0.5 ml per tube. It was done. These fractions were sequentially deposited on a thin layer of silica gel and then developed to divide the fractions (developing solvent; butanol: acetic acid: water = 4: 1: 1, color reaction; spraying sulfuric acid followed by heating). The results are shown in FIG. In FIG. 2, PT1 (139 mg, 24.8%) was collected in vitro Nos. 26-66 with four main spots and the lower one reacting with sulfuric acid and yellow color. PT2 (344 mg, 61.4%) is a collection of test tubes Nos. 66-91 and consists of two major spots. PT3 (61 mg, 10.9%) is a collection of test tubes 91-111, initially red when heated after sulfuric acid spray, and then blue when time passes, and have a spot with an average Rf value between 0.48 and 0.50. It is a fraction made into. PT4 (15.7 mg, 2.8%) is a collection of test tubes 111-138, and SPX3 and SPX4 are relatively pure fractions with one spot on the thin film. Fractions are shown in Figure 1.

Sephadex LH20 column chromatography gave SPX1, SPX2, SPX3 and SPX4.

SPX3 is a relatively pure fraction that is dissolved in 0.5 ml of water, dissolved and left to produce white crystals, which are dried (PTpur).

Example 5

Determination of the structure of PTpur

The isolated substance PTpur was identified as a glycoside in the white amorphous form of mp 239∼241 ° C., [α] _D = + 23.6 ° (c, 0.2, MeOH), in the Liberman-Bukart reaction. In addition, in IR (cm ^-1 ), it was observed at 3400 (br, -OH), 2940 (br, CH), 1695 (C = O), 1455, 1040 (CO), and absorption band of 1000-1100, 3000-3400 In our view, it is likely that it is a glycoside.

¹ H-NMR follows the NMR pattern of typical saponins, where 6 -CH ₃ groups were observed at 0.91, 0.92, 0.98, 1.00, 1.07, 1.21 ppm, and another -CH ₃ group was doublet at 1.64 ppm was observed as a doublet, from which it was estimated that there was one of rhamnos per constituent, with anomer protons of 6.25 (br.), 5.11 (1H, J = 7.80 Hz) and 4.97 ppm (1H, J = 6.66 Hz). Thus, PTpur was found to be a glycoside linked to three sugars.

^{In 13} C-NMR, hydroxymethyl group was observed at 65.4 ppm (C-23), and the three anomer carbon signals were 140.2 (C-1 ′), 106.7 (C-1 ′ ') and 101.7 ppm (C-1), respectively. ′), Two olefin carbons were observed at 122.5 ppm (C-12) and 144.8 ppm (C-13) and one carboxy carbon was observed at 180.2 ppm (C-28). Typically, glycosylation upfield shifts of about 4 Hz appear when the sugars are bound at the 28 position (180.2 ppm → 176.2 ppm), but the above compound is not found in the compound. I could be sure that this was not a combined glycoside.

Next, hydrolysis was performed in ethanol / sulfuric acid to confirm the composition of sugar and the structure of aglycone. Comparing the physicochemical data of the hydrolyzate, aglycone, and ¹³ C-NMR and ¹ H-NMR, PTpur was identified as hederagenin. In addition, the hydrolyzed sugars were found to be rhamnose, arabinose and glucose by comparative TLC.

As a result of comprehensive analysis of the above results and published literature, PTpur is a heparagenin 3- O- α-L-rhamnopyranosyl (1 → 2)-[β-, a saponin that has already been isolated from the plant. D-glucopyranosyl (1 → 4)]-α-L-arabinofyranoside was confirmed.

¹ H-NMR and ¹³ C-NMR data of PTpur are shown in Table 1 below.

Table 1.

Example 6

Isolation of PTculin, a Tyrosinase Inhibitor, from PT Fraction

When PT and ranunculin were developed on the silica gel thin film, it was confirmed that the same spot appeared at Rf = 0.3. (Solvent; methylene chloride / methanol = 4: 1).

Example 7

Isolation of PApur, a Tyrosinase Inhibitor, from PA.

30 mg of PA fraction was dissolved in 2 ml of methanol, and 3 ml of hexane was added thereto. Pour the hexane dissolve after standing. This extraction is repeated two more times. Hexanes were combined, dried, and then chromatographed with deoxypodophyllotoxin on a silica gel thin film. In the hexane lysate of PA extract, spots appearing at the same position as Deoxypodophyllotoxin were identified, and separated into preparative Silica gel thin films to obtain 3mg of deoxypodophyllotoxin, and the NMR measurement was the same as the standard product.

Next, the present invention will be described in more detail as formulation examples.

Formulation Example 1

emulsion

Composition (g) of emulsion;

Cetyl Octanoate 4.0

Cyclomethicone 3.0

Floating paraffin 4.0

Polysorbate 60 5.0

Sorbitan Stearate 3.0

The ET fraction 100mg, 400mg, 800mg, 1000mg each was homogeneously mixed in the above-mentioned agent (19g) and represented as Formulation P100, Formulation P400, Formulation P800, Formulation P1000mg.

Formulation Example 2

Injection

The subject is Baekduong, and the formula is supplemented with skin, ginseng and licorice, and extracted with 50% ethanol solution. The extract was lyophilized according to the preparation method of the injection.

Formulation Example 3

100 mg of the composition of Example 3

Starch 100mg

Lactose 50mg

Magnesium stearate proper amount

The above ingredients were filled into gelatin capsules according to the conventional method for preparing capsules to prepare capsules.

Formulation Example 4

100 mg of the composition of Example 3

Lactose 50mg

Magnesium stearate proper amount

Talc proper amount

Tablets were prepared by tableting the above components according to the conventional method for producing tablets.

Formulation Example 5

3.0 g of the composition of Example 3

Isomerized sugar 50.0g

Sodium Alginate 50.0mg

Sodium benzoate

Purified water

The above components were prepared according to a conventional method for preparing a liquid, and then filled into a 100 ml brown bottle to prepare a liquid.

Formulation Example 6

50 mg of the composition of Example 3

Ranunculus 10mg

Suitable amount of distilled water for injection

The above ingredients were filled into 1 ml ampoules according to a conventional method for preparing an injection and tightly prepared to prepare an injection.

Formulation Example 7

100 mg of the composition of Example 3

Deoxypodophyllotoxin 10mg

SB 365 10mg

Starch 100mg

Lactose 50mg

Magnesium stearate proper amount

The above ingredients were filled into gelatin capsules according to the conventional method for preparing capsules to prepare capsules.

Formulation Example 8

100 mg of the composition of Example 3

Deoxypodophyllotoxin 10mg

Lactose 50mg

Magnesium stearate proper amount

Talc proper amount

Tablets were prepared by tableting the above components according to the conventional method for producing tablets.

Formulation Example 9

3.0 g of the composition of Example 3

SB 365 100mg

Isomerized sugar 50.0g

Sodium Alginate 50.0mg

Sodium benzoate

Purified water

The above components were prepared according to a conventional method for preparing a liquid, and then filled into a 100 ml brown bottle to prepare a liquid.

Experimental Example 1

Clinical Whitening Effect of Baekduong Extract

The whitening effect was performed on 20 female volunteers. Ten of them had freckles on their face and ten were women with blemishes.

Ten women with freckles were labeled as A, A ', B, B', C, C ', D, D', E, E ', Formulation P100 for Group A, Formulation P400 for Group B, and Formulation P800 for Group C. , Formulation P1000 was applied to Group D, and finally only base was applied to Group E. The application location was a freckled face.

Ten women with blemishes were classified in the same way and the drugs and mechanisms were correct.

The application dose was 500 mg per 5x5 cm ² of formulation and base, with a frequency of application of 3 times a day. Each time you apply the drug, you have to wash your face. Drug application days were 3 weeks.

The effect was judged to be valid, somewhat effective and invalid by three observers comparing and observing from the beginning of the experiment.

The results are shown in Table 2.

Table 2. Effect of Pasqueflower Formulation on Freckles and Spotted Women

Freckles

A, A '(100 mg) A invalid, A' invalid G, G '(100 mg) G invalid, G' invalid

B, B '(400mg) B effective, B ′, somewhat effective H, H' (400mg) H somewhat effective, H 'somewhat effective

C, C '(600mg) C Treatment, C' Effective I, I '(600mg) I Treatment, I' Treatment

D, D '(800mg) D Treatment, D' Treatment J, J '(800mg) J Treatment, J' Treatment

E, E '(1000mg) E Treatment, E' Treatment K, K '(1000mg) K Treatment, K' Treatment

F, F '(base) invalid All L, L' (base) invalid

cure; Lack of freckles or blemishes; available; Overall reduction in freckles or blemishes; Freckles and blemishes slightly reduced

As can be seen in Table 2, the formulation P100 mixed with 100 mg of baekduong extract with 19 g of base did not show an effect in freckles group A, A '(100 mg), blemish group G, G' (100 mg), and B, B '( 400mg), H, H '(400mg) group is somewhat effective. At higher doses it is effective. In the case of blemishes, the treatment effect was more than 600mg group, and in the case of freckles, the treatment effect was more than 800g group.

Experimental Example 2

Clinical Whitening Effect of Injectables

Five women with blemishes were selected and labeled as A, B, C, D, and E. Each of them continued to inject 5.0 ml of the injection once a day for 3 days and then dosed the same again after 14 days. At the same time the study was administered. Females A, B, and C were cured at 5 days after injection and ointment application, and D and E were valid.

It can be seen that the administration of the injection is faster than the method of application to the skin without injection.

On the other hand, the whitening effect was confirmed in human clinical trials. Separation of the whitening effect material was not carried out by humans, and was performed by a commonly used tyrosinease inhibitory effect measuring method.

PT and PA fractions reduced their activity by 10% and 73% for tyrosinase at 5 mg / ml, respectively.

Pure ranunculin showed an inhibitory effect of 51% against tyrosinase.

Deoxypodophyllotoxin inhibited 38% of tyrosinase activity at 0.03 ug / ml. The low solubility in water made it difficult to experiment at higher concentrations.

Experimental Example 3

Clinical Study on Whitening Effect of Ranunculin, Deoxypodophyllotoxin, and SB365

Drug composition; Ranunculin 10 mg, Deoxypodophyllotoxin 50 mg, and SB365 15 mg are mixed with the above base (19 g) and rubbed on the spot area. Apply it three times a day until the blemish is cured.

A and B were treated at 10 days after drug administration and C was treated at 19 days after D. E was slightly effective but not treated.

Next, the drug was applied to five women (F, G, I, J, K) with the exception of SB365, which was ineffective in tyrosinase. FG was effective in 26 days, I was 35 days, and J and K were effective.

The clinical results show that SB365 does not inhibit tyrosinase but exhibits a whitening effect. SB365 may also act on melanocytes and inhibit the migration of melanin to keratinocytes. The identification of certain mechanisms will be of academic value.

By adding ginseng, licorice and milk white skin to the whitening whitening agent, the synergistic effect on the whitening whitening effect, as well as the skin protection through the bactericidal effect of saponin, and the skin cleaning and penetration effect are expected. Can be.

Experimental Example 4

Tyrosinase Inhibitory Activity

Tyrosinase was purchased from Sigma. It is dissolved in 3,4-dihydrophenylalanine sodium phosphate buffer (0.1M, pH 6.0), an enzyme substrate, and adjusted to a concentration of 1.6 mg / ml. The sample is dissolved in sodium phosphate buffer and the undissolved portion is removed by centrifugation. 0.7 ml of buffer, 0.2 ml of sample, and 0.1 ml of enzyme solution (15.7 unit / ml) were mixed, and after 60 seconds, transferred to a spectrophotometer to measure absorbance at 475 nm.

The tyrosinase inhibition rate (%) was calculated by the following equation.

% Inhibition of tyrosinase activity = [1- (S-B) / C] x 100

Where S; Enzyme and sample addition test tubes, B; Test tubes excluding enzymes from S, C; Test tubes with only sample removed from S.

Experimental Example 5

Inoculated with B-16 culture cells derived from mouse melanoma in an eagle MEM culture medium containing 10% of fetal bovine serum added to the concentration shown in Table 1 of the composition of Example 3 of the present invention. After culturing for 5 days under% CO2 conditions, the cells were dispersed with trypsin and centrifuged at 1,000 rpm for 5 minutes to collect the cells, and the blackness was determined by visual observation.

The criteria for determination are as follows.

-To the same extent as not adding the composition of Example 3

+: Slightly whitening

++: fairly white

+++: mostly whitening

TABLE 3

Amount (Concentration: Weight%) of the Composition of Example 3 Results

Control group--

0.01 +-

0.1 ++

0.3 ++

0.5 ++

0.7 +++

1.0 +++

As confirmed by the above results, it can be seen that the composition of the present invention is excellent in the whitening effect of melanin derived melanoma.

Experimental Example 6

Set up a 2x2cm area on the upper right side of the upper right arm of the test subjects (15 healthy males and 30 female volunteers, respectively) and clean the area under warm water with aluminum foil so that UV light is irradiated only on the site under test. The other parts were masked and irradiated with Toshiba Corporation's FL20SBLB lamps and FL20S E-30 lamps at a distance of 10 cm ^three times at a time of 0.8 × ¹⁰ erg / cm ³ / time / day. After irradiation, the site was coated with the sample shown in Table 2 three times a day (morning, day, night). In the evaluation, the degree of pigmentation after three weeks was visually determined, and the degree of improvement was evaluated in three stages of remarkable effect, effectiveness, and invalidity. The results are shown in Table 4 below.

Table 4

Component concentration (parts by weight)

Composition 0.5 of Example 3

Polyoxyethylene (40) monostearate 2.0

Glycerol Monostearate 5.0

Stearic Acid 5.0

Behenyl Alcohol 1.0

Liquid Paraffin 1.0

Griseryl Trioctanoate 10.0

1.3-bunylene glycol 5.0

Purified water

The components other than 1,3-butylene glycol and purified water were heated and dissolved (iii), and the mixed mixture was heated separately (iii) with 1,3-butylene glycol and purified water. The liquid phase was added to the oil phase, stirred, emulsified, and cooled to prepare a varnishing cream. (Baekduong extract varnishing cream)

Separately, except for Baekduong extract in Table 4 to prepare a varnishing cream was used as a control.

The results are shown in Table 5 below.

Table 5

Validity (person) Valid (person) Invalid (person)

Baekduong extract 17 10 3

Teen strip 0 3 27

From the above experimental results, it can be seen that the cosmetic containing the baekduong extract of the present invention has an excellent melanogenesis inhibitory effect.

The present invention contains the extract of baekduong as a main component, if necessary, ranunculin (ranunculin), deoxypodophyllotoxin obtained from the extract of baekduong, 3- O- α-L-lamnopyranosyl (1 → 2)-[β-D-glucopyranosyl (1 → 4)]-α-L-arabinofyranoside (SB365) containing at least one component selected from the skin and licorice extract 1 It is to provide a whitening cosmetic composition containing a paper-based auxiliary component, the composition of the present invention has an excellent whitening effect.

Claims (4)

Whitening cosmetic composition containing Baekduong extract as an active ingredient. In the extract of baekduong, ranunculin, deoxypodophyllotoxin, 3- O- α-L-rhamnopyranosyl (1 → 2)-[β-D-glucopyranosyl (1 →) 4)]-A-L-arabinopyranoside (SB365) A whitening cosmetic composition further comprising at least one component selected from. The whitening cosmetic composition according to claim 1 or 2, comprising an extract of at least one auxiliary ingredient selected from dermis, ginseng and licorice. A whitening cosmetic preparation prepared by adding a conventional excipient to the composition of claims 1 to 3 and formulated in a conventional formulation.